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Ding X, Li Y, Wang X, Li F, Xu Z, Zhu Y, Chen Z. Down-regulation of YAP prevents smoking- and alcohol-induced carcinogenesis of esophageal paracancerous tissue by promoting cellular pyroptosis. Sci Rep 2025; 15:14766. [PMID: 40295580 PMCID: PMC12037795 DOI: 10.1038/s41598-025-98952-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2024] [Accepted: 04/16/2025] [Indexed: 04/30/2025] Open
Abstract
Paracancerous tissues (PCTs) were previously considered benign regions, but recent findings reveal genomic instability in these areas. Smoking and alcohol consumption are closely associated with esophageal cancer (EC) development. This study explored the interplay between the Hippo pathway and pyroptosis in EC, PCTs, and distal normal tissues (DNTs). We used molecular epidemiological methods to analyze the effects of smoking and alcohol on these pathways. We found that key genes in both pathways were more altered in smokers and/or drinkers compared to non-smokers and non-drinkers. Additionally, we observed changes in some genes and proteins in PCTs, while the Hippo pathway and pyroptosis had not yet been influenced. We applied 4.0% alcohol combined with various concentrations of cigarette smoke extract (CSE) to PCTs cultured in vitro to observe carcinogenesis and changes in these pathways. Verteporfin, as an inhibitor of YAP, was also used in vitro culture experiments to observe its effects on cellular carcinogenesis. Among 56 EC patients, 41 had a history of smoking and/or alcohol consumption in this study. Compared to DNTs, Hippo pathway genes (Lats1, Yap, and Taz) and pyroptosis genes (Nlrp3, Asc, Gsdmd, and Caspase-1) were altered in 49 EC tissues, while changes of Lats1, Nlrp3, and Asc were observed in 47 PCTs. Additionally, 4.0% alcohol combined with 3.2%, 4.0%, and 5.8% CSE, respectively, not only induced cellular heterogeneity and even cancerous transformation, but also suppressed the Hippo pathway and pyroptosis in the PCTs cultured in vitro. Furthermore, in vitro, 9 μM verteporfin inhibited cellular heterogeneity/carcinogenesis in PCTs induced by 4.0% alcohol combined with 5.8% CSE through inhibiting YAP and promoting pyroptosis. It is speculated that the downregulation of YAP could prevent smoking- and alcohol-induced carcinogenesis in esophageal PCTs by promoting pyroptosis, which may offer new insights for the treatment of esophageal squamous carcinoma.
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Affiliation(s)
- Xinyu Ding
- Department of Thoracic Surgery, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, 200433, China
| | - Ying Li
- Department of Pathology, Second Affiliated Hospital of Naval Medical University, Shanghai, 200003, China
| | - Xuning Wang
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha, 410013, China
| | - Fan Li
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha, 410013, China
| | - Zhifei Xu
- Department of Thoracic Surgery, Second Affiliated Hospital of Naval Medical University, Shanghai, 200003, China
| | - Yongfei Zhu
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha, 410013, China.
| | - Zihao Chen
- Department of Thoracic Surgery, Second Affiliated Hospital of Naval Medical University, Shanghai, 200003, China.
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Perrotta G, Condrea D, Ghyselinck NB. Meiosis and retinoic acid in the mouse fetal gonads: An unforeseen twist. Curr Top Dev Biol 2024; 161:59-88. [PMID: 39870439 DOI: 10.1016/bs.ctdb.2024.10.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2025]
Abstract
In mammals, differentiation of germ cells is crucial for sexual reproduction, involving complex signaling pathways and environmental cues defined by the somatic cells of the gonads. This review examines the long-standing model positing that all-trans retinoic acid (ATRA) acts as a meiosis-inducing substance (MIS) in the fetal ovary by inducing expression of STRA8 in female germ cells, while CYP26B1 serves as a meiosis-preventing substance (MPS) in the fetal testis by degrading ATRA and preventing STRA8 expression in the male germ cells until postnatal development. Recent genetic studies in the mouse challenge this paradigm, revealing that meiosis initiation in female germ cells can occur independently of ATRA signaling, with key roles played by other intrinsic factors like DAZL and DMRT1, and extrinsic signals such as BMPs and vitamin C. Thus, ATRA can no longer be considered as 'the' long-searched MIS. Furthermore, evidence indicates that CYP26B1 does not prevent meiosis by degrading ATRA in the fetal testis, but acts by degrading an unidentified MIS or synthesizing an equally unknown MPS. By emphasizing the necessity of genetic loss-of-function approaches to accurately delineate the roles of signaling molecules such ATRA in vivo, this chapter calls for a reevaluation of the mechanisms instructing and preventing meiosis initiation in the fetal ovary and testis, respectively. It highlights the need for further research into the molecular identities of the signals involved in these processes.
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Affiliation(s)
- Giulia Perrotta
- Université de Strasbourg, IGBMC UMR 7104, Illkirch, France; CNRS, UMR 7104, Illkirch, France; Inserm, UMR-S 1258, Illkirch, France; IGBMC, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France
| | - Diana Condrea
- Université de Strasbourg, IGBMC UMR 7104, Illkirch, France; CNRS, UMR 7104, Illkirch, France; Inserm, UMR-S 1258, Illkirch, France; IGBMC, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France
| | - Norbert B Ghyselinck
- Université de Strasbourg, IGBMC UMR 7104, Illkirch, France; CNRS, UMR 7104, Illkirch, France; Inserm, UMR-S 1258, Illkirch, France; IGBMC, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.
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Bai M, Lei J, Li F, Wang X, Fu H, Yan Z, Huang X, Zhu Y. Short-chain chlorinated paraffins may induce thymic aging in mice by activating PERK-CHOP. ENVIRONMENTAL POLLUTION (BARKING, ESSEX : 1987) 2024; 357:124438. [PMID: 38942270 DOI: 10.1016/j.envpol.2024.124438] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/25/2024] [Revised: 06/10/2024] [Accepted: 06/23/2024] [Indexed: 06/30/2024]
Abstract
Humans indirectly consume approximately 0.02 mg/kg/day of short-chained chlorinated paraffins (SCCPs) through the environment; however, the thymic senescence/damage induced by SCCPs has not been assessed. In this study, 16 female mice (4-week-old) per group were orally administered 0, 0.01, 0.1, and 1 mg/kg/day of SCCPs for 21 days, and the phenotypes and levels of superoxide dismutase (SOD), malondialdehyde (MDA), Tβ4, αβ TCR, SA-β-Gal, GRP78, PERK/CHOP, P53/P21, and CASPASE-1 of the thymus were assessed as indicators. Another group comprising 16 mice was killed at 4-week-old and these indicators were assessed. Thereafter, the thymuses cultured in vitro were exposed to 0, 14, 140, and 1400 μg/L SCCPs, respectively, and the above indicators were measured after 7-day. Based on the results, the oral administration of ≥0.01 mg/kg/day SCCPs to mice and ≥14 μg/L of SCCPs in medium caused thymic aging features, such as a decrease in the ratio of cortex to medulla, gradual blurring of the boundary between the cortex and medulla, dose-dependent oxidative stress (decreased SOD and increased MDA), and decreased levels of Tβ4 and αβ TCRs in the thymus. The oral administration of ≥1 mg/kg/day of SCCPs also impeded the growth and development of female mice and their thymuses. Exposure to the low levels of SCCPs activated PERK-CHOP in the mouse thymus, which modulated increases in SA-β-Gal, IL-1β, P53, and CASPASE-1 in vivo and in vitro. Overall, environmental levels and human blood concentrations (14.8-1400 μg/L) of SCCPs may induce mouse thymus senescence by activating PERK-CHOP in vivo and in vitro, respectively.
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Affiliation(s)
- Mingxin Bai
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha, 410013, Hunan, China
| | - Jiawei Lei
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha, 410013, Hunan, China
| | - Fan Li
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha, 410013, Hunan, China
| | - Xuning Wang
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha, 410013, Hunan, China
| | - Hu Fu
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha, 410013, Hunan, China; Key Laboratory of Protein Chemistry and Fish Developmental Biology of Ministry of Education, Hunan Normal University, Changsha, 410081, China
| | - Zhengli Yan
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha, 410013, Hunan, China; Key Laboratory of Protein Chemistry and Fish Developmental Biology of Ministry of Education, Hunan Normal University, Changsha, 410081, China
| | - Xin Huang
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha, 410013, Hunan, China.
| | - Yongfei Zhu
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha, 410013, Hunan, China; Key Laboratory of Protein Chemistry and Fish Developmental Biology of Ministry of Education, Hunan Normal University, Changsha, 410081, China.
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Baalbaki G, Lim V, Gillet AP, Verner MA, Vaillancourt C, Caron-Beaudoin E, Delbes G. Trace elements alone or in mixtures associated with unconventional natural gas exploitation affect rat fetal steroidogenesis and testicular development in vitro. ENVIRONMENTAL POLLUTION (BARKING, ESSEX : 1987) 2024; 357:124393. [PMID: 38901820 DOI: 10.1016/j.envpol.2024.124393] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/06/2024] [Revised: 05/30/2024] [Accepted: 06/17/2024] [Indexed: 06/22/2024]
Abstract
Biomonitoring studies have shown that pregnant women living in regions of unconventional natural gas (UNG) exploitation have higher levels of trace elements. Whether developmental endocrine disruption can be expected at these exposure levels during pregnancy is unclear. In this study, we aimed to test the impact of five trace elements alone or in mixtures using in vitro cell- and tissue-based assays relevant to endocrine disruption and development. Manganese, aluminum, strontium, barium, and cobalt were tested at concentrations including those representatives of human fetal exposure. Using transactivation assays, none of the tested elements nor their mixture altered the human estrogen receptor 1 or androgen receptor genomic signalling. In the rat fetal testis assay, an organ culture system, cobalt (5 μg/l), barium (500 μg/l) and strontium (500 μg/l) significantly increased testosterone secretion. Cobalt and strontium were associated with hyperplasia and/or hypertrophy of fetal Leydig cells. Mixing the five elements at concentrations where none had an effect individually stimulated testosterone secretion by the rat fetal testis paralleled by the significant increase of 3β-hydroxysteroid dehydrogenase protein level in comparison to the vehicle control. The mechanisms involved may be specific to the fetal testis as no effect was observed in the steroidogenic H295R cells. Our data suggest that some trace elements in mixture at concentrations representative of human fetal exposure can impact testis development and function. This study highlights the potential risk posed by UNG operations, especially for the most vulnerable populations, pregnant individuals, and their fetus.
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Affiliation(s)
- Ghida Baalbaki
- Institut National de la Recherche Scientifique (INRS), Centre Armand Frappier Santé Biotechnologie, Laval, QC, Canada
| | - Victoria Lim
- Institut National de la Recherche Scientifique (INRS), Centre Armand Frappier Santé Biotechnologie, Laval, QC, Canada
| | - Antoine P Gillet
- Institut National de la Recherche Scientifique (INRS), Centre Armand Frappier Santé Biotechnologie, Laval, QC, Canada
| | - Marc-André Verner
- Université de Montréal, Department of Occupational and Environmental Health, Montreal, QC, Canada; Centre de Recherche en Santé Publique, Université de Montréal et CIUSSS du Centre-Sud-de-l'Île-de-Montréal, Montreal, QC, Canada
| | - Cathy Vaillancourt
- Institut National de la Recherche Scientifique (INRS), Centre Armand Frappier Santé Biotechnologie, Laval, QC, Canada; Research Center, CIUSSS du Nord-de-l'Île-de-Montréal, Montreal, QC, Canada
| | - Elyse Caron-Beaudoin
- Department of Health and Society, University of Toronto Scarborough, Toronto, ON, Canada; Department of Physical and Environmental Sciences, University of Toronto Scarborough, Toronto, ON, Canada; Department of Pharmacology & Toxicology, University of Toronto, Toronto, ON, Canada
| | - Geraldine Delbes
- Institut National de la Recherche Scientifique (INRS), Centre Armand Frappier Santé Biotechnologie, Laval, QC, Canada.
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Li F, Wang X, Zhang J, Nie H, He S, Li Y, Xia R, Zhu Y. Low levels of Cd 2+ combined with procymidone may cause ovarian damage in mice via unfolded protein response. ENVIRONMENTAL TOXICOLOGY 2024; 39:3160-3171. [PMID: 38323353 DOI: 10.1002/tox.24169] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/14/2023] [Revised: 10/28/2023] [Accepted: 01/26/2024] [Indexed: 02/08/2024]
Abstract
As no study about the combined effect of low levels of Cd2+ with procymidone (PCM) on organs and organisms, we investigated their actions on mouse-ovary in vivo and in vitro. Four-week mice were treated with corn oil for the control group, corn oil + 0.0045 mg/L Cd2+ (CdCl2 was dissolved in ultrapure water and freely consumed by mice) for Cd2+ group, 50 mg/kg/d PCM (suspended in corn oil and administered orally to mice) for PCM group, and 50 mg/kg/d PCM + 0.0015 (0.0045 and 0.0135) mg/L Cd2+ for L+ (M+ and H+) PCM group for 21 days. For in vitro experiment, the cultured ovaries were treated with acetone for the control group, 0.1% acetone + 8.4 μg/L Cd2+ for the Cd2+ group, 0.63 mg/L PCM (dissolved in acetone) for the PCM-group, and 0.63 mg/L PCM + 2.8 (8.4 and 25.2) μg/L Cd2+ for L+ (M+ and H+) PCM group for 7 days. Mouse body weight in each treatment group, the weight and volume of ovaries in all PCM groups were lower than the control. Both in vivo and in vitro, all-stage follicle numbers were lower in M+PCM and H+PCM groups, whereas the atretic follicles and CASPASE3/8 were higher; meanwhile, lower estradiol and progesterone and higher unfolded protein response (UPR) members in all PCM groups. L+, M+, and H+PCM groups had further ovarian damage and stronger UPR than PCM groups, as did M+PCM groups over Cd2+ groups. It is hypothesized low-level PCM and Cd2+ may mutually promote each other's triggered UPR and exacerbate ovarian damage.
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Affiliation(s)
- Fan Li
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha, China
| | - Xuning Wang
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha, China
| | - Jiaxin Zhang
- Department of Clinical Medicine, Medical School, Hunan Normal University, Changsha, China
| | - Hui Nie
- Department of Clinical Medicine, Medical School, Hunan Normal University, Changsha, China
| | - Shiyun He
- Department of Clinical Medicine, Medical School, Hunan Normal University, Changsha, China
| | - Yushan Li
- Department of Clinical Medicine, Medical School, Hunan Normal University, Changsha, China
| | - Ruowen Xia
- Department of Clinical Medicine, Medical School, Hunan Normal University, Changsha, China
| | - Yongfei Zhu
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha, China
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Li R, Li F, Wang X, Bai M, Fu H, Yan Z, Yang X, Zhu Y. 4-Phenylbutyric acid may prevent mouse ovarian and uterine damage due to procymidone-induced alteration of circRNA Scar and circZc3h4 levels by controlling excessive unfolded protein response. PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 2023; 196:105631. [PMID: 37945263 DOI: 10.1016/j.pestbp.2023.105631] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/30/2023] [Revised: 09/17/2023] [Accepted: 09/26/2023] [Indexed: 11/12/2023]
Abstract
Procymidone (PCM) below the no-observed-adverse-effect-level (NOAEL) has previously been proven to induce ovarian and uterine damage in adolescent mice due to its raised circRNA Scar, decreased circZc3h4, and overactivated unfolded protein response (UPR). Also, 4-phenylbutyric acid (4-PBA) inhibits histone deacetylase and endoplasmic reticulum stress, reduces UPR, improves metabolism, and ensures homeostasis within the endoplasmic reticulum. In this study, 20, 40 and 80 mM of 4-PBA were utilized respectively to intervene the damage caused by 1.0 × 10-5 M PCM to ovaries and uterus in vitro culture. Besides, 100 mg/kg /d 4-PBA was intraperitoneally injected to female adolescent mice before, during and after oral administration of 100 mg/kg /d PCM for prevention and cure to observe tissue changes in the ovaries and uteri, and levels of circRNA Scar, circZc3h4 and UPR members. Our findings demonstrated that in vitro experiments, all doses of 4-PBA could inhibit ovarian and uterine damage caused by PCM, and the effect of 80 mM was especially noticeable. In the in vivo experiments, the best results were obtained when PCM was given with simultaneous 4-PBA intervention, i.e., minimal ovarian and uterine damage. Both in vivo and in vitro, 4-PBA in the ovary and uterus resulted in decreased circRNA Scar levels, increased circZc3h4 abundance, and moderately elevated levels of UPR members. So, it is suggested that 4-PBA moderately activates UPR, partially or completely antagonizing the elevated circRNA Scar and decreased circZc3h4 and consequently preventing PCM-induced ovarian and uterine damage effectively in adolescent mice.
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Affiliation(s)
- Rui Li
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical school, Hunan Normal University, Changsha 410013, Hunan, China; College of Medicine, Yichun University, Yichun 336000, Jiangxi, China
| | - Fan Li
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical school, Hunan Normal University, Changsha 410013, Hunan, China
| | - Xuning Wang
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical school, Hunan Normal University, Changsha 410013, Hunan, China
| | - Mingxin Bai
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical school, Hunan Normal University, Changsha 410013, Hunan, China
| | - Hu Fu
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical school, Hunan Normal University, Changsha 410013, Hunan, China; Key Laboratory of Protein Chemistry and Fish Developmental Biology of Ministry of Education, Hunan Normal University, Changsha, 410081, China
| | - Zhengli Yan
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical school, Hunan Normal University, Changsha 410013, Hunan, China; Key Laboratory of Protein Chemistry and Fish Developmental Biology of Ministry of Education, Hunan Normal University, Changsha, 410081, China
| | - Xiaoping Yang
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research of Ministry of Education, Department of Pharmacy, School of Medicine, Hunan Normal University, Changsha, Hunan, China; Key Laboratory of Protein Chemistry and Fish Developmental Biology of Ministry of Education, Hunan Normal University, Changsha, 410081, China.
| | - Yongfei Zhu
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical school, Hunan Normal University, Changsha 410013, Hunan, China; Key Laboratory of Protein Chemistry and Fish Developmental Biology of Ministry of Education, Hunan Normal University, Changsha, 410081, China.
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Hau RK, Wright SH, Cherrington NJ. In Vitro and In Vivo Models for Drug Transport Across the Blood-Testis Barrier. Drug Metab Dispos 2023; 51:1157-1168. [PMID: 37258305 PMCID: PMC10449102 DOI: 10.1124/dmd.123.001288] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2023] [Revised: 05/10/2023] [Accepted: 05/25/2023] [Indexed: 06/02/2023] Open
Abstract
The blood-testis barrier (BTB) is a selectively permeable membrane barrier formed by adjacent Sertoli cells (SCs) in the seminiferous tubules of the testes that develops intercellular junctional complexes to protect developing germ cells from external pressures. However, due to this inherent defense mechanism, the seminiferous tubule lumen can act as a pharmacological sanctuary site for latent viruses (e.g., Ebola, Zika) and cancers (e.g., leukemia). Therefore, it is critical to identify and evaluate BTB carrier-mediated drug delivery pathways to successfully treat these viruses and cancers. Many drugs are unable to effectively cross cell membranes without assistance from carrier proteins like transporters because they are large, polar, and often carry a charge at physiologic pH. SCs express transporters that selectively permit endogenous compounds, such as carnitine or nucleosides, across the BTB to support normal physiologic activity, although reproductive toxicants can also use these pathways, thereby circumventing the BTB. Certain xenobiotics, including select cancer therapeutics, antivirals, contraceptives, and environmental toxicants, are known to accumulate within the male genital tract and cause testicular toxicity; however, the transport pathways by which these compounds circumvent the BTB are largely unknown. Consequently, there is a need to identify the clinically relevant BTB transport pathways in in vitro and in vivo BTB models that recapitulate human pharmacokinetics and pharmacodynamics for these xenobiotics. This review summarizes the various in vitro and in vivo models of the BTB reported in the literature and highlights the strengths and weaknesses of certain models for drug disposition studies. SIGNIFICANCE STATEMENT: Drug disposition to the testes is influenced by the physical, physiological, and immunological components of the blood-testis barrier (BTB). But many compounds are known to cross the BTB by transporters, resulting in pharmacological and/or toxicological effects in the testes. Therefore, models that assess drug transport across the human BTB must adequately account for these confounding factors. This review identifies and discusses the benefits and limitations of various in vitro and in vivo BTB models for preclinical drug disposition studies.
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Affiliation(s)
- Raymond K Hau
- College of Pharmacy, Department of Pharmacology & Toxicology, (R.K.H., N.J.C.) and College of Medicine, Department of Physiology, The University of Arizona, Tucson, Arizona (S.H.W.)
| | - Stephen H Wright
- College of Pharmacy, Department of Pharmacology & Toxicology, (R.K.H., N.J.C.) and College of Medicine, Department of Physiology, The University of Arizona, Tucson, Arizona (S.H.W.)
| | - Nathan J Cherrington
- College of Pharmacy, Department of Pharmacology & Toxicology, (R.K.H., N.J.C.) and College of Medicine, Department of Physiology, The University of Arizona, Tucson, Arizona (S.H.W.)
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Wang Q, Xin B, Wang X, Li F, Fu H, Yan Z, Zhu Y. TT-10 may elevate YAP and repair mouse uterine damage resulting from the inhibition effect of ibuprofen on COX2-PGE2 and YAP. Toxicol Lett 2023; 383:215-226. [PMID: 37453669 DOI: 10.1016/j.toxlet.2023.07.008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2022] [Revised: 06/23/2023] [Accepted: 07/11/2023] [Indexed: 07/18/2023]
Abstract
Ibuprofen (IBU) is an emerging environmental contaminant that, in high doses, can damage reproductive organs in humans and other mammals. Recently, its effects on the uterus have been investigated. It is known that the COX2-PGE2 pathway and Yes-associated protein (YAP) are involved in female reproductive organ development and form a COX2-PGE2-EP2-Gas-β-catenin-YAP-COX2 positive feedback loop, in addition, TT-10, a pharmacological product, has been found to increase YAP. In this study, IBU was orally administrated to female mice for 7 d at doses of 0, 50, 100, and 200 mg/kg·bw/day (control, low, medium, and high doses, respectively). In addition, 0, 50, 100, and 200 μmol/L IBU was added in vitro to cultured uterine cells for 7 d at control, low, medium, and high doses, respectively; then, 0, 5, 10, and 20 μmol/L TT-10 were given to the in vitro uterine culture containing 100 μmol/L IBU to observe the effect of YAP activation. The results showed that medium and high doses of IBU inhibited the COX2-PGE2 pathway, decreasing YAP and increasing pYAP, leading to reduced circPVT1, elevated miR-149, and increased apoptosis, ultimately damaging the uterus. Conversely, 10 μmol/L TT-10 maximally enhanced YAP, which regulated COX2-PGE2 pathway activation, increased circPVT1, and decreased miR-149, and promoted cell proliferation, preventing uterine damage. This suggests that IBU may cause uterine damage by inhibiting the COX2-PGE2 pathway and YAP, and that appropriate doses of TT-10 may repair this damage by elevating YAP and stimulating COX2 via the feedback loop.
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Affiliation(s)
- Qing Wang
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical school, Hunan Normal University, Changsha 410013, China
| | - Bingyan Xin
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical school, Hunan Normal University, Changsha 410013, China
| | - Xuning Wang
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical school, Hunan Normal University, Changsha 410013, China
| | - Fan Li
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical school, Hunan Normal University, Changsha 410013, China
| | - Hu Fu
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical school, Hunan Normal University, Changsha 410013, China
| | - Zhengli Yan
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical school, Hunan Normal University, Changsha 410013, China
| | - Yongfei Zhu
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Department of Preventive Medicine, Medical school, Hunan Normal University, Changsha 410013, China.
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Pryzhkova MV, Boers R, Jordan PW. Modeling Human Gonad Development in Organoids. Tissue Eng Regen Med 2022; 19:1185-1206. [PMID: 36350469 PMCID: PMC9679106 DOI: 10.1007/s13770-022-00492-y] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2022] [Revised: 09/08/2022] [Accepted: 09/17/2022] [Indexed: 11/11/2022] Open
Abstract
BACKGROUND Our learning about human reproductive development is greatly hampered due to the absence of an adequate model. Animal studies cannot truthfully recapitulate human developmental processes, and studies of human fetal tissues are limited by their availability and ethical restrictions. Innovative three-dimensional (3D) organoid technology utilizing human pluripotent stem cells (hPSCs) offered a new approach to study tissue and organ development in vitro. However, a system for modeling human gonad development has not been established, thus, limiting our ability to study causes of infertility. METHODS In our study we utilized the 3D hPSC organoid culture in mini-spin bioreactors. Relying on intrinsic self-organizing and differentiation capabilities of stem cells, we explored whether organoids could mimic the development of human embryonic and fetal gonad. RESULTS We have developed a simple, bioreactor-based organoid system for modeling early human gonad development. Male hPSC-derived organoids follow the embryonic gonad developmental trajectory and differentiate into multipotent progenitors, which further specialize into testicular supporting and interstitial cells. We demonstrated functional activity of the generated cell types by analyzing the expression of cell type-specific markers. Furthermore, the specification of gonadal progenitors in organoid culture was accompanied by the characteristic architectural tissue organization. CONCLUSION This organoid system opens the opportunity for detailed studies of human gonad and germ cell development that can advance our understanding of sex development disorders. Implementation of human gonad organoid technology could be extended to modeling causes of infertility and regenerative medicine applications.
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Affiliation(s)
- Marina V Pryzhkova
- Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, MD, 21205, USA.
- Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA.
| | - Romina Boers
- Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, MD, 21205, USA
- Department of Molecular Cell Biology and Immunology, Amsterdam Universitair Medische Centra, 1117 HV, Amsterdam, The Netherlands
| | - Philip W Jordan
- Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, MD, 21205, USA.
- Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, MD, 20814, USA.
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10
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Alhasnani MA, Loeb S, Hall SJ, Caruolo Z, Simmonds F, Solano AE, Spade DJ. Interaction between mono-(2-ethylhexyl) phthalate and retinoic acid alters Sertoli cell development during fetal mouse testis cord morphogenesis. Curr Res Toxicol 2022; 3:100087. [PMID: 36189433 PMCID: PMC9520016 DOI: 10.1016/j.crtox.2022.100087] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2022] [Revised: 09/17/2022] [Accepted: 09/17/2022] [Indexed: 11/24/2022] Open
Abstract
Phthalic acid esters (phthalates) are a class of industrial chemicals that cause developmental and reproductive toxicity, but there are significant gaps in knowledge of phthalate toxicity mechanisms. There is evidence that phthalates disrupt retinoic acid signaling in the fetal testis, potentially disrupting control of spatial and temporal patterns of testis development. Our goal was to determine how a phthalate would interact with retinoic acid signaling during fetal mouse testis development. We hypothesized that mono-(2-ethylhexyl) phthalate (MEHP) would exacerbate the adverse effect of all-trans retinoic acid (ATRA) on seminiferous cord development in the mouse fetal testis. To test this hypothesis, gestational day (GD) 14 C57BL/6 mouse testes were isolated and cultured on media containing MEHP, ATRA, or a combination of both compounds. Cultured testes were collected for global transcriptome analysis after one day in culture and for histology and immunofluorescent analysis of Sertoli cell differentiation after three days in culture. ATRA disrupted seminiferous cord morphogenesis and induced aberrant FOXL2 expression. MEHP alone had no significant effect on cord development, but combined exposure to MEHP and ATRA increased the number of FOXL2-positive cells, reduced seminiferous cord number, and increased testosterone levels, beyond the effect of ATRA alone. In RNA-seq analysis, ATRA treatment and MEHP treatment resulted in differential expression of genes 510 and 134 genes, respectively, including 70 common differentially expressed genes (DEGs) between the two treatments, including genes with known roles in fetal testis development. MEHP DEGs included RAR target genes, genes involved in angiogenesis, and developmental patterning genes, including members of the homeobox superfamily. These results support the hypothesis that MEHP modulates retinoic acid signaling in the mouse fetal testis and provide insight into potential mechanisms by which phthalates disrupt seminiferous cord morphogenesis.
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Key Words
- ATRA, All-trans retinoic acid. CAS # 302-79-4
- DMSO, dimethyl sulfoxide
- Fetal testis development
- GD, gestational day
- GO, Gene Ontology
- IPA, Ingenuity Pathway Analysis
- ITCN, Image-based Tool for Counting Nuclei
- MEHP, mono-(2-ethylheyxl) phthalate. CAS # 4376-20-9
- MNGs, multinucleated germ cells
- PVC, polyvinyl chloride
- Phthalate toxicity
- Retinoic acid
- Sertoli cell
- TDS, testicular dysgenesis syndrome
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Affiliation(s)
- Maha A. Alhasnani
- Department of Pathology and Laboratory Medicine, Brown University, Box G-E5, Providence, RI 02912, USA
| | - Skylar Loeb
- Department of Pathology and Laboratory Medicine, Brown University, Box G-E5, Providence, RI 02912, USA
| | - Susan J. Hall
- Department of Pathology and Laboratory Medicine, Brown University, Box G-E5, Providence, RI 02912, USA
| | - Zachary Caruolo
- Department of Pathology and Laboratory Medicine, Brown University, Box G-E5, Providence, RI 02912, USA
| | - Faith Simmonds
- Department of Pathology and Laboratory Medicine, Brown University, Box G-E5, Providence, RI 02912, USA
| | - Amanda E. Solano
- Department of Pathology and Laboratory Medicine, Brown University, Box G-E5, Providence, RI 02912, USA
| | - Daniel J. Spade
- Department of Pathology and Laboratory Medicine, Brown University, Box G-E5, Providence, RI 02912, USA
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11
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Planinić A, Marić T, Bojanac AK, Ježek D. Reinke crystals: Hallmarks of adult Leydig cells in humans. Andrology 2022; 10:1107-1120. [DOI: 10.1111/andr.13201] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2022] [Revised: 04/26/2022] [Accepted: 05/23/2022] [Indexed: 11/30/2022]
Affiliation(s)
- Ana Planinić
- Department of Histology and Embryology University of Zagreb School of Medicine
- Scientific Centre of Excellence for Reproductive and Regenerative Medicine University of Zagreb School of Medicine
| | - Tihana Marić
- Scientific Centre of Excellence for Reproductive and Regenerative Medicine University of Zagreb School of Medicine
- Department of Medical Biology University of Zagreb School of Medicine
| | - Ana Katušić Bojanac
- Scientific Centre of Excellence for Reproductive and Regenerative Medicine University of Zagreb School of Medicine
- Department of Medical Biology University of Zagreb School of Medicine
| | - Davor Ježek
- Department of Histology and Embryology University of Zagreb School of Medicine
- Scientific Centre of Excellence for Reproductive and Regenerative Medicine University of Zagreb School of Medicine
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12
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Tran KTD, Valli-Pulaski H, Colvin A, Orwig KE. Male fertility preservation and restoration strategies for patients undergoing gonadotoxic therapies†. Biol Reprod 2022; 107:382-405. [PMID: 35403667 PMCID: PMC9382377 DOI: 10.1093/biolre/ioac072] [Citation(s) in RCA: 29] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2021] [Revised: 03/29/2022] [Accepted: 04/04/2022] [Indexed: 11/22/2022] Open
Abstract
Medical treatments for cancers or other conditions can lead to permanent infertility. Infertility is an insidious disease that impacts not only the ability to have a biological child but also the emotional well-being of the infertile individuals, relationships, finances, and overall health. Therefore, all patients should be educated about the effects of their medical treatments on future fertility and about fertility preservation options. The standard fertility preservation option for adolescent and adult men is sperm cryopreservation. Sperms can be frozen and stored for a long period, thawed at a later date, and used to achieve pregnancy with existing assisted reproductive technologies. However, sperm cryopreservation is not applicable for prepubertal patients who do not yet produce sperm. The only fertility preservation option available to prepubertal boys is testicular tissue cryopreservation. Next-generation technologies are being developed to mature those testicular cells or tissues to produce fertilization-competent sperms. When sperm and testicular tissues are not available for fertility preservation, inducing pluripotent stem cells derived from somatic cells, such as blood or skin, may provide an alternative path to produce sperms through a process call in vitro gametogenesis. This review describes standard and experimental options to preserve male fertility as well as the experimental options to produce functional spermatids or sperms from immature cryopreserved testicular tissues or somatic cells.
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Affiliation(s)
- Kien T D Tran
- Molecular Genetics and Developmental Biology Graduate Program, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA,Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA,Magee-Womens Research Institute, Pittsburgh, PA, USA
| | - Hanna Valli-Pulaski
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA,Magee-Womens Research Institute, Pittsburgh, PA, USA
| | - Amanda Colvin
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA,Magee-Womens Research Institute, Pittsburgh, PA, USA
| | - Kyle E Orwig
- Correspondence: Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, PA 15213, USA. Tel: 412-641-2460; E-mail:
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13
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Li R, Xin B, Wang Q, Wang Z, Fu H, Yan Z, Zhu Y. Combined effect of unfolded protein response and circZc3h4, circRNA Scar in mouse ovary and uterus damage induced by procymidone. ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 2022; 229:113068. [PMID: 34902777 DOI: 10.1016/j.ecoenv.2021.113068] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/24/2021] [Revised: 11/22/2021] [Accepted: 12/05/2021] [Indexed: 06/14/2023]
Abstract
Procymidone (PCM) is a fungicide commonly used to prevent and control plant diseases, and it is also an environmental endocrine disruptor that has a typical anti-androgen effect on the function and/or structure of the vertebrate reproductive system. The activation of the unfolded protein response (UPR) will fold the protein correctly to ensure the cell's survival. PCM regulates GRP78 by affecting the level of hormones, and there is a regulatory relationship between the UPR, the circRNAs and the miRNAs. In vivo experiments, PCM (suspended in soybean oil) was orally administered to adolescent female mice for 21 days in 3 different doses of 50 mg kg-1 day-1 (low dose), 100 mg kg-1 day-1 (medium dose) and 200 mg kg-1 day-1 (high dose) to cause ovaries and uteruses damage, and in vitro experiments, various doses of PCM from 0.33 × 10-5 (low dose) to 1 × 10-5 (medium dose) then 3 × 10-5 M (high dose) were used to induce injury on the ovaries and uteri of the mice. We found out that both in vivo and in vitro, PCM caused dose-dependent damages to the ovaries and uteri, increased their circRNA Scar levels and decreased circZc3h4 abundance. Also, all UPR signaling pathways in the low-dose group and some in the middle-dose group were activated. It is speculated that UPR may antagonize the partial ovarian and uterine damage in adolescent mice induced by PCM at doses less than NOAEL via changes in circZc3h4 and circRNA Scar.
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Affiliation(s)
- Rui Li
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha 410013, China; Key Laboratory of Protein Chemistry and Developmental Biology of Fish of Ministry of Education, Hunan Normal University, Changsha 410081, China; Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha 410013, China
| | - Bingyan Xin
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha 410013, China; Key Laboratory of Protein Chemistry and Developmental Biology of Fish of Ministry of Education, Hunan Normal University, Changsha 410081, China; Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha 410013, China
| | - Qing Wang
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha 410013, China; Key Laboratory of Protein Chemistry and Developmental Biology of Fish of Ministry of Education, Hunan Normal University, Changsha 410081, China; Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha 410013, China
| | - Zhen Wang
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha 410013, China; Key Laboratory of Protein Chemistry and Developmental Biology of Fish of Ministry of Education, Hunan Normal University, Changsha 410081, China; Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha 410013, China
| | - Hu Fu
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha 410013, China; Key Laboratory of Protein Chemistry and Developmental Biology of Fish of Ministry of Education, Hunan Normal University, Changsha 410081, China; Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha 410013, China
| | - Zhengli Yan
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha 410013, China; Key Laboratory of Protein Chemistry and Developmental Biology of Fish of Ministry of Education, Hunan Normal University, Changsha 410081, China; Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha 410013, China
| | - Yongfei Zhu
- Key Laboratory of Study and Discovery of Small Targeted Molecules of Hunan Province, Medical School, Hunan Normal University, Changsha 410013, China; Key Laboratory of Protein Chemistry and Developmental Biology of Fish of Ministry of Education, Hunan Normal University, Changsha 410081, China; Department of Preventive Medicine, Medical School, Hunan Normal University, Changsha 410013, China.
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14
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Jorgensen A, Svingen T, Miles H, Chetty T, Stukenborg JB, Mitchell RT. Environmental Impacts on Male Reproductive Development: Lessons from Experimental Models. Horm Res Paediatr 2021; 96:190-206. [PMID: 34607330 DOI: 10.1159/000519964] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/29/2021] [Accepted: 09/11/2021] [Indexed: 11/19/2022] Open
Abstract
BACKGROUND Male reproductive development in mammals can be divided into a gonadal formation phase followed by a hormone-driven differentiation phase. Failure of these processes may result in Differences in Sex Development (DSD), which may include abnormalities of the male reproductive tract, including cryptorchidism, hypospadias, infertility, and testicular germ cell cancer (TGCC). These disorders are also considered to be part of a testicular dysgenesis syndrome (TDS) in males. Whilst DSDs are considered to result primarily from genetic abnormalities, the development of TDS disorders is frequently associated with environmental factors. SUMMARY In this review, we will discuss the development of the male reproductive system in relation to DSD and TDS. We will also describe the experimental systems, including studies involving animals and human tissues or cells that can be used to investigate the role of environmental factors in inducing male reproductive disorders. We will discuss recent studies investigating the impact of environmental chemicals (e.g., phthalates and bisphenols), lifestyle factors (e.g., smoking) and pharmaceuticals (e.g., analgesics) on foetal testis development. Finally, we will describe the evidence, involving experimental and epidemiologic approaches, for a role of environmental factors in the development of specific male reproductive disorders, including cryptorchidism, hypospadias, and TGCC. KEY MESSAGES Environmental exposures can impact the development and function of the male reproductive system in humans. Epidemiology studies and experimental approaches using human tissues are important to translate findings from animal studies and account for species differences in response to environmental exposures.
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Affiliation(s)
- Anne Jorgensen
- Department of Growth and Reproduction, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark
| | - Terje Svingen
- Division of Diet, Disease Prevention and Toxicology, National Food Institute, Technical University of Denmark, Kongens Lyngby, Denmark
| | - Harriet Miles
- Royal Hospital for Children and Young People, Edinburgh, UK
| | - Tarini Chetty
- Royal Hospital for Children and Young People, Edinburgh, UK
| | - Jan-Bernd Stukenborg
- NORDFERTIL Research Lab Stockholm, Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden
| | - Rod T Mitchell
- Royal Hospital for Children and Young People, Edinburgh, UK
- MRC Centre for Reproductive Health, The Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, UK
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15
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Connan-Perrot S, Léger T, Lelandais P, Desdoits-Lethimonier C, David A, Fowler PA, Mazaud-Guittot S. Six Decades of Research on Human Fetal Gonadal Steroids. Int J Mol Sci 2021; 22:ijms22136681. [PMID: 34206462 PMCID: PMC8268622 DOI: 10.3390/ijms22136681] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2021] [Revised: 06/14/2021] [Accepted: 06/18/2021] [Indexed: 11/16/2022] Open
Abstract
Human fetal gonads acquire endocrine steroidogenic capabilities early during their differentiation. Genetic studies show that this endocrine function plays a central role in the sexually dimorphic development of the external genitalia during fetal development. When this endocrine function is dysregulated, congenital malformations and pathologies are the result. In this review, we explain how the current knowledge of steroidogenesis in human fetal gonads has benefited from both the technological advances in steroid measurements and the assembly of detailed knowledge of steroidogenesis machinery and its expression in human fetal gonads. We summarise how the conversion of radiolabelled steroid precursors, antibody-based assays, mass spectrometry, ultrastructural studies, and the in situ labelling of proteins and mRNA have all provided complementary information. In this review, our discussion goes beyond the debate on recommendations concerning the best choice between the different available technologies, and their degrees of reproducibility and sensitivity. The available technologies and techniques can be used for different purposes and, as long as all quality controls are rigorously employed, the question is how to maximise the generation of robust, reproducible data on steroid hormones and their crucial roles in human fetal development and subsequent functions.
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Affiliation(s)
- Stéphane Connan-Perrot
- Univ Rennes, Inserm, EHESP, Irset (Institut de Recherche en Santé, Environnement et Travail), UMR_S 1085, 35000 Rennes, France; (S.C.-P.); (P.L.); (C.D.-L.); (A.D.)
| | - Thibaut Léger
- Fougères Laboratory, French Agency for Food, Environmental and Occupational Health & Safety (ANSES), CEDEX, 35306 Fougères, France;
| | - Pauline Lelandais
- Univ Rennes, Inserm, EHESP, Irset (Institut de Recherche en Santé, Environnement et Travail), UMR_S 1085, 35000 Rennes, France; (S.C.-P.); (P.L.); (C.D.-L.); (A.D.)
| | - Christèle Desdoits-Lethimonier
- Univ Rennes, Inserm, EHESP, Irset (Institut de Recherche en Santé, Environnement et Travail), UMR_S 1085, 35000 Rennes, France; (S.C.-P.); (P.L.); (C.D.-L.); (A.D.)
| | - Arthur David
- Univ Rennes, Inserm, EHESP, Irset (Institut de Recherche en Santé, Environnement et Travail), UMR_S 1085, 35000 Rennes, France; (S.C.-P.); (P.L.); (C.D.-L.); (A.D.)
| | - Paul A. Fowler
- Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK;
| | - Séverine Mazaud-Guittot
- Univ Rennes, Inserm, EHESP, Irset (Institut de Recherche en Santé, Environnement et Travail), UMR_S 1085, 35000 Rennes, France; (S.C.-P.); (P.L.); (C.D.-L.); (A.D.)
- Correspondence: ; Tel.: +33-2-23-23-58-86
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16
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Doungkamchan C, Orwig KE. Recent advances: fertility preservation and fertility restoration options for males and females. Fac Rev 2021; 10:55. [PMID: 34195694 PMCID: PMC8204761 DOI: 10.12703/r/10-55] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Fertility preservation is the process of saving gametes, embryos, gonadal tissues and/or gonadal cells for individuals who are at risk of infertility due to disease, medical treatments, age, genetics, or other circumstances. Adult patients have the options to preserve eggs, sperm, or embryos that can be used in the future to produce biologically related offspring with assisted reproductive technologies. These options are not available to all adults or to children who are not yet producing mature eggs or sperm. Gonadal cells/tissues have been frozen for several thousands of those patients worldwide with anticipation that new reproductive technologies will be available in the future. Therefore, the fertility preservation medical and research communities are obligated to responsibly develop next-generation reproductive technologies and translate them into clinical practice. We briefly describe standard options to preserve and restore fertility, but the emphasis of this review is on experimental options, including an assessment of readiness for translation to the human fertility clinic.
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Affiliation(s)
- Chatchanan Doungkamchan
- Molecular Genetics and Developmental Biology Graduate Program, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA
- Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
- Department of Anatomy, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
| | - Kyle E Orwig
- Molecular Genetics and Developmental Biology Graduate Program, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA
- Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
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17
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Mouilleau V, Vaslin C, Robert R, Gribaudo S, Nicolas N, Jarrige M, Terray A, Lesueur L, Mathis MW, Croft G, Daynac M, Rouiller-Fabre V, Wichterle H, Ribes V, Martinat C, Nedelec S. Dynamic extrinsic pacing of the HOX clock in human axial progenitors controls motor neuron subtype specification. Development 2021; 148:148/6/dev194514. [PMID: 33782043 DOI: 10.1242/dev.194514] [Citation(s) in RCA: 33] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Accepted: 02/16/2021] [Indexed: 12/17/2022]
Abstract
Rostro-caudal patterning of vertebrates depends on the temporally progressive activation of HOX genes within axial stem cells that fuel axial embryo elongation. Whether the pace of sequential activation of HOX genes, the 'HOX clock', is controlled by intrinsic chromatin-based timing mechanisms or by temporal changes in extrinsic cues remains unclear. Here, we studied HOX clock pacing in human pluripotent stem cell-derived axial progenitors differentiating into diverse spinal cord motor neuron subtypes. We show that the progressive activation of caudal HOX genes is controlled by a dynamic increase in FGF signaling. Blocking the FGF pathway stalled induction of HOX genes, while a precocious increase of FGF, alone or with GDF11 ligand, accelerated the HOX clock. Cells differentiated under accelerated HOX induction generated appropriate posterior motor neuron subtypes found along the human embryonic spinal cord. The pacing of the HOX clock is thus dynamically regulated by exposure to secreted cues. Its manipulation by extrinsic factors provides synchronized access to multiple human neuronal subtypes of distinct rostro-caudal identities for basic and translational applications.This article has an associated 'The people behind the papers' interview.
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Affiliation(s)
- Vincent Mouilleau
- Institut du Fer à Moulin, 75005 Paris, France.,Inserm, UMR-S 1270, 75005 Paris, France.,Sorbonne Université, Science and Engineering Faculty, 75005 Paris, France.,I-STEM, UMR 861, Inserm, UEPS, 91100 Corbeil-Essonnes, France
| | - Célia Vaslin
- Institut du Fer à Moulin, 75005 Paris, France.,Inserm, UMR-S 1270, 75005 Paris, France.,Sorbonne Université, Science and Engineering Faculty, 75005 Paris, France
| | - Rémi Robert
- Institut du Fer à Moulin, 75005 Paris, France.,Inserm, UMR-S 1270, 75005 Paris, France.,Sorbonne Université, Science and Engineering Faculty, 75005 Paris, France
| | - Simona Gribaudo
- Institut du Fer à Moulin, 75005 Paris, France.,Inserm, UMR-S 1270, 75005 Paris, France.,Sorbonne Université, Science and Engineering Faculty, 75005 Paris, France
| | - Nour Nicolas
- Laboratory of Development of the Gonads, Unit of Genetic Stability, Stem Cells and Radiation, UMR 967, INSERM, CEA/DSV/iRCM/SCSR, Université Paris Diderot, Sorbonne Paris Cité, Université Paris-Sud, Université Paris-Saclay, Fontenay aux Roses F-92265, France
| | - Margot Jarrige
- I-STEM, UMR 861, Inserm, UEPS, 91100 Corbeil-Essonnes, France
| | - Angélique Terray
- Institut du Fer à Moulin, 75005 Paris, France.,Inserm, UMR-S 1270, 75005 Paris, France.,Sorbonne Université, Science and Engineering Faculty, 75005 Paris, France
| | - Léa Lesueur
- I-STEM, UMR 861, Inserm, UEPS, 91100 Corbeil-Essonnes, France
| | - Mackenzie W Mathis
- Departments of Pathology and Cell Biology, Neuroscience, and Neurology, Center for Motor Neuron Biology and Disease, Columbia Stem Cell Initiative, Columbia University Medical Center, New York, NY 10032, USA
| | - Gist Croft
- Departments of Pathology and Cell Biology, Neuroscience, and Neurology, Center for Motor Neuron Biology and Disease, Columbia Stem Cell Initiative, Columbia University Medical Center, New York, NY 10032, USA
| | - Mathieu Daynac
- Institut du Fer à Moulin, 75005 Paris, France.,Inserm, UMR-S 1270, 75005 Paris, France.,Sorbonne Université, Science and Engineering Faculty, 75005 Paris, France
| | - Virginie Rouiller-Fabre
- Laboratory of Development of the Gonads, Unit of Genetic Stability, Stem Cells and Radiation, UMR 967, INSERM, CEA/DSV/iRCM/SCSR, Université Paris Diderot, Sorbonne Paris Cité, Université Paris-Sud, Université Paris-Saclay, Fontenay aux Roses F-92265, France
| | - Hynek Wichterle
- Departments of Pathology and Cell Biology, Neuroscience, and Neurology, Center for Motor Neuron Biology and Disease, Columbia Stem Cell Initiative, Columbia University Medical Center, New York, NY 10032, USA
| | - Vanessa Ribes
- Université de Paris, CNRS, Institut Jacques Monod, 15 rue Hélène Brion, 75013 Paris, France
| | - Cécile Martinat
- I-STEM, UMR 861, Inserm, UEPS, 91100 Corbeil-Essonnes, France
| | - Stéphane Nedelec
- Institut du Fer à Moulin, 75005 Paris, France .,Inserm, UMR-S 1270, 75005 Paris, France.,Sorbonne Université, Science and Engineering Faculty, 75005 Paris, France
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18
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Sharma S, Venzac B, Burgers T, Le Gac S, Schlatt S. Microfluidics in male reproduction: is ex vivo culture of primate testis tissue a future strategy for ART or toxicology research? Mol Hum Reprod 2021; 26:179-192. [PMID: 31977028 DOI: 10.1093/molehr/gaaa006] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2019] [Revised: 01/03/2020] [Indexed: 01/09/2023] Open
Abstract
The significant rise in male infertility disorders over the years has led to extensive research efforts to recapitulate the process of male gametogenesis in vitro and to identify essential mechanisms involved in spermatogenesis, notably for clinical applications. A promising technology to bridge this research gap is organ-on-chip (OoC) technology, which has gradually transformed the research landscape in ART and offers new opportunities to develop advanced in vitro culture systems. With exquisite control on a cell or tissue microenvironment, customized organ-specific structures can be fabricated in in vitro OoC platforms, which can also simulate the effect of in vivo vascularization. Dynamic cultures using microfluidic devices enable us to create stimulatory effect and non-stimulatory culture conditions. Noteworthy is that recent studies demonstrated the potential of continuous perfusion in OoC systems using ex vivo mouse testis tissues. Here we review the existing literature and potential applications of such OoC systems for male reproduction in combination with novel bio-engineering and analytical tools. We first introduce OoC technology and highlight the opportunities offered in reproductive biology in general. In the subsequent section, we discuss the complex structural and functional organization of the testis and the role of the vasculature-associated testicular niche and fluid dynamics in modulating testis function. Next, we review significant technological breakthroughs in achieving in vitro spermatogenesis in various species and discuss the evidence from microfluidics-based testes culture studies in mouse. Lastly, we discuss a roadmap for the potential applications of the proposed testis-on-chip culture system in the field of primate male infertility, ART and reproductive toxicology.
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Affiliation(s)
- Swati Sharma
- Centre for Reproductive Medicine and Andrology, University of Münster, Münster, Germany
| | - Bastien Venzac
- Applied Microfluidics for BioEngineering Research, MESA+ Institute for Nanotechnology and TechMed Centre, University of Twente, Enschede, The Netherlands
| | - Thomas Burgers
- Applied Microfluidics for BioEngineering Research, MESA+ Institute for Nanotechnology and TechMed Centre, University of Twente, Enschede, The Netherlands
| | - Séverine Le Gac
- Applied Microfluidics for BioEngineering Research, MESA+ Institute for Nanotechnology and TechMed Centre, University of Twente, Enschede, The Netherlands
| | - Stefan Schlatt
- Centre for Reproductive Medicine and Andrology, University of Münster, Münster, Germany
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19
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Neto FTL, Flannigan R, Goldstein M. Regulation of Human Spermatogenesis. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2021; 1288:255-286. [PMID: 34453741 DOI: 10.1007/978-3-030-77779-1_13] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Human spermatogenesis (HS) is an intricate network of sequential processes responsible for the production of the male gamete, the spermatozoon. These processes take place in the seminiferous tubules (ST) of the testis, which are small tubular structures considered the functional units of the testes. Each human testicle contains approximately 600-1200 STs [1], and are capable of producing up to 275 million spermatozoa per day [2].
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Affiliation(s)
| | - Ryan Flannigan
- Department of Urology, Weill Cornell Medicine, New York, NY, USA.,University of British Columbia, Vancouver, BC, Canada
| | - Marc Goldstein
- Department of Urology, Weill Cornell Medicine, New York, NY, USA.
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20
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Liu HC, Xie Y, Deng CH, Liu GH. Stem cell-based therapies for fertility preservation in males: Current status and future prospects. World J Stem Cells 2020; 12:1097-1112. [PMID: 33178394 PMCID: PMC7596443 DOI: 10.4252/wjsc.v12.i10.1097] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/26/2020] [Revised: 05/13/2020] [Accepted: 08/26/2020] [Indexed: 02/06/2023] Open
Abstract
With the decline in male fertility in recent years, strategies for male fertility preservation have received increasing attention. In this study, by reviewing current treatments and recent publications, we describe research progress in and the future directions of stem cell-based therapies for male fertility preservation, focusing on the use of spermatogonial stem cells (SSCs), SSC niches, SSC-based testicular organoids, other stem cell types such as mesenchymal stem cells, and stem cell-derived extracellular vesicles. In conclusion, a more comprehensive understanding of the germ cell microenvironment, stem cell-derived extracellular vesicles, and testicular organoids will play an important role in achieving male fertility preservation.
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Affiliation(s)
- Han-Chao Liu
- Department of Andrology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
| | - Yun Xie
- Department of Andrology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
| | - Chun-Hua Deng
- Department of Andrology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
| | - Gui-Hua Liu
- Reproductive Medicine Research Center, The Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou 510655, Guangdong Province, China
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21
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Kotula-Balak M, Duliban M, Pawlicki P, Tuz R, Bilinska B, Płachno BJ, Arent ZJ, Krakowska I, Tarasiuk K. The meaning of non-classical estrogen receptors and peroxisome proliferator-activated receptor for boar Leydig cell of immature testis. Acta Histochem 2020; 122:151526. [PMID: 32094002 DOI: 10.1016/j.acthis.2020.151526] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2019] [Revised: 02/10/2020] [Accepted: 02/10/2020] [Indexed: 01/09/2023]
Abstract
Communication in biological systems involves diverse-types of cell-cell interaction including cross-talk between receptors expressed by the target cells. Recently, novel sort of estrogen receptors (G protein - coupled estrogen receptor; GPER and estrogen-related receptor; ERR) that signal directly via estrogen binding and/or via mutual interaction-regulated estrogen signaling were reported in various organs including testis. Peroxisome proliferator - activated receptor (PPAR) is responsible for maintaining of lipid homeostasis that is critical for sex steroid production in the testis. Here, we investigated the role of interaction between GPER, ERRβ and PPARγ in steroidogenic Leydig cells of immature boar testis. Testicular fragments cultured ex vivo were treated with GPER or PPARγ antagonists. Then, cell ultrastructure, expression and localization of GPER, ERRβ, PPARγ together with the molecular receptor mechanism, through cyclic AMP and Raf/Ras/extracellular signal activated kinases (ERK), in the control of cholesterol concentration and estrogen production by Leydig cells were studied. In the ultrastructure of antagonist-treated Leydig cells, mitochondria were not branched and not bifurcated as they were found in control. Additionally, in PPARγ-blocked Leydig cells changes in the number of lipid droplets were revealed. Independent of used antagonist, western blot revealed decreased co-expression of GPER, ERRβ, PPARγ with exception of increased expression of ERRβ after PPARγ blockage. Immunohistochemistry confirmed presence of all receptors partially located in the nucleus or cytoplasm of Leydig cells of both control and treated testes. Changes in receptor expression, decreased cholesterol and increased estradiol tissue concentrations occurred through decreased cAMP level (with exception after GPER blockage) as well as Raf/Ras/ERK pathway expression. These all findings indicate that GPER-ERRβ-PPARγ interaction exists in immature boar testis and regulates Leydig cell function. Further detailed studies and considerations on GPER-ERRβ-PPARγ as possible diagnosis/therapy target in disturbances of testis steroidogenic function are needed.
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Affiliation(s)
- M Kotula-Balak
- University Centre of Veterinary Medicine, University of Agriculture in Krakow, Mickiewicza 24/28, 30-059, Krakow, Poland.
| | - M Duliban
- Department of Endocrinology, Institute of Zoology, Jagiellonian University in Krakow, Gronostajowa 9, 30-387 Krakow, Poland
| | - P Pawlicki
- Department of Endocrinology, Institute of Zoology, Jagiellonian University in Krakow, Gronostajowa 9, 30-387 Krakow, Poland
| | - R Tuz
- Department of Swine and Small Animal Breeding, Institute of Animal Sciences, University of Agriculture in Krakow, Mickiewicza 24/28, 30-059 Krakow, Poland
| | - B Bilinska
- Department of Endocrinology, Institute of Zoology, Jagiellonian University in Krakow, Gronostajowa 9, 30-387 Krakow, Poland
| | - B J Płachno
- Department of Plant Cytology and Embryology, Institute of Botany, Jagiellonian University in Kraków, Poland, Gronostajowa 9, 30-387 Krakow, Poland
| | - Z J Arent
- University Centre of Veterinary Medicine, University of Agriculture in Krakow, Mickiewicza 24/28, 30-059, Krakow, Poland
| | - I Krakowska
- University Centre of Veterinary Medicine, University of Agriculture in Krakow, Mickiewicza 24/28, 30-059, Krakow, Poland
| | - K Tarasiuk
- University Centre of Veterinary Medicine, University of Agriculture in Krakow, Mickiewicza 24/28, 30-059, Krakow, Poland
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22
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Liu Y, Fan X, Yue M, Yue W, Zhang X, Zhang J, Ren G, He J. Expression and localization of meiosis-associated protein in gonads of female rats at different stages. Acta Histochem 2020; 122:151509. [PMID: 31964534 DOI: 10.1016/j.acthis.2020.151509] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2019] [Revised: 01/12/2020] [Accepted: 01/13/2020] [Indexed: 11/15/2022]
Abstract
It was well known that a critical process of oogenesis in the female mammalian was the entry of mitotic oogonia into meiosis. Early studies from model animal mice suggested that the retinoic acid (RA) response signal protein STRA8 (stimulated by retinoic acid gene 8) and the meiosis-specific chromosomal behavior marker protein SCP3 (Synaptonemal Complex Protein 3) were two crucial molecular markers during meiosis. The expression of STRA8 and SCP3 at different stages in rat ovaries was investigated by immunohistochemistry, qRT-PCR and Western Blot. Immunohistochemistry results showed that STRA8 and SCP3 were mainly expressed in embryonic stage. And STRA8 was expressed in the cytoplasm and nucleus of the ovaries after birth. qRT-PCR and Western Blot results showed that the mRNA and protein levels of STRA8 and SCP3 were expressed in embryonic stage. The expression of STRA8 and SCP3 indicated germ cells enter meiosis in rats embryo, and STRA8 and SCP3 could serve as molecular markers for the meiosis in rats. The localization of STRA8 in the nucleus increased the possibility that STRA8 might act as transcription factor or activate transcription to function after birth.
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Affiliation(s)
- Yihui Liu
- College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, 030801, PR China
| | - Xiaorui Fan
- College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, 030801, PR China
| | - Meishan Yue
- College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, 030801, PR China
| | - Weidong Yue
- College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, 030801, PR China
| | - Xinrong Zhang
- College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, 030801, PR China
| | - Jingwen Zhang
- College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, 030801, PR China
| | - Gaoya Ren
- College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, 030801, PR China
| | - Junping He
- College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi, 030801, PR China.
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23
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Spade DJ, Hall SJ, Wortzel JD, Reyes G, Boekelheide K. All-trans Retinoic Acid Disrupts Development in Ex Vivo Cultured Fetal Rat Testes. II: Modulation of Mono-(2-ethylhexyl) Phthalate Toxicity. Toxicol Sci 2020; 168:149-159. [PMID: 30476341 DOI: 10.1093/toxsci/kfy283] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Humans are universally exposed to low levels of phthalate esters (phthalates), which are used to plasticize polyvinyl chloride. Phthalates exert adverse effects on the development of seminiferous cords in the fetal testis through unknown toxicity pathways. To investigate the hypothesis that phthalates alter seminiferous cord development by disrupting retinoic acid (RA) signaling in the fetal testis, gestational day 15 fetal rat testes were exposed for 1-3 days to 10-6 M all-trans retinoic acid (ATRA) alone or in combination with 10-6-10-4 M mono-(2-ethylhexyl) phthalate (MEHP) in ex vivo culture. As previously reported, exogenous ATRA reduced seminiferous cord number. This effect was attenuated in a concentration-dependent fashion by MEHP co-exposure. ATRA and MEHP-exposed testes were depleted of DDX4-positive germ cells but not Sertoli cells. MEHP alone enhanced the expression of the RA receptor target Rbp1 and the ovary development-associated genes Wnt4 and Nr0b1, and suppressed expression of the Leydig cell marker, Star, and the germ cell markers, Ddx4 and Pou5f1. In co-exposures, MEHP predominantly enhanced the gene expression effects of ATRA, but the Wnt4 and Nr0b1 concentration-responses were nonlinear. Similarly, ATRA increased the number of cells expressing the granulosa cell marker FOXL2 in testis cultures, but this induction was attenuated by addition of MEHP. These results indicate that MEHP can both enhance and inhibit actions of ATRA during fetal testis development and provide evidence that RA signaling is a target for phthalate toxicity in the fetal testis.
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Affiliation(s)
- Daniel J Spade
- Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
| | - Susan J Hall
- Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
| | - Jeremy D Wortzel
- Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
| | - Gerardo Reyes
- Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912.,Division of Natural Sciences, College of Mount Saint Vincent, Riverdale, New York 10471
| | - Kim Boekelheide
- Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
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24
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Bowles J, Feng CW, Ineson J, Miles K, Spiller CM, Harley VR, Sinclair AH, Koopman P. Retinoic Acid Antagonizes Testis Development in Mice. Cell Rep 2019; 24:1330-1341. [PMID: 30067986 DOI: 10.1016/j.celrep.2018.06.111] [Citation(s) in RCA: 45] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2017] [Revised: 04/26/2018] [Accepted: 06/27/2018] [Indexed: 12/29/2022] Open
Abstract
Mammalian sex determination depends on a complex interplay of signals that promote the bipotential fetal gonad to develop as either a testis or an ovary, but the details are incompletely understood. Here, we investigated whether removal of the signaling molecule retinoic acid (RA) by the degradative enzyme CYP26B1 is necessary for proper development of somatic cells of the testes. Gonadal organ culture experiments suggested that RA promotes expression of some ovarian markers and suppresses expression of some testicular markers, acting downstream of Sox9. XY Cyp26b1-null embryos, in which endogenous RA is not degraded, develop mild ovotestes, but more important, steroidogenesis is impaired and the reproductive tract feminized. Experiments involving purified gonadal cells showed that these effects are independent of germ cells and suggest the direct involvement of the orphan nuclear receptor DAX1. Our results reveal that active removal of endogenous RA is required for normal testis development in the mouse.
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Affiliation(s)
- Josephine Bowles
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia; School of Biomedical Sciences, The University of Queensland, Brisbane, QLD 4072, Australia.
| | - Chun-Wei Feng
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia; School of Biomedical Sciences, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Jessica Ineson
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia; School of Biomedical Sciences, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Kim Miles
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Cassy M Spiller
- School of Biomedical Sciences, The University of Queensland, Brisbane, QLD 4072, Australia
| | - Vincent R Harley
- Hudson Institute of Medical Research, Clayton, Melbourne, VIC 3168, Australia
| | - Andrew H Sinclair
- Murdoch Children's Research Institute and Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Melbourne, VIC 3052, Australia
| | - Peter Koopman
- Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
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25
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Divergent Roles of CYP26B1 and Endogenous Retinoic Acid in Mouse Fetal Gonads. Biomolecules 2019; 9:biom9100536. [PMID: 31561560 PMCID: PMC6843241 DOI: 10.3390/biom9100536] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2019] [Revised: 09/17/2019] [Accepted: 09/24/2019] [Indexed: 11/19/2022] Open
Abstract
In female mammals, germ cells enter meiosis in the fetal ovaries, while in males, meiosis is prevented until postnatal development. Retinoic acid (RA) is considered the main inducer of meiotic entry, as it stimulates Stra8 which is required for the mitotic/meiotic switch. In fetal testes, the RA-degrading enzyme CYP26B1 prevents meiosis initiation. However, the role of endogenous RA in female meiosis entry has never been demonstrated in vivo. In this study, we demonstrate that some effects of RA in mouse fetal gonads are not recapitulated by the invalidation or up-regulation of CYP26B1. In organ culture of fetal testes, RA stimulates testosterone production and inhibits Sertoli cell proliferation. In the ovaries, short-term inhibition of RA-signaling does not decrease Stra8 expression. We develop a gain-of-function model to express CYP26A1 or CYP26B1. Only CYP26B1 fully prevents STRA8 induction in female germ cells, confirming its role as part of the meiotic prevention machinery. CYP26A1, a very potent RA degrading enzyme, does not impair the formation of STRA8-positive cells, but decreases Stra8 transcription. Collectively, our data reveal that CYP26B1 has other activities apart from metabolizing RA in fetal gonads and suggest a role of endogenous RA in amplifying Stra8, rather than being the initial inducer of Stra8. These findings should reactivate the quest to identify meiotic preventing or inducing substances.
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26
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Bhattacharya I, Sen Sharma S, Majumdar SS. Pubertal orchestration of hormones and testis in primates. Mol Reprod Dev 2019; 86:1505-1530. [DOI: 10.1002/mrd.23246] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2019] [Accepted: 07/15/2019] [Indexed: 12/14/2022]
Affiliation(s)
- Indrashis Bhattacharya
- Department of Zoology & BiotechnologyHNB Garhwal University, Srinagar CampusSrinagar India
- Cellular Endocrinology LabNational Institute of ImmunologyNew Delhi India
| | - Souvik Sen Sharma
- Cellular Endocrinology LabNational Institute of ImmunologyNew Delhi India
| | - Subeer S. Majumdar
- Cellular Endocrinology LabNational Institute of ImmunologyNew Delhi India
- Gene and Protein Engineering LabNational Institute of Animal BiotechnologyHyderabad India
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27
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Differentiation of human induced pluripotent stem cells into Leydig-like cells with molecular compounds. Cell Death Dis 2019; 10:220. [PMID: 30833541 PMCID: PMC6399252 DOI: 10.1038/s41419-019-1461-0] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2018] [Revised: 02/01/2019] [Accepted: 02/18/2019] [Indexed: 12/28/2022]
Abstract
Leydig cells (LCs) play crucial roles in producing testosterone, which is critical in the regulation of male reproduction and development. Low levels of testosterone will lead to male hypogonadism. LC transplantation is a promising alternative therapy for male hypogonadism. However, the source of LCs limits this strategy for clinical applications. Thus far, others have reported that LCs can be derived from stem cells by gene transfection, but the safe and effective induction method has not yet been reported. Here, we report that Leydig-like cells can be derived from human induced pluripotent stem cells (iPSCs) using a novel differentiation protocol based on molecular compounds. The iPSCs-derived Leydig-like cells (iPSC-LCs) acquired testosterone synthesis capabilities, had the similar gene expression profiles with LCs, and positively expressed Leydig cell lineage-specific protein markers LHCGR, STAR, SCARB1, SF-1, CYP11A1, HSD3B1, and HSD17B3 as well as negatively expressed iPSC-specific markers NANOG, OCT4, and SOX2. When iPSC-LCs labeled with lipophilic red dye (PKH26) were transplanted into rat testes that were selectively eliminated endogenous LCs using EDS (75 mg/kg), the transplanted iPSC-LCs could survive and function in the interstitium of testes, and accelerate the recovery of serum testosterone levels and testis weights. Collectively, these findings demonstrated that the iPSCs were able to be differentiated into Leydig-like cells by few defined molecular compounds, which may lay the safer groundwork for further clinical application of iPSC-LCs for hypogonadism.
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28
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Spade DJ, Dere E, Hall SJ, Schorl C, Freiman RN, Boekelheide K. All-Trans Retinoic Acid Disrupts Development in Ex Vivo Cultured Fetal Rat Testes. I: Altered Seminiferous Cord Maturation and Testicular Cell Fate. Toxicol Sci 2019; 167:546-558. [PMID: 30329139 PMCID: PMC6358251 DOI: 10.1093/toxsci/kfy260] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
Exposure to excess retinoic acid (RA) disrupts the development of the mammalian testicular seminiferous cord. However, the molecular events surrounding RA-driven loss of cord structure have not previously been examined. To investigate the mechanisms associated with this adverse developmental effect, fetal rat testes were isolated on gestational day 15, after testis determination and the initiation of cord development, and cultured in media containing all-trans RA (ATRA; 10-8 to 10-6 M) or vehicle for 3 days. ATRA exposure resulted in a concentration-dependent decrease in the number of seminiferous cords per testis section and number of germ cells, assessed by histopathology and immunohistochemistry. Following 1 day of culture, genome-wide expression profiling by microarray demonstrated that ATRA exposure altered biological processes related to retinoid metabolism and gonadal sex determination. Real-time RT-PCR analysis confirmed that ATRA enhanced the expression of the key ovarian development gene Wnt4 and the antitestis gene Nr0b1 in a concentration-dependent manner. After 3 days of culture, ATRA-treated testes contained both immunohistochemically DMRT1-positive and FOXL2-positive somatic cells, providing evidence of disrupted testicular cell fate maintenance following ATRA exposure. We conclude that exogenous RA disrupts seminiferous cord development in ex vivo cultured fetal rat testes, resulting in a reduction in seminiferous cord number, and interferes with maintenance of somatic cell fate by enhancing expression of factors that promote ovarian development.
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Affiliation(s)
- Daniel J Spade
- Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
| | - Edward Dere
- Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
- Division of Urology, Rhode Island Hospital, Providence, Rhode Island 02903
| | - Susan J Hall
- Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
| | - Christoph Schorl
- Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912
| | - Richard N Freiman
- Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912
| | - Kim Boekelheide
- Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912
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29
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Savchuk I, Morvan ML, Antignac JP, Kurek M, Le Bizec B, Söder O, Svechnikov K. Ontogenesis of human fetal testicular steroidogenesis at early gestational age. Steroids 2019; 141:96-103. [PMID: 30529237 DOI: 10.1016/j.steroids.2018.12.001] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/04/2018] [Revised: 11/28/2018] [Accepted: 12/02/2018] [Indexed: 11/29/2022]
Abstract
The onset of steroidogenesis in human fetal testes (HFT) during the first trimester is poorly investigated. One important unresolved question is the ontogeny of steroidogenic enzymes and formation of steroidogenic pathways in the HFT at early pregnancy. Our aim was to explore steroidogenesis, the expression of steroidogenic enzymes and their maturation in the HFT at gestational weeks (GW) 8-12. Steroids in the HFT were analyzed by gas chromatography/coupled to tandem mass spectrometry. The expression of steroidogenic enzymes in the HFT at GW8-12 was investigated by qPCR, automated Western blotting and immunohistochemistry. We demonstrated that the HFT at GW8-9 produced low level of testosterone via the Δ4 pathway and progesterone was the major steroid found in the testicular tissue. In contrast, more mature Leydig cells from the HFT at GW11-12 synthesized high levels of androgens via the Δ5 pathway. We also observed a significant upregulation of the expression of StAR, CYP11A1, CYP17A1 and its accessory proteins, P450 oxidoreductase (POR) and cytochrome b5 in the HFT at GW11-12 compared to GW8-9. Altogether, our data suggest that that human fetal Leydig cells differentiate rapidly at the end of the first trimester by acquiring capacity to express high levels of steroidogenic enzymes and switch from the Δ4 to the Δ5 pathways to synthesize high levels of androgens due to maturation of the CYP17-POR-b5 complex.
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Affiliation(s)
- I Savchuk
- Department of Women's and Children's Health, Pediatric Endocrinology Unit, Karolinska Institute & University Hospital, Stockholm, Sweden
| | - M L Morvan
- École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (Oniris), Laboratoire d'Étude des Résidus et Contaminants dans les aliments (LABERCA), UMR INRA 1329, Nantes, France
| | - J P Antignac
- École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (Oniris), Laboratoire d'Étude des Résidus et Contaminants dans les aliments (LABERCA), UMR INRA 1329, Nantes, France
| | - M Kurek
- Department of Women's and Children's Health, Pediatric Endocrinology Unit, NORDFERTIL Research Lab, Karolinska Institute & University Hospital, Stockholm, Sweden
| | - B Le Bizec
- École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (Oniris), Laboratoire d'Étude des Résidus et Contaminants dans les aliments (LABERCA), UMR INRA 1329, Nantes, France
| | - O Söder
- Department of Women's and Children's Health, Pediatric Endocrinology Unit, Karolinska Institute & University Hospital, Stockholm, Sweden
| | - K Svechnikov
- Department of Women's and Children's Health, Pediatric Endocrinology Unit, Karolinska Institute & University Hospital, Stockholm, Sweden.
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30
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Albalushi H, Sahlin L, Åkesson E, Kurek M, Kjartansdóttir KR, Lindh R, Söder O, Rotstein E, Hovatta O, Stukenborg JB. Hormone Production by Human First-Trimester Gonads in a Functional In Vitro System. Endocrinology 2019; 160:133-142. [PMID: 30418555 DOI: 10.1210/en.2018-00734] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/15/2018] [Accepted: 11/03/2018] [Indexed: 01/01/2023]
Abstract
In the past, explant tissue-culture methodologies have been used to grow gonads and study their development. Results from in vitro cultures of human gonads showed limited progress toward gonadal cell differentiation and were focused mainly on germ-cell differentiation. Thus, detailed studies focusing on human first-trimester gonadal tissue functionality in vitro are still missing. In this study we investigated the endocrine function of human first-trimester gonads in vitro. We included 27 female and 28 male gonadal samples, derived from a total of 55 cases, at postconceptional ages of 4.5 to 10.5 weeks. Tissues were cultured using an explant tissue-culture system for 14 days. Assays for testosterone (liquid chromatography-tandem mass spectrometry), anti-Müllerian hormone (AMH; ELISA), and inhibin B (ELISA) were performed using media collected after 7 and 14 days of culture. We demonstrated sex- and age-dependent secretion profiles of testosterone, AMH, and inhibin B in the culture media, which resemble the pattern of hormone production in human gonads in vivo, from the few available studies at the same age range. Our study shows that explant tissue-culture conditions are robust for culture of human first-trimester gonadal somatic cells. Thus, it can be used to study human gonadal development and related diseases as well as the effect of potentially hormone-disturbing substances in human gonads during development. However, detailed molecular studies are needed for better understanding of the mechanistic control of the endocrine function of human first-trimester gonads.
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Affiliation(s)
- Halima Albalushi
- NORDFERTIL Research Laboratory Stockholm, Solna, Sweden
- Pediatric Endocrinology Unit, Department of Women's and Children's Health, Karolinska Institutet and University Hospital, Stockholm, Sweden
- Sultan Qaboos University, College of Medicine and Health Sciences, Muscat, Oman
| | - Lena Sahlin
- NORDFERTIL Research Laboratory Stockholm, Solna, Sweden
- Pediatric Endocrinology Unit, Department of Women's and Children's Health, Karolinska Institutet and University Hospital, Stockholm, Sweden
| | - Elisabet Åkesson
- Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Huddinge, Sweden
- R&D Unit, Stockholms Sjukhem, Stockholm, Sweden
| | - Magdalena Kurek
- NORDFERTIL Research Laboratory Stockholm, Solna, Sweden
- Pediatric Endocrinology Unit, Department of Women's and Children's Health, Karolinska Institutet and University Hospital, Stockholm, Sweden
| | - Kristín Rós Kjartansdóttir
- Pediatric Endocrinology Unit, Department of Women's and Children's Health, Karolinska Institutet and University Hospital, Stockholm, Sweden
| | - Rika Lindh
- Pediatric Endocrinology Unit, Department of Women's and Children's Health, Karolinska Institutet and University Hospital, Stockholm, Sweden
| | - Olle Söder
- Pediatric Endocrinology Unit, Department of Women's and Children's Health, Karolinska Institutet and University Hospital, Stockholm, Sweden
| | - Emilia Rotstein
- Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet and University Hospital Karolinska Institutet, Huddinge, Sweden
| | - Outi Hovatta
- Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet and University Hospital Karolinska Institutet, Huddinge, Sweden
| | - Jan-Bernd Stukenborg
- NORDFERTIL Research Laboratory Stockholm, Solna, Sweden
- Pediatric Endocrinology Unit, Department of Women's and Children's Health, Karolinska Institutet and University Hospital, Stockholm, Sweden
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Tharmalingam MD, Jorgensen A, Mitchell RT. Experimental models of testicular development and function using human tissue and cells. Mol Cell Endocrinol 2018; 468:95-110. [PMID: 29309804 DOI: 10.1016/j.mce.2017.12.011] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/01/2017] [Revised: 12/21/2017] [Accepted: 12/21/2017] [Indexed: 12/17/2022]
Abstract
The mammalian testis has two main roles, production of gametes for reproduction and synthesis of steroid- and peptide hormones for masculinization. These processes are tightly regulated and involve complex interactions between a number of germ and somatic cell-types that comprise a unique microenvironment known as the germ stem cell niche. In humans, failure of normal testicular development or function is associated with susceptibility to a variety of male reproductive disorders including disorders of sex development, infertility and testicular cancer. Whilst studies in rodent models have provided detailed insight into the signaling pathways and molecular mechanisms that regulate the testis, there are important species differences in testicular development, function and reproductive disorders that highlight the need for suitable experimental models utilising human testicular tissues or cells. In this review, we outline experimental approaches used to sustain cells and tissue from human testis at different developmental time-points and discuss relevant end-points. These include survival, proliferation and differentiation of cell lineages within the testis as well as autocrine, paracrine and endocrine function. We also highlight the utility of these experimental approaches for modelling the effects of environmental exposures on testicular development and function.
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Affiliation(s)
- Melissa D Tharmalingam
- MRC Centre for Reproductive Health, The University of Edinburgh, The Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh, EH16 4TJ, Scotland, UK
| | - Anne Jorgensen
- Department of Growth and Reproduction, University Hospital of Copenhagen, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark
| | - Rod T Mitchell
- MRC Centre for Reproductive Health, The University of Edinburgh, The Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh, EH16 4TJ, Scotland, UK; Department of Endocrinology and Diabetes, Edinburgh Royal Hospital for Sick Children, 9 Sciennes Road, Edinburgh, EH9 1LF, Scotland, UK.
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Eladak S, Moison D, Guerquin MJ, Matilionyte G, Kilcoyne K, N’Tumba-Byn T, Messiaen S, Deceuninck Y, Pozzi-Gaudin S, Benachi A, Livera G, Antignac JP, Mitchell R, Rouiller-Fabre V, Habert R. Effects of environmental Bisphenol A exposures on germ cell development and Leydig cell function in the human fetal testis. PLoS One 2018; 13:e0191934. [PMID: 29385186 PMCID: PMC5791995 DOI: 10.1371/journal.pone.0191934] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2017] [Accepted: 01/15/2018] [Indexed: 11/20/2022] Open
Abstract
BACKGROUND Using an organotypic culture system termed human Fetal Testis Assay (hFeTA) we previously showed that 0.01 μM BPA decreases basal, but not LH-stimulated, testosterone secreted by the first trimester human fetal testis. The present study was conducted to determine the potential for a long-term antiandrogenic effect of BPA using a xenograft model, and also to study the effect of BPA on germ cell development using both the hFETA and xenograft models. METHODS Using the hFeTA system, first trimester testes were cultured for 3 days with 0.01 to 10 μM BPA. For xenografts, adult castrate male nude mice were injected with hCG and grafted with first trimester testes. Host mice received 10 μM BPA (~ 500 μg/kg/day) in their drinking water for 5 weeks. Plasma levels of total and unconjugated BPA were 0.10 μM and 0.038 μM respectively. Mice grafted with second trimester testes received 0.5 and 50 μg/kg/day BPA by oral gavage for 5 weeks. RESULTS With first trimester human testes, using the hFeTA model, 10 μM BPA increased germ cell apoptosis. In xenografts, germ cell density was also reduced by BPA exposure. Importantly, BPA exposure significantly decreased the percentage of germ cells expressing the pluripotency marker AP-2γ, whilst the percentage of those expressing the pre-spermatogonial marker MAGE-A4 significantly increased. BPA exposure did not affect hCG-stimulated androgen production in first and second trimester xenografts as evaluated by both plasma testosterone level and seminal vesicle weight in host mice. CONCLUSIONS Exposure to BPA at environmentally relevant concentrations impairs germ cell development in first trimester human fetal testis, whilst gonadotrophin-stimulated testosterone production was unaffected in both first and second trimester testis. Studies using first trimester human fetal testis demonstrate the complementarity of the FeTA and xenograft models for determining the respective short-term and long term effects of environmental exposures.
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Affiliation(s)
- Soria Eladak
- Univ. Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Genetic Stability, Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, Fontenay-aux-Roses, France
- INSERM, Unité 967, Fontenay aux Roses, France
| | - Delphine Moison
- Univ. Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Genetic Stability, Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, Fontenay-aux-Roses, France
- INSERM, Unité 967, Fontenay aux Roses, France
| | - Marie-Justine Guerquin
- Univ. Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Genetic Stability, Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, Fontenay-aux-Roses, France
- INSERM, Unité 967, Fontenay aux Roses, France
| | - Gabriele Matilionyte
- MRC Centre for Reproductive Health, The University of Edinburgh, The Queen's Medical Research Institute, Edinburgh, Scotland, United Kingdom
| | - Karen Kilcoyne
- MRC Centre for Reproductive Health, The University of Edinburgh, The Queen's Medical Research Institute, Edinburgh, Scotland, United Kingdom
| | - Thierry N’Tumba-Byn
- Univ. Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Genetic Stability, Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, Fontenay-aux-Roses, France
- INSERM, Unité 967, Fontenay aux Roses, France
| | - Sébastien Messiaen
- Univ. Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Genetic Stability, Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, Fontenay-aux-Roses, France
- INSERM, Unité 967, Fontenay aux Roses, France
| | - Yoann Deceuninck
- Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA), Ecole Nationale Vétérinaire Agroalimentaire et de l’Alimentation Nantes Atlantique (ONIRIS), Nantes, France
| | - Stéphanie Pozzi-Gaudin
- Service de Gynécologie-Obstétrique et Médecine de la Reproduction, Hôpital A. Béclère, Université Paris Sud, Clamart, France
| | - Alexandra Benachi
- Service de Gynécologie-Obstétrique et Médecine de la Reproduction, Hôpital A. Béclère, Université Paris Sud, Clamart, France
| | - Gabriel Livera
- Univ. Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Genetic Stability, Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, Fontenay-aux-Roses, France
- INSERM, Unité 967, Fontenay aux Roses, France
| | - Jean-Philippe Antignac
- Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA), Ecole Nationale Vétérinaire Agroalimentaire et de l’Alimentation Nantes Atlantique (ONIRIS), Nantes, France
| | - Rod Mitchell
- MRC Centre for Reproductive Health, The University of Edinburgh, The Queen's Medical Research Institute, Edinburgh, Scotland, United Kingdom
| | - Virginie Rouiller-Fabre
- Univ. Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Genetic Stability, Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, Fontenay-aux-Roses, France
- INSERM, Unité 967, Fontenay aux Roses, France
| | - René Habert
- Univ. Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Genetic Stability, Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, Fontenay-aux-Roses, France
- INSERM, Unité 967, Fontenay aux Roses, France
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Alves-Lopes JP, Stukenborg JB. Testicular organoids: a new model to study the testicular microenvironment in vitro? Hum Reprod Update 2017; 24:176-191. [PMID: 29281008 DOI: 10.1093/humupd/dmx036] [Citation(s) in RCA: 51] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2017] [Revised: 11/20/2017] [Accepted: 11/27/2017] [Indexed: 12/11/2022] Open
Abstract
BACKGROUND In recent decades, a broad range of strategies have been applied to model the testicular microenvironment in vitro. These models have been utilized to study testicular physiology and development. However, a system that allows investigations into testicular organogenesis and its impact in the spermatogonial stem-cell (SSC) niche in vitro has not been developed yet. Recently, the creation of tissue-specific organ-like structures called organoids has resurged, helping researchers to answer scientific questions that previous in vitro models could not help to elucidate. So far, a small number of publications have concerned the generation of testicular organoids and their application in the field of reproductive medicine and biology. OBJECTIVE AND RATIONALE Here, we aim to elucidate whether testicular organoids might be useful in answering current scientific questions about the regulation and function of the SSC niche as well as germ cell proliferation and differentiation, and whether or not the existing in vitro models are already sufficient to address them. Moreover, we would like to discuss how an organoid system can be a better solution to address these prominent scientific problems in our field, by the creation of a rationale parallel to those in other areas where organoid systems have been successfully utilized. SEARCH METHODS We comprehensively reviewed publications regarding testicular organoids and the methods that most closely led to the formation of these organ-like structures in vitro by searching for the following terms in both PubMed and the Web of Science database: testicular organoid, seminiferous tubule 3D culture, Sertoli cell 3D culture, testicular cord formation in vitro, testicular morphogenesis in vitro, germ cell 3D culture, in vitro spermatogenesis, testicular de novo morphogenesis, seminiferous tubule de novo morphogenesis, seminiferous tubule-like structures, testicular in vitro model and male germ cell niche in vitro, with no restrictions to any publishing year. The inclusion criteria were based on the relation with the main topic (i.e. testicular organoids, testicular- and seminiferous-like structures as in vitro models), methodology applied (i.e. in vitro culture, culture dimensions (2D, 3D), testicular cell suspension or fragments) and outcome of interest (i.e. organization in vitro). Publications about grafting of testicular tissue, germ-cell transplantation and female germ-cell culture were excluded. OUTCOMES The application of organoid systems is making its first steps in the field of reproductive medicine and biology. A restricted number of publications have reported and characterized testicular organoids and even fewer have denominated such structures by this method. However, we detected that a clear improvement in testicular cell reorganization is recognized when 3D culture conditions are utilized instead of 2D conditions. Depending on the scientific question, testicular organoids might offer a more appropriate in vitro model to investigate testicular development and physiology because of the easy manipulation of cell suspensions (inclusion or exclusion of a specific cell population), the fast reorganization of these structures and the controlled in vitro conditions, to the same extent as with other organoid strategies reported in other fields. WIDER IMPLICATIONS By way of appropriate research questions, we might use testicular organoids to deepen our basic understanding of testicular development and the SSC niche, leading to new methodologies for male infertility treatment.
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Affiliation(s)
- João Pedro Alves-Lopes
- Department of Women's and Children's Health, NORDFERTIL Research Lab Stockholm, Paediatric Endocrinology Unit, Q2:08, Karolinska Institutet and Karolinska University Hospital, SE-17176 Stockholm, Sweden
| | - Jan-Bernd Stukenborg
- Department of Women's and Children's Health, NORDFERTIL Research Lab Stockholm, Paediatric Endocrinology Unit, Q2:08, Karolinska Institutet and Karolinska University Hospital, SE-17176 Stockholm, Sweden
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Picut CA, Ziejewski MK, Stanislaus D. Comparative Aspects of Pre- and Postnatal Development of the Male Reproductive System. Birth Defects Res 2017; 110:190-227. [PMID: 29063715 DOI: 10.1002/bdr2.1133] [Citation(s) in RCA: 51] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2017] [Revised: 08/29/2017] [Accepted: 08/30/2017] [Indexed: 01/01/2023]
Abstract
This review describes pre- and postnatal development of the male reproductive system in humans and laboratory animals, and highlights species differences in the timing and control of hormonal and morphologic events. Major differences are that the fetal testis is dependent on gonadotropins in humans, but is independent of such in rats; humans have an extended postnatal quiescent period, whereas rats exhibit no quiescence; and events such as secretion by the prostate and seminal vesicles, testicular descent, and the appearance of spermatogonia are all prenatal events in humans, but are postnatal events in rats. Major differences in the timing of the developmental sequence between rats and humans include: gonocyte transformation period (rat: postnatal day 0-9; human: includes gestational week 22 to 9 months of age); masculinization programming window (rat: gestational day 15.5-17.5; human: gestational week 9-14); and mini-puberty (rat: 0-6 hr after birth; human: 3-6 months of age). Endocrine disruptors can cause unique lesions in the prenatal and early postnatal testis; therefore, it is important to consider the differences in the timing of the developmental sequence when designing preclinical studies as identification of windows of sensitivity for endocrine disruption or toxicants will aid in interpretation of results and provide clues to a mode of action. Birth Defects Research 110:190-227, 2018. © 2017 Wiley Periodicals, Inc.
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Affiliation(s)
- Catherine A Picut
- Charles River Laboratories, Pathology Associates, Durham, North Carolina
| | - Mary K Ziejewski
- GlaxoSmithKline Research & Development, King of Prussia, Pennsylvania
| | - D Stanislaus
- GlaxoSmithKline Research & Development, King of Prussia, Pennsylvania
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Lim J, Kong W, Lu M, Luderer U. The Mouse Fetal Ovary Has Greater Sensitivity Than the Fetal Testis to Benzo[a]pyrene-Induced Germ Cell Death. Toxicol Sci 2016; 152:372-81. [PMID: 27208085 DOI: 10.1093/toxsci/kfw083] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023] Open
Abstract
The polycyclic aromatic hydrocarbon pollutant benzo[a]pyrene (BaP) is a known developmental gonadotoxicant. However, the mechanism of BaP-induced germ cell death is unclear. We investigated whether exposure to BaP induces apoptotic germ cell death in the mouse fetal ovary or testis. Mouse fetal gonads were dissected at embryonic day 13.5 days postcoitum (dpc) and fixed immediately or cultured for 6, 24, 48, or 72 h with various concentrations of BaP (1-1000 ng/ml). Germ cells numbers, apoptosis, and proliferation were evaluated by immunostaining. Treatment of fetal ovaries with BaP for 72 h concentration-dependently depleted germ cells. Treatment with BaP elevated the expression of BAX protein at 6 h and activated downstream caspases-9 and -3 at 24 h in a concentration-dependent manner in germ cells of fetal ovaries. As a consequence, ovarian germ cell numbers were significantly and concentration-dependently decreased at 48 h. Pretreatment with z-VAD-fmk, a pan-caspase inhibitor, prior to exposure to 1000 ng/ml BaP prevented BaP-mediated ovarian germ cell death; there were no effects of BaP or z-VAD-fmk on germ cell proliferation. No significant effects of BaP exposure on caspase 3 activation or germ cell numbers were observed in fetal testes after 48 h of culture. Our findings show that BaP exposure increases caspase-dependent and BAX-associated germ cell apoptosis in the mouse fetal ovary, leading to germ cell depletion. In contrast, the cultured 13.5 dpc fetal testis is relatively resistant to BaP-induced germ cell death. This study provides a novel insight into molecular mechanisms by which BaP has direct gonadotoxicity in the mouse fetal ovary.
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Affiliation(s)
| | | | | | - Ulrike Luderer
- *Departments of Medicine Developmental and Cell Biology Program in Public Health, University of California, Irvine 92617
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Spermatogenesis in humans and its affecting factors. Semin Cell Dev Biol 2016; 59:10-26. [PMID: 27143445 DOI: 10.1016/j.semcdb.2016.04.009] [Citation(s) in RCA: 313] [Impact Index Per Article: 34.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2016] [Revised: 04/13/2016] [Accepted: 04/15/2016] [Indexed: 12/13/2022]
Abstract
Spermatogenesis is an extraordinary complex process. The differentiation of spermatogonia into spermatozoa requires the participation of several cell types, hormones, paracrine factors, genes and epigenetic regulators. Recent researches in animals and humans have furthered our understanding of the male gamete differentiation, and led to clinical tools for the better management of male infertility. There is still much to be learned about this intricate process. In this review, the critical steps of human spermatogenesis are discussed together with its main affecting factors.
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Abstract
The two major functions of the testis, steroidogenesis and gametogenesis, take place during fetal life. These two functions have been extensively studied in rodents and adult humans. However, their onset during fetal life is poorly documented in humans. In the first part of this work we presented both our experimental data and some data of literature concerning the development of the human fetal testis. In the second part of this article, using the organ culture system we previously developed, we have investigated the regulations or perturbations of fetal testis development both in rodent and human models. Our findings provide important insight into the potential role of exposure to environmental pollutants (physical factors, in particular ionizing radiation, cadmium and endocrine disruptors such as phthalates) during fetal testicular development and their potential deleterious effects on male fertility in adulthood. Our results highlight the specificity of the human model compared with rodent models.
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Adverse effects of endocrine disruptors on the foetal testis development: focus on the phthalates. Folia Histochem Cytobiol 2016; 47:S67-74. [PMID: 20067897 DOI: 10.2478/v10042-009-0056-5] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
There are great concerns about the increasing incidence of abnormalities in male reproductive function. Human sperm counts have markedly dropped and the rate of testicular cancer has clearly augmented over the past four decades. Moreover, the prevalence rates of cryptorchidism and hypospadias are also probably increasing. It has been hypothesized that all these adverse trends in male reproduction result from abnormalities in the development of the testis during foetal and neonatal life. Furthermore, many recent epidemiological, clinical and experimental data suggest that these male reproductive disorders could be due to the effects of xenobiotics termed endocrine disruptors, which are becoming more and more concentrated and prevalent in our environment. Among these endocrine disruptors, we chose to focus this review on the phthalates for different reasons: 1) they are widespread in the environment; 2) their concentrations in many human biological fluids have been measured; 3) the experimental data using rodent models suggesting a reprotoxicity are numerous and are the most convincing; 4) their deleterious effects on the in vivo and in vitro development and function of the rat foetal testis have been largely studied; 5) some epidemiological data in humans suggest a reprotoxic effect at environmental concentrations at least during neonatal life. However, the direct effects of phthalates on human foetal testis have never been explored. Thus, as we did for the rat in the 1990s, we recently developed and validated an organ culture system which allows maintenance of the development of the different cell types of human foetal testis. In this system, addition of 10-4 M MEHP (mono-2-ethylhexyl phthalate), the most produced phthalate, had no effect on basal or LH-stimulated production of testosterone, but it reduced the number of germ cells by increasing their apoptosis, without modification of their proliferation. This is the first experimental demonstration that phthalates alter the development of the foetal testis in humans. Using our organotypic culture system, we and others are currently investigating the effect of MEHP in the mouse and the rat, and it will be interesting to compare the results between these species to analyse the relevance of toxicological tests based on rodent models.
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Chen SR, Liu YX. Testis Cord Maintenance in Mouse Embryos: Genes and Signaling1. Biol Reprod 2016; 94:42. [DOI: 10.1095/biolreprod.115.137117] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2015] [Accepted: 01/12/2016] [Indexed: 12/12/2022] Open
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Jørgensen A, Lindhardt Johansen M, Juul A, Skakkebaek NE, Main KM, Rajpert-De Meyts E. Pathogenesis of germ cell neoplasia in testicular dysgenesis and disorders of sex development. Semin Cell Dev Biol 2015; 45:124-37. [PMID: 26410164 DOI: 10.1016/j.semcdb.2015.09.013] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2015] [Accepted: 09/21/2015] [Indexed: 12/29/2022]
Abstract
Development of human gonads is a sex-dimorphic process which evolved to produce sex-specific types of germ cells. The process of gonadal sex differentiation is directed by the action of the somatic cells and ultimately results in germ cells differentiating to become functional gametes through spermatogenesis or oogenesis. This tightly controlled process depends on the proper sequential expression of many genes and signalling pathways. Disturbances of this process can be manifested as a large spectrum of disorders, ranging from severe disorders of sex development (DSD) to - in the genetic male - mild reproductive problems within the testicular dysgenesis syndrome (TDS), with large overlap between the syndromes. These disorders carry an increased but variable risk of germ cell neoplasia. In this review, we discuss the pathogenesis of germ cell neoplasia associated with gonadal dysgenesis, especially in individuals with 46,XY DSD. We summarise knowledge concerning development and sex differentiation of human gonads, with focus on sex-dimorphic steps of germ cell maturation, including meiosis. We also briefly outline the histopathology of germ cell neoplasia in situ (GCNIS) and gonadoblastoma (GDB), which are essentially the same precursor lesion but with different morphological structure dependent upon the masculinisation of the somatic niche. To assess the risk of germ cell neoplasia in different types of DSD, we have performed a PubMed search and provide here a synthesis of the evidence from studies published since 2006. We present a model for pathogenesis of GCNIS/GDB in TDS/DSD, with the risk of malignancy determined by the presence of the testis-inducing Y chromosome and the degree of masculinisation. The associations between phenotype and the risk of neoplasia are likely further modulated in each individual by the constellation of the gene polymorphisms and environmental factors.
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Affiliation(s)
- Anne Jørgensen
- Department of Growth & Reproduction and International Center for Research and Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, University of Copenhagen, Denmark.
| | - Marie Lindhardt Johansen
- Department of Growth & Reproduction and International Center for Research and Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, University of Copenhagen, Denmark.
| | - Anders Juul
- Department of Growth & Reproduction and International Center for Research and Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, University of Copenhagen, Denmark.
| | - Niels E Skakkebaek
- Department of Growth & Reproduction and International Center for Research and Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, University of Copenhagen, Denmark.
| | - Katharina M Main
- Department of Growth & Reproduction and International Center for Research and Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, University of Copenhagen, Denmark.
| | - Ewa Rajpert-De Meyts
- Department of Growth & Reproduction and International Center for Research and Training in Endocrine Disruption of Male Reproduction and Child Health (EDMaRC), Rigshospitalet, University of Copenhagen, Denmark.
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Jørgensen A, Nielsen JE, Perlman S, Lundvall L, Mitchell RT, Juul A, Rajpert-De Meyts E. Ex vivo culture of human fetal gonads: manipulation of meiosis signalling by retinoic acid treatment disrupts testis development. Hum Reprod 2015; 30:2351-63. [PMID: 26251460 DOI: 10.1093/humrep/dev194] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2015] [Accepted: 07/13/2015] [Indexed: 01/01/2023] Open
Abstract
STUDY QUESTION What are the effects of experimentally manipulating meiosis signalling by addition of retinoic acid (RA) in cultured human fetal gonads? SUMMARY ANSWER RA-treatment accelerated meiotic entry in cultured fetal ovary samples, while addition of RA resulted in a dysgenetic gonadal phenotype in fetal testis cultures. WHAT IS KNOWN ALREADY One of the first manifestations of sex differentiation is the initiation of meiosis in fetal ovaries. In contrast, meiotic entry is actively prevented in the fetal testis at this developmental time-point. It has previously been shown that RA-treatment mediates initiation of meiosis in human fetal ovary ex vivo. STUDY DESIGN, SIZE, DURATION This was a controlled ex vivo study of human fetal gonads treated with RA in 'hanging-drop' tissue cultures. The applied experimental set-up preserves germ cell-somatic niche interactions and the investigated outcomes included tissue integrity and morphology, cell proliferation and survival and the expression of markers of meiosis and sex differentiation. PARTICIPANTS/MATERIALS, SETTING, METHODS Tissue from 24 first trimester human fetuses was included in this study, all from elective terminations at gestational week (GW) 7-12. Gonads were cultured for 2 weeks with and without addition of 1 µM RA. Samples were subsequently formalin-fixed and investigated by immunohistochemistry and cell counting. Proteins investigated and quantified included; octamer-binding transcription factor 4 (OCT4), transcription factor AP-2 gamma (AP2γ) (embryonic germ cell markers), SRY (sex determining region Y)-box 9 (SOX9), anti-Müllerian hormone (AMH) (immature Sertoli cell markers), COUP transcription factor 2 (COUP-TFII) (marker of interstitial cells), forkhead box L2 (FOXL2) (granulosa cell marker), H2A histone family, member X (γH2AX) (meiosis marker), doublesex and mab-3 related transcription factor 1 (DMRT1) (meiosis regulator), cleaved poly ADP ribose polymerase (PARP), cleaved Caspase 3 (apoptosis markers) and Ki-67 antigen (Ki-67) (proliferation marker). Also, proliferation was determined using a 5'-bromo-2'-deoxyuridine (BrdU) incorporation assay. MAIN RESULTS AND THE ROLE OF CHANCE A novel ex vivo 'hanging-drop' culture model for human fetal gonads was successfully established. Continued proliferation of cells without signs of increased apoptosis was observed after 2 weeks of culture. In cultured fetal ovaries treated with RA, an increased number of meiotic germ cells (P < 0.05) and DMRT1-positive oogonia initiating meiosis (P < 0.05) was observed, which is in agreement with a previous study. In fetal testes, RA-treatment resulted in a decreased number of gonocytes (P < 0.05), a reduced percentage of proliferating gonocytes (P < 0.05), altered expression pattern of the somatic cell markers AMH and COUP-TFII, as well as disrupted seminiferous cord structure and testis morphology. LIMITATIONS, REASONS FOR CAUTION The number of samples included in this study was relatively small due to the limited availability of human fetal tissue. WIDER IMPLICATIONS OF THE FINDINGS The hanging-drop culture, similarly to other organ culture approaches, allows studies of germ cell-somatic niche interactions and determination of effects after manipulating specific signalling pathways. Our novel finding of disrupted fetal testis development after treatment with RA indicates that abnormal meiosis regulation can potentially cause gonadal dysgenesis. Further studies will elucidate the exact mechanisms and timing of observed effects. STUDY FUNDING/COMPETING INTERESTS This work was supported in part by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S to A.Jø. Additional funding for this project was obtained from The Research Council of the Capital Region of Denmark (E.R.-D.M.), The Research Fund at Rigshospitalet (A.Ju. and J.E.N.), Familien Erichssens Fund (A.Jø.), Dagmar Marshalls Fund (A.Jø.) and Aase & Ejnar Danielsens Fund (A.Jø.). The authors have no conflicts of interest.
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Affiliation(s)
- A Jørgensen
- University Department of Growth and Reproduction and EDMaRC, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
| | - J E Nielsen
- University Department of Growth and Reproduction and EDMaRC, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
| | - S Perlman
- Department of Gynaecology, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, Copenhagen DK-2100, Denmark
| | - L Lundvall
- Department of Gynaecology, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, Copenhagen DK-2100, Denmark
| | - R T Mitchell
- MRC Centre for Reproductive Health, The University of Edinburgh, The Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK Edinburgh Royal Hospital for Sick Children, 9 Sciennes Road, Edinburgh EH9 1LF, UK
| | - A Juul
- University Department of Growth and Reproduction and EDMaRC, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
| | - E Rajpert-De Meyts
- University Department of Growth and Reproduction and EDMaRC, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
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Manku G, Culty M. Dynamic changes in the expression of apoptosis-related genes in differentiating gonocytes and in seminomas. Asian J Androl 2015; 17:403-14. [PMID: 25677133 PMCID: PMC4430938 DOI: 10.4103/1008-682x.146101] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2014] [Revised: 11/24/2014] [Accepted: 11/26/2014] [Indexed: 01/25/2023] Open
Abstract
Apoptosis is an integral part of the spermatogenic process, necessary to maintain a proper ratio of Sertoli to germ cell numbers and provide an adequate microenvironment to germ cells. Apoptosis may also represent a protective mechanism mediating the elimination of abnormal germ cells. Extensive apoptosis occurs between the first and second postnatal weeks, at the point when gonocytes, precursors of spermatogonial stem cells, should have migrated toward the basement membrane of the tubules and differentiated into spermatogonia. The mechanisms regulating this process are not well-understood. Gonocytes undergo phases of proliferation, migration, and differentiation which occur in a timely and closely regulated manner. Gonocytes failing to migrate and differentiate properly undergo apoptosis. Inadequate gonocyte differentiation has been suggested to lead to testicular germ cell tumor (TGCT) formation. Here, we examined the expression levels of apoptosis-related genes during gonocyte differentiation by quantitative real-time polymerase chain reaction, identifying 48 pro- and anti-apoptotic genes increased by at least two-fold in rat gonocytes induced to differentiate by retinoic acid, when compared to untreated gonocytes. Further analysis of the most highly expressed genes identified the pro-apoptotic genes Gadd45a and Cycs as upregulated in differentiating gonocytes and in spermatogonia compared with gonocytes. These genes were also significantly downregulated in seminomas, the most common type of TGCT, compared with normal human testicular tissues. These results indicate that apoptosis-related genes are actively regulated during gonocyte differentiation. Moreover, the down-regulation of pro-apoptotic genes in seminomas suggests that they could represent new therapeutic targets in the treatment of TGCTs.
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Affiliation(s)
- Gurpreet Manku
- The Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec, Canada
- Department of Medicine, McGill University, Montreal, Quebec, Canada
- Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada
| | - Martine Culty
- The Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec, Canada
- Department of Medicine, McGill University, Montreal, Quebec, Canada
- Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada
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Eladak S, Grisin T, Moison D, Guerquin MJ, N'Tumba-Byn T, Pozzi-Gaudin S, Benachi A, Livera G, Rouiller-Fabre V, Habert R. A new chapter in the bisphenol A story: bisphenol S and bisphenol F are not safe alternatives to this compound. Fertil Steril 2014; 103:11-21. [PMID: 25475787 DOI: 10.1016/j.fertnstert.2014.11.005] [Citation(s) in RCA: 452] [Impact Index Per Article: 41.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2014] [Revised: 11/04/2014] [Accepted: 11/05/2014] [Indexed: 10/24/2022]
Abstract
Bisphenol A (BPA) is a widely studied typical endocrine-disrupting chemical, and one of the major new issues is the safe replacement of this commonly used compound. Bisphenol S (BPS) and bisphenol F (BPF) are already or are planned to be used as BPA alternatives. With the use of a culture system that we developed (fetal testis assay [FeTA]), we previously showed that 10 nmol/L BPA reduces basal testosterone secretion of human fetal testis explants and that the susceptibility to BPA is at least 100-fold lower in rat and mouse fetal testes. Here, we show that addition of LH in the FeTA system considerably enhances BPA minimum effective concentration in mouse and human but not in rat fetal testes. Then, using the FeTA system without LH (the experimental conditions in which mouse and human fetal testes are most sensitive to BPA), we found that, as for BPA, 10 nmol/L BPS or BPF is sufficient to decrease basal testosterone secretion by human fetal testes with often nonmonotonic dose-response curves. In fetal mouse testes, the dose-response curves were mostly monotonic and the minimum effective concentrations were 1,000 nmol/L for BPA and BPF and 100 nmol/L for BPS. Finally, 10,000 nmol/L BPA, BPS, or BPF reduced Insl3 expression in cultured mouse fetal testes. This is the first report describing BPS and BPF adverse effects on a physiologic function in humans and rodents.
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Affiliation(s)
- Soria Eladak
- Unit of Genetic Stability, Stem Cells, and Radiation, Laboratory of Development of the Gonads, Université Paris Diderot, Sorbonne Paris Cité, Fontenay-aux-Roses, France; Commissariat à l'Energie Atomique, Fontenay-aux-Roses, France; Institut National de la Santé et de la Recherche Médicale, Unité 967, Fontenay-aux-Roses, France
| | - Tiphany Grisin
- Unit of Genetic Stability, Stem Cells, and Radiation, Laboratory of Development of the Gonads, Université Paris Diderot, Sorbonne Paris Cité, Fontenay-aux-Roses, France; Commissariat à l'Energie Atomique, Fontenay-aux-Roses, France; Institut National de la Santé et de la Recherche Médicale, Unité 967, Fontenay-aux-Roses, France
| | - Delphine Moison
- Unit of Genetic Stability, Stem Cells, and Radiation, Laboratory of Development of the Gonads, Université Paris Diderot, Sorbonne Paris Cité, Fontenay-aux-Roses, France; Commissariat à l'Energie Atomique, Fontenay-aux-Roses, France; Institut National de la Santé et de la Recherche Médicale, Unité 967, Fontenay-aux-Roses, France
| | - Marie-Justine Guerquin
- Unit of Genetic Stability, Stem Cells, and Radiation, Laboratory of Development of the Gonads, Université Paris Diderot, Sorbonne Paris Cité, Fontenay-aux-Roses, France; Commissariat à l'Energie Atomique, Fontenay-aux-Roses, France; Institut National de la Santé et de la Recherche Médicale, Unité 967, Fontenay-aux-Roses, France
| | - Thierry N'Tumba-Byn
- Unit of Genetic Stability, Stem Cells, and Radiation, Laboratory of Development of the Gonads, Université Paris Diderot, Sorbonne Paris Cité, Fontenay-aux-Roses, France; Commissariat à l'Energie Atomique, Fontenay-aux-Roses, France; Institut National de la Santé et de la Recherche Médicale, Unité 967, Fontenay-aux-Roses, France
| | - Stéphanie Pozzi-Gaudin
- Service de Gynécologie-Obstétrique et Médecine de la Reproduction, Hôpital A. Béclère, Université Paris Sud, Clamart, France
| | - Alexandra Benachi
- Service de Gynécologie-Obstétrique et Médecine de la Reproduction, Hôpital A. Béclère, Université Paris Sud, Clamart, France
| | - Gabriel Livera
- Unit of Genetic Stability, Stem Cells, and Radiation, Laboratory of Development of the Gonads, Université Paris Diderot, Sorbonne Paris Cité, Fontenay-aux-Roses, France; Commissariat à l'Energie Atomique, Fontenay-aux-Roses, France; Institut National de la Santé et de la Recherche Médicale, Unité 967, Fontenay-aux-Roses, France
| | - Virginie Rouiller-Fabre
- Unit of Genetic Stability, Stem Cells, and Radiation, Laboratory of Development of the Gonads, Université Paris Diderot, Sorbonne Paris Cité, Fontenay-aux-Roses, France; Commissariat à l'Energie Atomique, Fontenay-aux-Roses, France; Institut National de la Santé et de la Recherche Médicale, Unité 967, Fontenay-aux-Roses, France
| | - René Habert
- Unit of Genetic Stability, Stem Cells, and Radiation, Laboratory of Development of the Gonads, Université Paris Diderot, Sorbonne Paris Cité, Fontenay-aux-Roses, France; Commissariat à l'Energie Atomique, Fontenay-aux-Roses, France; Institut National de la Santé et de la Recherche Médicale, Unité 967, Fontenay-aux-Roses, France.
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Habert R, Livera G, Rouiller-Fabre V. Man is not a big rat: concerns with traditional human risk assessment of phthalates based on their anti-androgenic effects observed in the rat foetus. Basic Clin Androl 2014; 24:14. [PMID: 25780587 PMCID: PMC4349750 DOI: 10.1186/2051-4190-24-14] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2014] [Accepted: 08/15/2014] [Indexed: 11/10/2022] Open
Abstract
Phthalates provide one of the most documented example evidencing how much we must be cautious when using the traditional paradigm based on extrapolation of experimental data from rodent studies for human health risk assessment of endocrine disruptors (EDs). Since foetal testis is known as one of the most sensitive targets of EDs, phthalate risk assessment is routinely based on the capacity of such compounds to decrease testosterone production by the testis or to impair masculinization in the rat during foetal life. In this paper, the well-established inhibiting effects of phthalates of the foetal Leydig cells function in the rat are briefly reviewed. Then, data obtained in humans and other species are carefully analysed. Already in January 2009, using the organotypic culture system named Fetal Testis Assay (FeTA) that we developed, we reported that phthalates might not affect testosterone production in human foetal testes. Several recent experimental studies using xenografts confirm the absence of detectable anti-androgenic effect of phthalates in the human foetal testes. Epidemiological studies led to contradictory results. Altogether, these findings suggest that phthalates effects on foetal Leydig cells are largely species-specific. Consequently, the phthalate threshold doses that disturb foetal steroidogenesis in rat testes and that are presently used to define the acceptable daily intake levels for human health protection must be questioned. This does not mean that phthalates are safe because these compounds have many deleterious effects upon germ cell development that may be common to the different studied species including human. More generally, the identification of common molecular, cellular or/and phenotypic targets in rat and human testes should precede the choice of the toxicological endpoint in rat to accurately assess the safety threshold of any ED in humans.
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Affiliation(s)
- René Habert
- Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, University Paris Diderot, BP 6, 92265 Fontenay-aux-Roses, France ; CEA, DSV, iRCM, SCSR, LDG, 92265 Fontenay-aux-Roses, France ; INSERM, Unité 967, F-92265 Fontenay aux Roses, France ; Stem Cells and Radiation Unit, LDG / SCSR / iRCM / DSV, Centre CEA, BP6, F-92265 Fontenay aux Roses, France
| | - Gabriel Livera
- Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, University Paris Diderot, BP 6, 92265 Fontenay-aux-Roses, France ; CEA, DSV, iRCM, SCSR, LDG, 92265 Fontenay-aux-Roses, France ; INSERM, Unité 967, F-92265 Fontenay aux Roses, France
| | - Virginie Rouiller-Fabre
- Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, University Paris Diderot, BP 6, 92265 Fontenay-aux-Roses, France ; CEA, DSV, iRCM, SCSR, LDG, 92265 Fontenay-aux-Roses, France ; INSERM, Unité 967, F-92265 Fontenay aux Roses, France
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Investigating the protective role of death receptor 3 (DR3) in renal injury using an organ culture model. Methods Mol Biol 2014; 1155:69-79. [PMID: 24788174 DOI: 10.1007/978-1-4939-0669-7_7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/21/2023]
Abstract
Death receptor 3 (DR3; also designated as Wsl-1, Apo3, LARD, TRAMP, TNFRSF25, and TR3) is a member of the tumor necrosis factor (TNF) receptor superfamily that has emerged as a major regulator of inflammation and autoimmune diseases. DR3 contains a homologous intracellular region called the death domain (DD) that can bind adaptor proteins, which also contain a DD, initiating cellular responses such as caspase activation and apoptotic cell death. However, in other circumstances DR3 can initiate induction of transcription genes and gene products that can prevent cell death from occurring. Our laboratory has reported an inducible expression of DR3 in human and mouse tubular epithelial cells in renal injury, but its function in these setting still remains unclear. To directly manipulate and evaluate the role of DR3 in vivo, I have used an in vitro organ culture (OC) model, which I have developed in our laboratory. In this chapter, I will describe in detail the OC model used to study the role of DR3 in renal injury and discuss its advantages and limitations. In my hands, the OC model has proven to be an efficient tool for studying human cell heterogeneity, basal and regulated receptor expression, signalling pathways, and various biological responses not readily achievable in traditional cell culture models. Various assays can be carried out on organ cultures including histology, biochemistry, cell biology, and molecular biology, which will not be described in detail in this chapter.
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46
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O'Shaughnessy PJ, Fowler PA. Development of the human fetal testis. ANNALES D'ENDOCRINOLOGIE 2014; 75:48-53. [DOI: 10.1016/j.ando.2014.03.009] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/24/2014] [Accepted: 03/25/2014] [Indexed: 10/25/2022]
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Habert R, Muczynski V, Grisin T, Moison D, Messiaen S, Frydman R, Benachi A, Delbes G, Lambrot R, Lehraiki A, N'tumba-Byn T, Guerquin MJ, Levacher C, Rouiller-Fabre V, Livera G. Concerns about the widespread use of rodent models for human risk assessments of endocrine disruptors. Reproduction 2014; 147:R119-29. [PMID: 24497529 PMCID: PMC3959776 DOI: 10.1530/rep-13-0497] [Citation(s) in RCA: 62] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Fetal testis is a major target of endocrine disruptors (EDs). During the last 20 years, we have developed an organotypic culture system that maintains the function of the different fetal testis cell types and have used this approach as a toxicological test to evaluate the effects of various compounds on gametogenesis and steroidogenesis in rat, mouse and human testes. We named this test rat, mouse and human fetal testis assay. With this approach, we compared the effects of six potential EDs ((mono-(2-ethylhexyl) phthalate (MEHP), cadmium, depleted uranium, diethylstilboestrol (DES), bisphenol A (BPA) and metformin) and one signalling molecule (retinoic acid (RA)) on the function of rat, mouse and human fetal testis at a comparable developmental stage. We found that the response is similar in humans and rodents for only one third of our analyses. For instance, RA and MEHP have similar negative effects on gametogenesis in the three species. For another third of our analyses, the threshold efficient concentrations that disturb gametogenesis and/or steroidogenesis differ as a function of the species. For instance, BPA and metformin have similar negative effects on steroidogenesis in human and rodents, but at different threshold doses. For the last third of our analyses, the qualitative response is species specific. For instance, MEHP and DES affect steroidogenesis in rodents, but not in human fetal testis. These species differences raise concerns about the extrapolation of data obtained in rodents to human health risk assessment and highlight the need of rigorous comparisons of the effects in human and rodent models, when assessing ED risk.
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Affiliation(s)
- René Habert
- Unit of Stem Cells and Radiation, Laboratory of Development of the Gonads, Sorbonne Paris Cité, Université Paris Diderot, BP 6, 92265 Fontenay-aux-Roses, France
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Effects of vitamin A on in vitro maturation of pre-pubertal mouse spermatogonial stem cells. PLoS One 2013; 8:e82819. [PMID: 24349372 PMCID: PMC3857286 DOI: 10.1371/journal.pone.0082819] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2012] [Accepted: 11/06/2013] [Indexed: 11/19/2022] Open
Abstract
Testicular tissue cryopreservation is the only potential option for fertility preservation in pre-pubertal boys exposed to gonadotoxic treatment. Completion of spermatogenesis after in vitro maturation is one of the future uses of harvested testicular tissue. The purpose of the current study was to evaluate the effects of vitamin A on in vitro maturation of fresh and frozen-thawed mouse pre-pubertal spermatogonial stem cells in an organ culture system. Pre-pubertal CD1 mouse fresh testes were cultured for 7 (D7), 9 (D9) and 11 (D11) days using an organ culture system. Basal medium was supplemented with different concentrations of retinol (Re) or retinoic acid (RA) alone or in combination. Seminiferous tubule morphology (tubule diameter, intra-tubular cell type), intra-tubular cell death and proliferation (PCNA antibody) and testosterone level were assessed at D7, D9 and D11. Pre-pubertal mouse testicular tissue were frozen after a soaking temperature performed at -7°C, -8°C or -9°C and after thawing, were cultured for 9 days, using the culture medium preserving the best fresh tissue functionality. Retinoic acid at 10-6M and retinol at 3.3.10-7M, as well as retinol 10-6M are favourable for seminiferous tubule growth, maintenance of intra-tubular cell proliferation and germ cell differentiation of fresh pre-pubertal mouse spermatogonia. Structural and functional integrity of frozen-thawed testicular tissue appeared to be well-preserved after soaking temperature at -8°C, after 9 days of organotypic culture using 10-6M retinol. RA and Re can control in vitro germ cell proliferation and differentiation. Re at a concentration of 10-6M maintains intra-tubular cell proliferation and the ability of spermatogonia to initiate spermatogenesis in fresh and frozen pre-pubertal mouse testicular tissue using a soaking temperature at -8°C. Our data suggested a possible human application for in vitro maturation of cryopreserved pre-pubertal testicular tissue.
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N'Tumba-Byn T, Moison D, Lacroix M, Lecureuil C, Lesage L, Prud'homme SM, Pozzi-Gaudin S, Frydman R, Benachi A, Livera G, Rouiller-Fabre V, Habert R. Differential effects of bisphenol A and diethylstilbestrol on human, rat and mouse fetal leydig cell function. PLoS One 2012; 7:e51579. [PMID: 23284716 PMCID: PMC3524173 DOI: 10.1371/journal.pone.0051579] [Citation(s) in RCA: 74] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2012] [Accepted: 11/01/2012] [Indexed: 01/23/2023] Open
Abstract
Endocrine disruptors (ED) have been incriminated in the current increase of male reproductive alterations. Bisphenol A (BPA) is a widely used weak estrogenic environmental ED and it is debated whether BPA concentrations within the average internal exposure are toxic. In the present study we investigated the effects of 10(-12) to 10(-5) M BPA concentrations on fetal Leydig cell function, as fetal life is a critical period of sensitivity to ED effects on male reproductive function. To this aim, fetal testes from human at 6.5-10.5 gestational weeks (GW) or from rat and mouse at a comparable critical period of development (14.5 days post-coitum (dpc) for rat and 12.5 dpc for mouse) were explanted and cultured using our validated organotypic culture system in the presence or absence of BPA for 1-3 days. BPA concentrations as low as 10(-8) M reduced testosterone secretion by human testes from day 1 of culture onwards, but not by mouse and rat testes where concentrations equal to 10(-5) M BPA were required. Similarly, 10(-8) M BPA reduced INSL3 mRNA levels only in human cultured testes. On the contrary, 10(-5) and 10(-6) M diethylstilbestrol (DES), a classical estrogenic compound, affected testosterone secretion only in rat and mouse testis cultures, but not in human testis cultures. Lastly, contrarily to the DES effect, the negative effect of BPA on testosterone produced by the mouse fetal testis was maintained after invalidation of estrogen receptor α (ERα). In conclusion, these results evidenced i) a deleterious effect of BPA on fetal Leydig cells function in human for concentrations from 10(-8) M upwards, ii) species-specific differences raising concerns about extrapolation of data from rodent studies to human risk assessment, iii) a specific signaling pathway for BPA which differs from the DES one and which does not involve ERα.
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Affiliation(s)
- Thierry N'Tumba-Byn
- Université Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, Fontenay-aux-Roses, France
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Muczynski V, Lecureuil C, Messiaen S, Guerquin MJ, N’Tumba-Byn T, Moison D, Hodroj W, Benjelloun H, Baijer J, Livera G, Frydman R, Benachi A, Habert R, Rouiller-Fabre V. Cellular and molecular effect of MEHP Involving LXRα in human fetal testis and ovary. PLoS One 2012; 7:e48266. [PMID: 23118965 PMCID: PMC3484128 DOI: 10.1371/journal.pone.0048266] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2012] [Accepted: 09/21/2012] [Indexed: 01/26/2023] Open
Abstract
Background Phthalates have been shown to have reprotoxic effects in rodents and human during fetal life. Previous studies indicate that some members of the nuclear receptor (NR) superfamilly potentially mediate phthalate effects. This study aimed to assess if expression of these nuclear receptors are modulated in the response to MEHP exposure on the human fetal gonads in vitro. Methodology/Principal Findings Testes and ovaries from 7 to 12 gestational weeks human fetuses were exposed to 10−4M MEHP for 72 h in vitro. Transcriptional level of NRs and of downstream genes was then investigated using TLDA (TaqMan Low Density Array) and qPCR approaches. To determine whether somatic or germ cells of the testis are involved in the response to MEHP exposure, we developed a highly efficient cytometric germ cell sorting approach. In vitro exposure of fetal testes and ovaries to MEHP up-regulated the expression of LXRα, SREBP members and of downstream genes involved in the lipid and cholesterol synthesis in the whole gonad. In sorted testicular cells, this effect is only observable in somatic cells but not in the gonocytes. Moreover, the germ cell loss induced by MEHP exposure, that we previously described, is restricted to the male gonad as oogonia density is not affected in vitro. Conclusions/Significance We evidenced for the first time that phthalate increases the levels of mRNA for LXRα, and SREBP members potentially deregulating lipids/cholesterol synthesis in human fetal gonads. Interestingly, this novel effect is observable in both male and female whereas the germ cell apoptosis is restricted to the male gonad. Furthermore, we presented here a novel and potentially very useful flow cytometric cell sorting method to analyse molecular changes in germ cells versus somatic cells.
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Affiliation(s)
- Vincent Muczynski
- University Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, 92265 Fontenay-aux-Roses, France
- INSERM, Unité 967, F-92265, Fontenay aux Roses, France
| | - Charlotte Lecureuil
- University Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, 92265 Fontenay-aux-Roses, France
- INSERM, Unité 967, F-92265, Fontenay aux Roses, France
| | - Sébastien Messiaen
- University Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, 92265 Fontenay-aux-Roses, France
- INSERM, Unité 967, F-92265, Fontenay aux Roses, France
| | - Marie-Justine Guerquin
- University Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, 92265 Fontenay-aux-Roses, France
- INSERM, Unité 967, F-92265, Fontenay aux Roses, France
| | - Thierry N’Tumba-Byn
- University Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, 92265 Fontenay-aux-Roses, France
- INSERM, Unité 967, F-92265, Fontenay aux Roses, France
| | - Delphine Moison
- University Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, 92265 Fontenay-aux-Roses, France
- INSERM, Unité 967, F-92265, Fontenay aux Roses, France
| | - Wassim Hodroj
- University Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, 92265 Fontenay-aux-Roses, France
- INSERM, Unité 967, F-92265, Fontenay aux Roses, France
| | - Hinde Benjelloun
- Flow Cytometry Facility, CEA – DSV/iRCM/SCSR, F-92265 Fontenay aux Roses, France
| | - Jan Baijer
- Flow Cytometry Facility, CEA – DSV/iRCM/SCSR, F-92265 Fontenay aux Roses, France
| | - Gabriel Livera
- University Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, 92265 Fontenay-aux-Roses, France
- INSERM, Unité 967, F-92265, Fontenay aux Roses, France
| | - René Frydman
- Service de Gynécologie-Obstétrique, Hôpital A. Béclère,- Université Paris Sud, F-92141 Clamart, France
| | - Alexandra Benachi
- Service de Gynécologie-Obstétrique, Hôpital A. Béclère,- Université Paris Sud, F-92141 Clamart, France
| | - René Habert
- University Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, 92265 Fontenay-aux-Roses, France
- INSERM, Unité 967, F-92265, Fontenay aux Roses, France
| | - Virginie Rouiller-Fabre
- University Paris Diderot, Sorbonne Paris Cité, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, Fontenay-aux-Roses, France
- CEA, DSV, iRCM, SCSR, LDG, 92265 Fontenay-aux-Roses, France
- INSERM, Unité 967, F-92265, Fontenay aux Roses, France
- * E-mail:
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