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Lord T, Oatley JM. Spermatogenic Stem Cells: Core Biology, Defining Features, and Utilities. Mol Reprod Dev 2024; 91:e23777. [PMID: 39392153 DOI: 10.1002/mrd.23777] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2024] [Accepted: 09/24/2024] [Indexed: 10/12/2024]
Abstract
The actions of spermatogenic stem cells (SSCs) provide the foundation for continual spermatogenesis and regeneration of the cognate lineage following cytotoxic insult or transplantation. Several decades of research with rodent models have yielded knowledge about the core biology, morphological features, and molecular profiles of mammalian SSCs. Translation of these discoveries to utilities for human fertility preservation, improving animal agriculture, and wildlife conservation are actively being pursued. Here, we provide overviews of these aspects covering both historical and current states of understanding.
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Affiliation(s)
- Tessa Lord
- Discipline of Biological Sciences, College of Engineering, Science and Environment, The University of Newcastle, Callaghan, New South Wales, Australia
- Infertility and Reproduction Program, Hunter Medical Research Institute, New Lambton Heights, New South Wales, Australia
| | - Jon M Oatley
- Center for Reproductive Biology, School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, Washington, USA
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Moeinzadeh A, Ashtari B, Garcia H, Koruji M, Velazquez CA, Bagher Z, Barati M, Shabani R, Davachi SM. The Effect of Chitosan/Alginate/Graphene Oxide Nanocomposites on Proliferation of Mouse Spermatogonial Stem Cells. J Funct Biomater 2023; 14:556. [PMID: 38132810 PMCID: PMC10744091 DOI: 10.3390/jfb14120556] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2023] [Revised: 10/30/2023] [Accepted: 11/17/2023] [Indexed: 12/23/2023] Open
Abstract
Male survivors of childhood cancer have been known to be afflicted with azoospermia. To combat this, the isolation and purification of spermatogonial stem cells (SSCs) are crucial. Implementing scaffolds that emulate the extracellular matrix environment is vital for promoting the regeneration and proliferation of SSCs. This research aimed to evaluate the efficiency of nanocomposite scaffolds based on alginate, chitosan, and graphene oxide (GO) in facilitating SSCs proliferation. To analyze the cytotoxicity of the scaffolds, an MTT assay was conducted at 1, 3, and 7 days, and the sample containing 30 µg/mL of GO (ALGCS/GO30) exhibited the most favorable results, indicating its optimal performance. The identity of the cells was confirmed using flow cytometry with C-Kit and GFRα1 markers. The scaffolds were subjected to various analyses to characterize their properties. FTIR was employed to assess the chemical structure, XRD to examine crystallinity, and SEM to visualize the morphology of the scaffolds. To evaluate the proliferation of SSCs, qRT-PCR was used. The study's results demonstrated that the ALGCS/GO30 nanocomposite scaffold exhibited biocompatibility and facilitated the attachment and proliferation of SSCs. Notably, the scaffold displayed a significant increase in proliferation markers compared to the control group, indicating its ability to support SSC growth. The expression level of the PLZF protein was assessed using the Immunocytochemistry method. The observations confirmed the qRT-PCR results, which indicated that the nanocomposite scaffolds had higher levels of PLZF protein expression than scaffolds without GO. The biocompatible ALGCS/GO30 is a promising alternative for promoting SSC proliferation in in vitro applications.
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Affiliation(s)
- Alaa Moeinzadeh
- Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
- Department of Tissue Engineering and Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Behnaz Ashtari
- Radiation Biology Research Center, Iran University of Medical Sciences, Tehran, Iran
- Department of Medical Nanotechnology, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Heriberto Garcia
- Department of Biology and Chemistry, Texas A&M International University, Laredo, TX 78041, USA
| | - Morteza Koruji
- Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
- Stem Cell and Regenerative Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Carlo Alberto Velazquez
- Department of Biology and Chemistry, Texas A&M International University, Laredo, TX 78041, USA
| | - Zohreh Bagher
- Department of Tissue Engineering and Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran
- ENT and Head & Neck Research Center and Department, The Five Senses Institute, Hazrat Rasoul Akram Hospital, Iran University of Medical Sciences, Tehran, Iran
| | - Mahmood Barati
- Department of Medical Biotechnology, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Ronak Shabani
- Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
- Reproductive Sciences and Technology Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Seyed Mohammad Davachi
- Department of Biology and Chemistry, Texas A&M International University, Laredo, TX 78041, USA
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Cai Y, Liu Z, Zhang G, Yang Y, Zhang Y, Wang F, Deng M. miR-101-5p overexpression suppresses the proliferation of goat spermatogonial stem cells by targeting EZH2. Anim Reprod Sci 2023; 255:107281. [PMID: 37352705 DOI: 10.1016/j.anireprosci.2023.107281] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2023] [Revised: 06/09/2023] [Accepted: 06/15/2023] [Indexed: 06/25/2023]
Abstract
MicroRNAs (miRNAs), as post-transcriptional gene mediators, regulate the biological characteristics of spermatogonial stem cells (SSCs), including proliferation, differentiation and apoptosis. However, the potential roles and mechanisms by which miR-101-5p affected the biological characters of goat SSCs have not been fully elucidated. Herein, we reported that miR-101-5p overexpression decreased cell viability (P < 0.01), arrested cell cycle in the G1 phase (P < 0.05), and aggravated apoptosis of goat SSCs (P < 0.01) compared with negative control (NC), as determined by CCK-8 assay and flow cytometry analysis. Additionally, PCNA protein expression was attenuated by miR-101-5p overexpression (P < 0.05). Notably, the expression of SSCs specific genes Oct4 (P < 0.05), PLZF (P < 0.01) and DAZL (P < 0.01) were decreased in miR-101-5p overexpressed SSCs. Furthermore, the dual luciferase reporter assay showed that, when co-transfected with miR-101-5p mimics, the relative luciferase activity of EZH2 wide-type (WT) was inhibited (P < 0.05) compared with the transfection of EZH2 mutant (MUT). EZH2 expression was negatively correlated with miR-101-5p expression in goat SSCs. Collectively, our data implicates that miR-101-5p overexpression aggravates cell apoptosis, and suppresses cell proliferation of goat SSCs via targeting EZH2, which may impair spermatogenesis.
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Affiliation(s)
- Yu Cai
- Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China
| | - Zifei Liu
- Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China
| | - Guomin Zhang
- College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
| | - Yingnan Yang
- Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China
| | - Yanli Zhang
- Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China
| | - Feng Wang
- Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China.
| | - Mingtian Deng
- Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China.
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WARDAK MOHAMMADKAZIM, KULATHUNGA KAUSHALYA, PRIYADARSHANA CHATHURA. Localization and characterization of SSCs from pre-pubertal bovine testes. THE INDIAN JOURNAL OF ANIMAL SCIENCES 2022. [DOI: 10.56093/ijans.v92i10.124617] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
Self renewal and proliferation ability of spermatogonial stem cells (SSCs) support spermatogenesis during adult life. Theoretically, these stem cells can be utilized for transmission of genetic information to descendants via testicular transplantation. However, lack of knowledge in methodologies for identification of SSCs limits the application of SSCs transplantation in domestic animals. Accumulated studies have shown that SSCs specific markers (DBA, UCHL1) and stem cell marker (Sox2, Oct4) are useful to screen SSCs that able to be used for transplantation. However, in cattle, less information is available on the expression status of these markers till date. Therefore, a study was carried out in 2019 at Tsukuba University, Japan where testes from 3, 5 and 7 months old calves were utilized to examine testicular localization and in vitro propogation of stem cell markers. SSCs were isolated by enzymatic digestion combined with centrifugal separation on discontinuous Percoll density gradient. Cell propagation and SSCs marker expression were determined at 5, 10 and 15 days post-culture. Immunostaining in conjunction with Western Blot analysis of cultured cells showed that stem cell markers (UCHL1, Oct4 and Sox2) were expressed in SSCs suggesting that differentiation of gonocyte started by 3 months and SSCs differentiation begins after 5 months of age. Taken together, these results demonstrated marker expression and localization of bull SSCs and showed that in vitro culturing of bull SSCs is implementable.
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Heidarizadi S, Rashidi Z, Jalili C, Gholami M. Overview of biological effects of melatonin on testis: A review. Andrologia 2022; 54:e14597. [PMID: 36168927 DOI: 10.1111/and.14597] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2022] [Revised: 08/31/2022] [Accepted: 09/05/2022] [Indexed: 11/29/2022] Open
Abstract
Infertility is a major global health issue and male factors account for half of all infertility cases. One of the causes of male infertility is the loss of spermatogonial stem cells, which may occur because of chemotherapy, radiotherapy or genetic defects. In numerous animal species, the evidence suggests the pineal gland and melatonin secretion in their reproductive activities are involved. Recently, considerable attention has pointed to the usage of melatonin in the treatment of diseases. Melatonin is associated with the regulation of circadian and seasonal rhythmic functions, immune system functions, retinal physiology, spermatogenesis and inhibition of tumour growth in different species. Several studies demonstrated that melatonin acts as an anti-apoptotic, anti-inflammatory, anticancer and antioxidant agent. Melatonin can also protect testicles and spermatogonia against oxidative damage, chemotherapy drugs, environmental radiation, toxic substances, hyperthermia, ischemia/reperfusion, diabetes-induced testicular damage, metal-induced testicular toxicity, improve sperm quality and it affects the testosterone secretion pathway by affecting Leydig cells. Therefore, the objective of this study is to investigate the biological effects of melatonin as a natural antioxidant on testicles and their disorders.
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Affiliation(s)
- Somayeh Heidarizadi
- Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
| | - Zahra Rashidi
- Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran.,Fertility and Infertility Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
| | - Cyrus Jalili
- Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran.,Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
| | - Mohammadreza Gholami
- Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
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Narimanpour Z, Bojnordi MN, Hamidabadi HG. Spermatogenic differentiation of spermatogonial stem cells on three-dimensional silk nanofiber scaffold. MIDDLE EAST FERTILITY SOCIETY JOURNAL 2022. [DOI: 10.1186/s43043-022-00107-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
Abstract
Background
Nano-fibrous scaffolds provide a three-dimensional matrix that guides sufficient orientation of seeded cells similar to a natural niche. In this research, we designed a silk scaffold to improve the differention of mouse spermatogonial stem cells to spermatogenic cell lines. Spermatogonial stem cells were collected from neonatal mouse (2–6 days) testes (n=60) using a two steps mechanical and enzymatic method. Cells were seeded on a silk scaffold and were cultured in Dulbecco’s modified Eagle’s medium, supplemented with 15 % fetal bovine serum and 1000 units/ml leukemia inhibitory factor, and incubated at 32°C in a humidified atmosphere of 5% CO2 in air. SEM technique was done for confirmation of seeding cells.
In this study two major groups (i.e., 2D and 3D culture groups) of 30 mice each. Isolated testicular cells from each group were cultured in the absence of silk scaffold or the presence of silk scaffold.
For induction of differentiation, seeded cells on a scaffold were exposed to 1 μM and 50 ng/ml BMP-4. The specific spermatogenic genes, e.g.; VASA, DAZL, PLZF, and Piwil2, were assessed via real-time PCR and immunocytochemistry techniques. P values less than 0.05 were assumed significant. All experiments were performed at least three times.
Results
SEM analysis confirmed the homogeneity of fabricated silk scaffold and average diameter of 450 nm for nanofibers fibers. Silk scaffold induces attachment of SSCs in comparison to the monolayer group. Spermatogonia stem cell colonies were observed gradually after 1 week of culture. Electrospun scaffold supports the differentiation of SSCs to spermatogenic lines. Dates of real-time PCR showed that the expression of meiotic markers, VASA, DAZL, and Piwil2 as related to specific spermatogenic genes, had a significant upregulation in cell-seeded silk scaffold compared to the control group (P < 0.05).
Immunocytochemistry founding approved the expression of specific spermatogenic markers; DAZL and PLZF were higher in the experiment group compared to the control (P < 0.05).
Conclusion
It is concluded silk scaffold induces spermatogenic differentiation of mouse spermatogonial stem cells in vitro.
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Arevalo D, Flores AC, Diaz RF, Garcia-Herreros M, Aponte PM. Filgrastim (r-met-hu G-CSF) enhances the efficiency of spermatogenesis in prepubertal Bos indicus bulls. Reprod Domest Anim 2021; 57:438-443. [PMID: 34897834 DOI: 10.1111/rda.14068] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2021] [Revised: 12/07/2021] [Accepted: 12/10/2021] [Indexed: 11/30/2022]
Abstract
This study aimed to test the effects of the drug r-met-hu-G-CSF (filgrastim) on spermatogenic efficiency in prepubertal Brahman bulls. Twelve intact, healthy prepubertal bulls were administered 0, 1 (LD = low dose) or 4 (HD = high dose) µg/Kg r-met-hu-G-CSF (daily for 4 days), and haematological analysis was performed. Bulls were castrated (D0 or D60). BW (body weight) and SC (scrotal circumference) were recorded. Testis weight and volume were taken at castration with samples for testis histology and stereology: germ cell types, spermatids count and DSP (daily sperm production per gram)/g of testicular parenchyma. Testicular weight, volume, BW, SC and gonadosomatic index (GSI) were NS (LD-HD; p > .05). At D0 (age 11 months), the most advanced germ cell types (maGCt) ranged from intermediate spermatogonia to pachytene spermatocytes. After 2 months, control animals had round spermatids as maGCt, LD animals 75% round spermatids and 25% elongated spermatids, and HD animals round spermatids. Spermatids/testis were higher in LD (1.23 ± 0.2 millions) than in controls (0.65 ± 0.1 millions, p < .05). Spermatogenic efficiency (DSP/g) was higher in LD (5.4 ± 0.4 million) than in controls (3.2 ± 0.2 million, p < .01). In conclusion, r-met-hu-G-CSF raises spermatogenic efficiency in prepubertal Brahman bulls.
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Affiliation(s)
- Dario Arevalo
- Colegio de Ciencias Biológicas y Ambientales, Universidad San Francisco de Quito (USFQ), Quito, Ecuador
| | - Andrea C Flores
- Colegio de Ciencias de la Salud, Escuela de Medicina Veterinaria, Universidad San Francisco de Quito (USFQ), Quito, Ecuador
| | - Ramiro F Diaz
- Colegio de Ciencias de la Salud, Escuela de Medicina Veterinaria, Universidad San Francisco de Quito (USFQ), Quito, Ecuador.,Instituto de Investigaciones en Biomedicina 'One-health', Universidad San Francisco de Quito (USFQ), Quito, Ecuador
| | - Manuel Garcia-Herreros
- Instituto Nacional de Investigação Agrária e Veterinária, I. P. (INIAV, I.P.), Polo de Santarém, Santarém, Portugal
| | - Pedro M Aponte
- Colegio de Ciencias Biológicas y Ambientales, Universidad San Francisco de Quito (USFQ), Quito, Ecuador.,Colegio de Ciencias de la Salud, Escuela de Medicina Veterinaria, Universidad San Francisco de Quito (USFQ), Quito, Ecuador.,Instituto de Investigaciones en Biomedicina 'One-health', Universidad San Francisco de Quito (USFQ), Quito, Ecuador
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Sokouti Nasimi F, Zahri S, Ahmadian S, Bagherzadeh A, Nazdikbin Yamchi N, Haghighi L, Bedate AM, Khalilzadeh B, Rahbarghazi R, Mahdipour M. Estradiol modulated differentiation and dynamic growth of CD90 + spermatogonial stem cells toward Sertoli-like cells. Life Sci 2021; 286:120041. [PMID: 34637796 DOI: 10.1016/j.lfs.2021.120041] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2021] [Revised: 09/28/2021] [Accepted: 10/06/2021] [Indexed: 11/28/2022]
Abstract
Mouse CD90+ SSCs were enriched using the MACS technique and incubated with different doses of estradiol, ranging from 0.01 ng/mL to 500 μg/mL, for 7 days. The viability of SSCs was determined using an MTT assay. The combined effects of estradiol plus Sertoli cell differentiation medium on the orientation of SSCs toward Sertoli-like cells were also assessed. Using immunofluorescence imaging, we monitored protein levels of Oct3/4 after being exposed to estradiol. In addition, protein levels of testosterone, TF, and ABP were measured using ELISA. The expression of Sertoli cell-specific genes such as SOX9, GATA4, FSHR, TF, and ESR-1 and -2 was monitored using real-time PCR assay, and the effects of 14-day injection of estradiol on sperm parameters and Oct3/4 positive progenitor cells in a model of mouse were determined. Data showed that estradiol increased the viability of mouse SSCs in a dose-dependent manner compared to the control (p < 0.05). Along with these changes, cells displayed morphological changes and reduced Oct3/4 transcription factor levels compared to the control SSCs. 7-day incubation of SSCs with estradiol led to the up-regulation of SOX9, GATA4, FSHR, TF, and ESR-1 and -2, and levels of testosterone, TF, and ABP were increased compared to the control group (p < 0.05). The in-vivo examination noted that estradiol reduced sperm parameters coincided with morphological abnormalities (p < 0.05). Histological examination revealed pathological changes in seminiferous tubules and reduction of testicular Oct3/4+ progenitor cells. In conclusion, estradiol treatment probably can induce Sertoli cell differentiation of SSCs while exogenous administration leads to testicular progenitor cell depletion and infertility in long term.
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Affiliation(s)
- Fatemeh Sokouti Nasimi
- Department of Biology, Faculty of Basic Sciences, Mohaghegh Ardabili University, Ardabil, Iran
| | - Saber Zahri
- Department of Biology, Faculty of Basic Sciences, Mohaghegh Ardabili University, Ardabil, Iran
| | - Shahin Ahmadian
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Afsaneh Bagherzadeh
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | | | - Leila Haghighi
- Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Alberto Miranda Bedate
- Department of Immune Mechanisms (IMM), Center for Immunology of Infectious Diseases and Vaccines (IIV), National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands
| | - Balal Khalilzadeh
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Reza Rahbarghazi
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Applied Cell Sciences, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Mahdi Mahdipour
- Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; Department of Reproductive Biology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
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Binsila B, Selvaraju S, Ranjithkumaran R, Archana SS, Krishnappa B, Ghosh SK, Kumar H, Subbarao RB, Arangasamy A, Bhatta R. Current scenario and challenges ahead in application of spermatogonial stem cell technology in livestock. J Assist Reprod Genet 2021; 38:3155-3173. [PMID: 34661801 DOI: 10.1007/s10815-021-02334-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2021] [Accepted: 09/27/2021] [Indexed: 11/28/2022] Open
Abstract
PURPOSE Spermatogonial stem cells (SSCs) are the source for the mature male gamete. SSC technology in humans is mainly focusing on preserving fertility in cancer patients. Whereas in livestock, it is used for mining the factors associated with male fertility. The review discusses the present status of SSC biology, methodologies developed for in vitro culture, and challenges ahead in establishing SSC technology for the propagation of superior germplasm with special reference to livestock. METHOD Published literatures from PubMed and Google Scholar on topics of SSCs isolation, purification, characterization, short and long-term culture of SSCs, stemness maintenance, epigenetic modifications of SSCs, growth factors, and SSC cryopreservation and transplantation were used for the study. RESULT The fine-tuning of SSC isolation and culture conditions with special reference to feeder cells, growth factors, and additives need to be refined for livestock. An insight into the molecular mechanisms involved in maintaining stemness and proliferation of SSCs could facilitate the dissemination of superior germplasm through transplantation and transgenesis. The epigenetic influence on the composition and expression of the biomolecules during in vitro differentiation of cultured cells is essential for sustaining fertility. The development of surrogate males through gene-editing will be historic achievement for the foothold of the SSCs technology. CONCLUSION Detailed studies on the species-specific factors regulating the stemness and differentiation of the SSCs are required for the development of a long-term culture system and in vitro spermatogenesis in livestock. Epigenetic changes in the SSCs during in vitro culture have to be elucidated for the successful application of SSCs for improving the productivity of the animals.
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Affiliation(s)
- Balakrishnan Binsila
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India.
| | - Sellappan Selvaraju
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Rajan Ranjithkumaran
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Santhanahalli Siddalingappa Archana
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Balaganur Krishnappa
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Subrata Kumar Ghosh
- Animal Reproduction Division, Indian Council of Agricultural Research-Indian Veterinary Research Institute, Izatnagar, 243 122, India
| | - Harendra Kumar
- Animal Reproduction Division, Indian Council of Agricultural Research-Indian Veterinary Research Institute, Izatnagar, 243 122, India
| | - Raghavendra B Subbarao
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Arunachalam Arangasamy
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - Raghavendra Bhatta
- Indian council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
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Use of alginate hydrogel to improve long-term 3D culture of spermatogonial stem cells: stemness gene expression and structural features. ZYGOTE 2021; 30:312-318. [PMID: 34641993 DOI: 10.1017/s0967199421000551] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
The quality and quantity of a spermatogonial stem-cell (SSC) culture can be measured in less time using a 3D culture in a scaffold. The present study investigated stemness gene expression and the morphological and structural characterization of SSCs encapsulated in alginate. SSCs were harvested from BALB/c neonatal mice testes through two-step mechanical and enzymatic digestion. The spermatogonial populations were separated using magnetic-activated cell sorting (MACS) using an anti-Thy1 antibody and c-Kit. The SSCs then were encapsulated in alginate hydrogel. After 2 months of SSC culturing, the alginate microbeads were extracted and stained to evaluate their histological properties. Real-time polymerase chain reaction (PCR) was performed to determine the stemness gene expression. Scanning electron microscopy (SEM) was performed to evaluate the SSC morphology, density and scaffold structure. The results showed that encapsulated SSCs had decreased expression of Oct4, Sox2 and Nanos2 genes, but the expression of Nanog, Bcl6b and Plzf genes was not significantly altered. Histological examination showed that SSCs with pale nuclei and numerous nucleolus formed colonies. SEM evaluation revealed that the alginate scaffold structure preserved the SSC morphology and density for more than 60 days. Cultivation of SSCs on alginate hydrogel can affect Oct4, Sox2 and Nanos2 expression.
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N'Tumba-Byn T, Yamada M, Seandel M. Loss of tyrosine kinase receptor Ephb2 impairs proliferation and stem cell activity of spermatogonia in culture†. Biol Reprod 2021; 102:950-962. [PMID: 31836902 DOI: 10.1093/biolre/ioz222] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2019] [Revised: 10/30/2019] [Accepted: 12/11/2019] [Indexed: 12/17/2022] Open
Abstract
Germline stem and progenitor cells can be extracted from the adult mouse testis and maintained long-term in vitro. Yet, the optimal culture conditions for preserving stem cell activity are unknown. Recently, multiple members of the Eph receptor family were detected in murine spermatogonia, but their roles remain obscure. One such gene, Ephb2, is crucial for maintenance of somatic stem cells and was previously found enriched at the level of mRNA in murine spermatogonia. We detected Ephb2 mRNA and protein in primary adult spermatogonial cultures and hypothesized that Ephb2 plays a role in maintenance of stem cells in vitro. We employed CRISPR-Cas9 targeting and generated stable mutant SSC lines with complete loss of Ephb2. The characteristics of Ephb2-KO cells were interrogated using phenotypic and functional assays. Ephb2-KO SSCs exhibited reduced proliferation compared to wild-type cells, while apoptosis was unaffected. Therefore, we examined whether Ephb2 loss correlates with activity of canonical pathways involved in stem cell self-renewal and proliferation. Ephb2-KO cells had reduced ERK MAPK signaling. Using a lentiviral transgene, Ephb2 expression was rescued in Ephb2-KO cells, which partially restored signaling and proliferation. Transplantation analysis revealed that Ephb2-KO SSCs cultures formed significantly fewer colonies than WT, indicating a role for Ephb2 in preserving stem cell activity of cultured cells. Transcriptome analysis of wild-type and Ephb2-KO SSCs identified Dppa4 and Bnc1 as differentially expressed, Ephb2-dependent genes that are potentially involved in stem cell function. These data uncover for the first time a crucial role for Ephb2 signaling in cultured SSCs.
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Affiliation(s)
- Thierry N'Tumba-Byn
- Department of Surgery, Weill Cornell Medical College, New York, NY, United States of America
| | - Makiko Yamada
- Department of Surgery, Weill Cornell Medical College, New York, NY, United States of America
| | - Marco Seandel
- Department of Surgery, Weill Cornell Medical College, New York, NY, United States of America
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Nabighadim A, Jafarnezhad-Ansariha F, Majidi Zolbin M, Daryabari SS, Fendereski K, Kajbafzadeh AM. Gene and histomorphology alteration analysis in spermatogenesis arrest mouse model: a probable novel approach for infertility. Cent European J Urol 2021; 74:99-108. [PMID: 33976924 PMCID: PMC8097657 DOI: 10.5173/ceju.2020.0175] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2020] [Revised: 06/14/2020] [Accepted: 12/06/2020] [Indexed: 11/22/2022] Open
Abstract
Introduction Approximately 15% of couples in the reproductive age are struggling with infertility which, in nearly half of them, is caused by male factors. Material and methods The present study comprised of two groups of sixteen C57BL/6 mice; each mouse received either an intraperitoneal injection of 30 mg/kg of an alkylating agent or the same amount of distilled water. Testes were harvested 30 days following the injection. Morphometric analysis of hematoxylin and eosin (H&E) stained slides including mean tubular area, diameter and intratubular particles were performed. Spermatogenesis rate was assessed by spermatogonial markers including promyelocytic leukemia zinc finger protein (PLZF) and neurogenin-3 (NGN3). Moreover, the expression rate of Wilms Tumor-1 (WT-1), A-Kinase Anchoring Protein 4 (AKAP4) and adenosine deaminase domain containing 1 (ADAD1) genes were evaluated via real-time polymerase chain reaction (RT-PCR). Results The body weight gradually increased in both groups after a period of 30 days, however, the increase was significantly (p-value = 0.023) lower in the chemically treated group. All the morphometric parameters were considerably decreased in the azoospermic mice. Also, promyelocytic leukemia zinc finger protein and neurogenin-3 expression dramatically declined (p-value <0.001 for both markers). In comparison with the negative control group, the expression rates of A-Kinase Anchoring Protein 4 and adenosine deaminase domain containing 1, two genes participating in the sperm structure, were remarkably reduced in the intervention group (p-value <0.001); however, our investigations demonstrated that the azoospermia model could induce a 5-fold upregulation in Wilms Tumor-1 gene expression. Conclusions Development of an azoospermia model can upregulate Wilms Tumor-1 gene expression in a higher rate after 30 days; however, expression of the testis-specific genes, A-Kinase Anchoring Protein 4 and adenosine deaminase domain containing 1, decreased after the intervention. To the best of our knowledge, this upregulation could be related to spermatogenesis recovery after the follow-up period.
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Affiliation(s)
- Amirreza Nabighadim
- Pediatric Urology and Regenerative Medicine Research Center, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran
| | - Fahimeh Jafarnezhad-Ansariha
- Pediatric Urology and Regenerative Medicine Research Center, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran
| | - Masoumeh Majidi Zolbin
- Pediatric Urology and Regenerative Medicine Research Center, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran
| | - Seyedeh Sima Daryabari
- Pediatric Urology and Regenerative Medicine Research Center, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran
| | - Kiarad Fendereski
- Pediatric Urology and Regenerative Medicine Research Center, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran
| | - Abdol Mohammad Kajbafzadeh
- Pediatric Urology and Regenerative Medicine Research Center, Children's Medical Center, Tehran University of Medical Sciences, Tehran, Iran
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13
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PATHAK MANISHA, KHARCHE SD, SINGH SP, JENA D, PATHAK JUHI, GUPTA DEEKSHA, SIKARWAR AKS, CHAUHAN MS. Fetal bovine serum (FBS) enhances proliferation and colonization of caprine spermatogonial stem cells. THE INDIAN JOURNAL OF ANIMAL SCIENCES 2020. [DOI: 10.56093/ijans.v90i5.104609] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/05/2022]
Abstract
Enrichment of cell suspension with germ cells prior to injection into recipient seminiferous tubules is of importance in spermatogonial stem cells (SSCs) transplantation. Fetal bovine serum (FBS) is the most widely used growth supplement for cell cultures, primarily because of its high levels of growth stimulatory factors and low levels of growth inhibitory factors. This study was undertaken to investigate the effect of serum concentration on colony formation and development of different types of SSC colonies with respect to passage number. Cells were isolated from pre-pubertal buck testes by two step enzymatic digestion method. The filtered cells were enriched by differential adherence selection method. Cells were then randomly divided into 8 groups, depending on concentration of FBS in culture medium ranging from 0% to 35%. In experiment 1, effect of different concentrations of FBS on total number pSSCs with reference to differential plating was observed while in experiment 2, effect of different concentrations of FBS on types of pSSC colonies with respect to passage number was observed. No colony formation was observed in control group (0% FBS) while significantly higher number of single, paired, cluster and rosette colonies observed were with 20% FBS group in differential 2 (D2) as compared to other groups. Alkaline phosphatase staining and immunocytochemistry staining (PGP9.5 and OCT4) were positive in SSCs colonies. The growth rate of the culture was significantly and consistently higher with 20% FBS.
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Binsila BK, Selvaraju S, Ghosh SK, Ramya L, Arangasamy A, Ranjithkumaran R, Bhatta R. EGF, GDNF, and IGF-1 influence the proliferation and stemness of ovine spermatogonial stem cells in vitro. J Assist Reprod Genet 2020; 37:2615-2630. [PMID: 32821972 DOI: 10.1007/s10815-020-01912-5] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2019] [Accepted: 08/03/2020] [Indexed: 11/25/2022] Open
Abstract
PURPOSE The objective of the present study was to purify sheep spermatogonial stem cells (SSCs) from testicular isolate using combined enrichment methods and to study the effect of growth factors on SSC stemness during culture. METHODS The testicular cells from prepubertal male sheep were isolated, and SSCs were purified using Ficoll gradients (10 and 12%) followed by differential plating (laminin with BSA). SSCs were cultured with StemPro®-34 SFM, additives, and FBS for 7 days. The various doses (ng/ml) of growth factors, EGF at 10, 15, and 20, GDNF at 40, 70, and 100 and IGF-1 at 50, 100, and 150 were tested for the proliferation and stemness of SSCs in vitro. The stemness in cultured cells was assessed using SSC markers PLZF, ITGA6, and GFRα1. RESULTS Ficoll density gradient separation significantly (p < 0.05) increased the percentage of SSCs in 12% fraction (35.1 ± 3.8 vs 11.2 ± 3.7). Subsequently, purification using laminin with BSA plating further enriched SSCs to 61.7 ± 4.7%. GDNF at 40 ng/ml, EGF at 15 and 20 ng/ml and IGF1 at 100 and 150 ng/ml significantly (p < 0.05) improved proliferation and stemness of SSCs up to 7 days in culture. GDNF at 40 ng/ml outperformed other growth factors tested and could maintain the ovine SSCs proliferation and stemness for 36 days. CONCLUSIONS The combined enrichment method employing density gradient centrifugation and laminin with BSA plating improves the purification efficiency of ovine SSCs. GDNF at 40 ng/ml is essential for optimal proliferation and sustenance of stemness of ovine SSCs in vitro.
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Affiliation(s)
- B K Binsila
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India.
| | - S Selvaraju
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - S K Ghosh
- Animal Reproduction Division, Indian Council of Agricultural Research-Indian Veterinary Research Institute, Izatnagar, 243 122, India
| | - L Ramya
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - A Arangasamy
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - R Ranjithkumaran
- Reproductive Physiology Laboratory, Animal Physiology Division, Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
| | - R Bhatta
- Indian Council of Agricultural Research-National Institute of Animal Nutrition and Physiology, Bengaluru, 560 030, India
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15
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Azarniad R, Razi M, Hasanzadeh S, Malekinejad H. Experimental diabetes negatively affects the spermatogonial stem cells' self-renewal by suppressing GDNF network interactions. Andrologia 2020; 52:e13710. [PMID: 32539191 DOI: 10.1111/and.13710] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2020] [Revised: 05/03/2020] [Accepted: 05/15/2020] [Indexed: 01/09/2023] Open
Abstract
The present study was done to analyse the time-dependent effects of diabetes on Sertoli cells-spermatogonial stem cells' (SSCs) network interaction by focusing on glial cell line-derived neurotrophic factor (GDNF) and its special receptors, gfrα1 and c-RET as well as the Bcl-6b. In total, 40 Wistar rats were considered in; control, 20, 45 and 60 days diabetes-induced groups. An experimental diabetes was induced by STZ. The GDNF, gfrα1, c-RET and Bcl-6b expressions were evaluated. The serum level of testosterone, tubular repopulation (RI) and spermiogenesis (SPI) indices, general histological alterations, germ cells, mRNA damage, sperm count and viability were assessed. The diabetes, in a time-dependent manner, diminished mRNA and protein levels of GDNF, gfrα1, c-RET and Bcl-6b versus control group (p < .05), enhanced percentage of seminiferous tubules with negative RI, SPI, and diminished Leydig and Sertoli cells distribution, serum levels of testosterone, sperm count and viability. Finally, the number, percentage of cells and seminiferous tubules with normal mRNA content were significantly (p < .05) diminished. In conclusion, as a new data, we showed that the diabetes by inducing severe mRNA damage and suppressing GDNF, gfrα1, c-RET and Bcl-6b expressions, potentially affects the Sertoli-SSCs' network and consequently inhibits the SSCs' self-renewal process.
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Affiliation(s)
- Rozita Azarniad
- Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
| | - Mazdak Razi
- Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
| | - Shapour Hasanzadeh
- Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
| | - Hassan Malekinejad
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, Urmia University of Medical Sciences, Urmia, Iran.,Food and Beverages Safety Research Center, Urmia University of Medical Sciences, Urmia, Iran
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16
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Sohni A, Tan K, Song HW, Burow D, de Rooij DG, Laurent L, Hsieh TC, Rabah R, Hammoud SS, Vicini E, Wilkinson MF. The Neonatal and Adult Human Testis Defined at the Single-Cell Level. Cell Rep 2020; 26:1501-1517.e4. [PMID: 30726734 PMCID: PMC6402825 DOI: 10.1016/j.celrep.2019.01.045] [Citation(s) in RCA: 224] [Impact Index Per Article: 44.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2018] [Revised: 11/21/2018] [Accepted: 01/10/2019] [Indexed: 12/14/2022] Open
Abstract
Spermatogenesis has been intensely studied in rodents but remains poorly understood in humans. Here, we used single-cell RNA sequencing to analyze human testes. Clustering analysis of neonatal testes reveals several cell subsets, including cell populations with characteristics of primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). In adult testes, we identify four undifferentiated spermatogonia (SPG) clusters, each of which expresses specific marker genes. We identify protein markers for the most primitive SPG state, allowing us to purify this likely SSC-enriched cell subset. We map the timeline of male germ cell development from PGCs through fetal germ cells to differentiating adult SPG stages. We also define somatic cell subsets in both neonatal and adult testes and trace their developmental trajectories. Our data provide a blueprint of the developing human male germline and supporting somatic cells. The PGC-like and SSC markers are candidates to be used for SSC therapy to treat infertility. Sohni et al. use scRNA-seq analysis to define cell subsets in the human testis. Highlights include the identification of primordial germ cell- and spermatogonial stem cell-like cell subsets in neonatal testes, numerous undifferentiated spermatogonial cell states in adult testes, and somatic cell subsets in both neonatal and adult testes.
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Affiliation(s)
- Abhishek Sohni
- Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Diego, La Jolla, CA 92093, USA
| | - Kun Tan
- Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Diego, La Jolla, CA 92093, USA
| | - Hye-Won Song
- Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Diego, La Jolla, CA 92093, USA
| | - Dana Burow
- Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Diego, La Jolla, CA 92093, USA
| | - Dirk G de Rooij
- Reproductive Biology Group, Division of Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, 3584 CH Utrecht, the Netherlands
| | - Louise Laurent
- Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Diego, La Jolla, CA 92093, USA
| | - Tung-Chin Hsieh
- Department of Urology, University of California, San Diego, La Jolla, CA 92103, USA
| | - Raja Rabah
- Pediatric and Perinatal Pathology, Michigan Medicine, CS Mott and VonVoigtlander Women's Hospitals, Ann Arbor, MI 48109-4272, USA
| | - Saher Sue Hammoud
- Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109, USA
| | - Elena Vicini
- Department of Anatomy, Histology, Forensic Medicine and Orthopedic, Section of Histology Sapienza University of Rome, 00161 Rome, Italy
| | - Miles F Wilkinson
- Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Diego, La Jolla, CA 92093, USA; Institute of Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
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17
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Ziloochi Kashani M, Bagher Z, Asgari HR, Najafi M, Koruji M, Mehraein F. Differentiation of neonate mouse spermatogonial stem cells on three-dimensional agar/polyvinyl alcohol nanofiber scaffold. Syst Biol Reprod Med 2020; 66:202-215. [PMID: 32138551 DOI: 10.1080/19396368.2020.1725927] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Electrospun nanofiber matrices sufficiently mimic the structural morphology of natural extracellular matrix. In this study, we aimed to examine the effects of agar/polyvinyl alcohol nanofiber (PVA) scaffold on the proliferation efficiency and differentiation potential of neonate mouse spermatogonial stem cells (SCCs). Testicular cells were isolated from testes of 40 mouse pups and were seeded in: 1) 2D cell culture plates in the absence (2D/-GF) or presence (2D/+GF) of growth factors and 2) onto agar/PVA scaffold in the absence (3D/-GF) or presence (3D/+GF) of growth factors. The cells were subsequently cultured for 4 weeks. First 2 weeks were dedicated to proliferative phase, whereas the next 2 weeks emphasized the differentiation phase. The identity of the SCCs was investigated at different time-points by flow cytometry and quantitative reverse transcription PCR (qRT-PCR) analyses against the germ cell markers, including PLZF, Id-4, Gfrα-1, Tekt-1, and Sycp-3. After 2 weeks of culture, the 3D/+GF group showed the highest percentage of PLZF-positive cells among culture systems (P < 0.05). The expression levels of pre-meiotic markers (Id-4 and Gfrα-1) decreased significantly in all groups, particularly in 3D/+GF group after 28 days of culture. Additionally, the cells in the 3D/+GF group displayed the highest expression of meiotic (Sycp-3) and post-meiotic markers (Tekt-1) 14 days after differentiation induction. Seemingly, the combination of the agar/PVA scaffold and growth factor-supplemented medium synergistically increased the differentiation rate of mouse SSCs into meiotic and post-meiotic cells. Thus, agar/PVA nanofiber scaffolds may have the potential for applications in the restoration of infertility, especially in azoospermic males. ABBREVIATIONS 2D: two dimentional; 3D: three dimentional; bFGF: basic fibroblast growth factor; BMP-4: bone morphogenetic protein 4; DMEM: Dulbecco's modified Eagle's medium; ECM: extracellular matrix; FCS: fetal calf serum; FTIR: Fourier-transform infrared spectroscopy; GDNF: glial cell line-derived neurotrophic factor; GF: growth factors; Gfrα-1, GDNF family co-receptor α1; Id-4, Inhibitor of DNA Binding 4; MTT: methylthiazoltetrazolium; PLZF: promyelocytic leukemia zinc finger; PVA: polyvinyl alcohol; qRT-PCR: quantitative reverse transcription PCR; RA: retinoic acid; SACS: soft agar culture system; SD: standard deviation; SEM: scanning electron microscope; SSCs: spermatogonial stem cells; Sycp-3, Synaptonemal complex protein 3; Tekt-1, Tektin 1.
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Affiliation(s)
- Marzieh Ziloochi Kashani
- Cellular and Molecular Research Center, Iran University of Medical Sciences , Tehran, Iran.,Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences , Tehran, Iran
| | - Zohreh Bagher
- ENT and Head & Neck Research Center and Department, the Five Senses Institute, Hazrat Rasoul Akram Hospital, Iran University of Medical Sciences , Tehran, Iran
| | - Hamid Reza Asgari
- Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences , Tehran, Iran
| | - Mohammad Najafi
- Department of Biochemistry, Faculty of Medicine, Iran University of Medical Sciences , Tehran, Iran
| | - Morteza Koruji
- Cellular and Molecular Research Center, Iran University of Medical Sciences , Tehran, Iran.,Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences , Tehran, Iran
| | - Fereshteh Mehraein
- Department of Anatomical Sciences, School of Medicine, Iran University of Medical Sciences , Tehran, Iran.,Minimally Invasive Surgery Research Center, Iran University of Medical Sciences , Tehran, Iran
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18
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Song H, Wang L, Chen D, Li F. The Function of Pre-mRNA Alternative Splicing in Mammal Spermatogenesis. Int J Biol Sci 2020; 16:38-48. [PMID: 31892844 PMCID: PMC6930371 DOI: 10.7150/ijbs.34422] [Citation(s) in RCA: 54] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2019] [Accepted: 09/20/2019] [Indexed: 01/05/2023] Open
Abstract
Alternative pre-mRNA splicing plays important roles in co-transcriptional and post-transcriptional regulation of gene expression functioned during many developmental processes, such as spermatogenesis. The studies focusing on alternative splicing on spermatogenesis supported the notion that the development of testis is regulated by a higher level of alternative splicing than other tissues. Here, we aim to review the mechanisms underlying alternative splicing, particularly the splicing variants functioned in the process of spermatogenesis and the male infertility. There are five points regarding the alternative splicing including ⅰ) a brief introduction of alternative pre-mRNA splicing; ⅱ) the alternative splicing events in spermatogenesis-associated genes enriched in different stages of spermatogenesis; ⅲ) the mechanisms of alternative splicing regulation, such as splicing factors and m6A demethylation; ⅳ) the splice site recognition and alternative splicing, including the production and degradation of abnormal transcripts caused by gene variations and nonsense-mediated mRNA decay, respectively; ⅴ) abnormal alternative splicing correlated with male infertility. Taking together, this review highlights the impacts of alternative splicing and splicing variants in mammal spermatogenesis and provides new insights of the potential application of the alternative splicing into the therapy of male infertility.
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Affiliation(s)
- Huibin Song
- Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs & Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, PR China
| | - Ling Wang
- Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs & Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, PR China
| | - Dake Chen
- Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs & Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, PR China
| | - Fenge Li
- Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs & Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, PR China.,The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, PR China
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19
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Ezirim CY, Abarikwu SO, Uwakwe AA, Mgbudom-Okah CJ. Protective effects of Anthocleista djalonensis A. Chev root extracts against induced testicular inflammation and impaired spermatogenesis in adult rats. Mol Biol Rep 2019; 46:5983-5994. [DOI: 10.1007/s11033-019-05033-w] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2019] [Accepted: 08/12/2019] [Indexed: 12/23/2022]
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20
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Ghorbani S, Eyni H, Khosrowpour Z, Salari Asl L, Shabani R, Nazari H, Mehdizadeh M, Ebrahimi Warkiani M, Amjadi F. Spermatogenesis induction of spermatogonial stem cells using nanofibrous poly(
l
‐lactic acid)/multi‐walled carbon nanotube scaffolds and naringenin. POLYM ADVAN TECHNOL 2019. [DOI: 10.1002/pat.4733] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Affiliation(s)
- Sadegh Ghorbani
- Department of Anatomical Sciences, School of Medical SciencesTarbiat Modares University Tehran Iran
- Interdisciplinary Nanoscience Center (iNANO)Aarhus University Aarhus Denmark
| | - Hossein Eyni
- Department of Anatomical Sciences, School of Medical SciencesTarbiat Modares University Tehran Iran
| | - Zahra Khosrowpour
- Department of Anatomical Sciences, School of Medical SciencesTarbiat Modares University Tehran Iran
| | - Leila Salari Asl
- Department of Anatomical Sciences, School of Medical SciencesTarbiat Modares University Tehran Iran
| | - Ronak Shabani
- Cellular and Molecular Research Center, School of MedicineIran University of Medical Sciences Tehran Iran
- Department of Anatomical Sciences, School of MedicineIran University of Medical Sciences Tehran Iran
| | - Hojjatollah Nazari
- Department of Cell Therapy and Hematology, Faculty of Medical SciencesTarbiat Modares University Tehran Iran
| | - Mehdi Mehdizadeh
- Cellular and Molecular Research Center, School of MedicineIran University of Medical Sciences Tehran Iran
- Department of Anatomical Sciences, School of MedicineIran University of Medical Sciences Tehran Iran
| | - Majid Ebrahimi Warkiani
- School of Biomedical EngineeringUniversity of Technology Sydney New South Wales Australia
- Institute of Molecular MedicineSechenov First Moscow State University Moscow Russia
| | - FatemehSadat Amjadi
- Cellular and Molecular Research Center, School of MedicineIran University of Medical Sciences Tehran Iran
- Department of Anatomical Sciences, School of MedicineIran University of Medical Sciences Tehran Iran
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21
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Li X, Long XY, Xie YJ, Zeng X, Chen X, Mo ZC. The roles of retinoic acid in the differentiation of spermatogonia and spermatogenic disorders. Clin Chim Acta 2019; 497:54-60. [PMID: 31302099 DOI: 10.1016/j.cca.2019.07.013] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2019] [Revised: 06/20/2019] [Accepted: 07/10/2019] [Indexed: 12/21/2022]
Abstract
Male fertility depends on the regulatory balance between germ cell self-renewal and differentiation, and the spatial and temporal patterns of this balance must be maintained throughout the life cycle. Retinoic acid and its receptors are important factors in spermatogenesis. Spermatogonia cells can self-proliferate and differentiate and have unique meiotic capabilities; they halve their genetic material and produce monomorphic sperm to pass genetic material to the next generation. A number of studies have found that the spermatogenesis process is halted in animals with vitamin A deficiency and that most germ cells are degraded, but they tend to recover after treatment with RA or vitamin A. This literature review discusses our understanding of how RA regulates sperm cell differentiation and meiosis and also reviews the functional information and details of RA.
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Affiliation(s)
- Xuan Li
- Clinical Anatomy & Reproductive Medicine Application Institute, Department of Histology and Embryology, Hengyang Medical School, University of South China, Hengyang 421001, China
| | - Xiang-Yang Long
- Department of Urology, The Second Affiliated Hospital, University of South China, Hengyang 421001, China
| | - Yuan-Jie Xie
- Clinical Anatomy & Reproductive Medicine Application Institute, Department of Histology and Embryology, Hengyang Medical School, University of South China, Hengyang 421001, China
| | - Xin Zeng
- Clinical Anatomy & Reproductive Medicine Application Institute, Department of Histology and Embryology, Hengyang Medical School, University of South China, Hengyang 421001, China
| | - Xi Chen
- Clinical Anatomy & Reproductive Medicine Application Institute, Department of Histology and Embryology, Hengyang Medical School, University of South China, Hengyang 421001, China.
| | - Zhong-Cheng Mo
- Clinical Anatomy & Reproductive Medicine Application Institute, Department of Histology and Embryology, Hengyang Medical School, University of South China, Hengyang 421001, China.
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22
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Park HJ, Lee WY, Park C, Hong K, Song H. CD14 is a unique membrane marker of porcine spermatogonial stem cells, regulating their differentiation. Sci Rep 2019; 9:9980. [PMID: 31292454 PMCID: PMC6620343 DOI: 10.1038/s41598-019-46000-6] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2018] [Accepted: 06/20/2019] [Indexed: 01/15/2023] Open
Abstract
Molecular markers of spermatogonia are necessary for studies on spermatogonial stem cells (SSCs) and improving our understanding of molecular and cellular biology of spermatogenesis. Although studies of germ cell surface marker have been extensively conducted in the testes of rodents, these markers have not been well studied in domestic animals. We aimed to determine the expression pattern of cluster of differentiation 14 (CD14) in developing porcine testes and cultured porcine SSCs (pSSCs), as well as its role in pSSC colony formation. Interestingly, expression of CD14 was observed in porcine testes with PGP9.5-positive undifferentiated spermatogonia at all developmental stages. In addition, in vitro cultured pSSCs expressed CD14 and showed successful colony formation, as determined by fluorescence-activated cell sorting and flow cytometry. PKH26 dye-stained CD14-positive cells transplants were performed into the testes of recipient mice, which were depleted of both testicular germ and somatic cells from immunodeficiency mice and were shown to colonise the recipient testes. Moreover, a colony-forming assay showed that the development of pSSC colonies was disrupted by a high concentration of lipopolysaccharide. These studies indicated that CD14 is surface marker of early spermatogonia in developing porcine testes and in pSSCs, suggesting a role for CD14 in porcine spermatogenesis.
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Affiliation(s)
- Hyun-Jung Park
- Department of Stem Cell and Regenerative Technology, KIT, Konkuk University, 120 Neungdongro, Gwangjin-gu, Seoul, 05029, Republic of Korea
| | - Won-Young Lee
- Department of Beef Science, Korea National College of Agricultures and Fisheries, Jeonju-si, Jeonbuk, 54874, Republic of Korea
| | - Chankyu Park
- Department of Stem Cell and Regenerative Technology, KIT, Konkuk University, 120 Neungdongro, Gwangjin-gu, Seoul, 05029, Republic of Korea
| | - Kwonho Hong
- Department of Stem Cell and Regenerative Technology, KIT, Konkuk University, 120 Neungdongro, Gwangjin-gu, Seoul, 05029, Republic of Korea
| | - Hyuk Song
- Department of Stem Cell and Regenerative Technology, KIT, Konkuk University, 120 Neungdongro, Gwangjin-gu, Seoul, 05029, Republic of Korea.
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Cytological analysis of pregnancy-associated plasma protein-A expression in porcine neonatal testis. JOURNAL OF ANIMAL REPRODUCTION AND BIOTECHNOLOGY 2018. [DOI: 10.12750/jet.2018.33.3.177] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
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Sinha D, Kalimutho M, Bowles J, Chan AL, Merriner DJ, Bain AL, Simmons JL, Freire R, Lopez JA, Hobbs RM, O'Bryan MK, Khanna KK. Cep55 overexpression causes male-specific sterility in mice by suppressing Foxo1 nuclear retention through sustained activation of PI3K/Akt signaling. FASEB J 2018; 32:4984-4999. [PMID: 29683733 DOI: 10.1096/fj.201701096rr] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Spermatogenesis is a dynamic process involving self-renewal and differentiation of spermatogonial stem cells, meiosis, and ultimately, the differentiation of haploid spermatids into sperm. Centrosomal protein 55 kDa (CEP55) is necessary for somatic cell abscission during cytokinesis. It facilitates equal segregation of cytoplasmic contents between daughter cells by recruiting endosomal sorting complex required for transport machinery (ESCRT) at the midbody. In germ cells, CEP55, in partnership with testes expressed-14 (TEX14) protein, has also been shown to be an integral component of intercellular bridge before meiosis. Various in vitro studies have demonstrated a role for CEP55 in multiple cancers and other diseases. However, its oncogenic potential in vivo remains elusive. To investigate, we generated ubiquitously overexpressing Cep55 transgenic ( Cep55Tg/Tg) mice aiming to characterize its oncogenic role in cancer. Unexpectedly, we found that Cep55Tg/Tg male mice were sterile and had severe and progressive defects in spermatogenesis related to spermatogenic arrest and lack of spermatids in the testes. In this study, we characterized this male-specific phenotype and showed that excessively high levels of Cep55 results in hyperactivation of PI3K/protein kinase B (Akt) signaling in testis. In line with this finding, we observed increased phosphorylation of forkhead box protein O1 (FoxO1), and suppression of its nuclear retention, along with the relative enrichment of promyelocytic leukemia zinc finger (PLZF) -positive cells. Independently, we observed that Cep55 amplification favored upregulation of ret ( Ret) proto-oncogene and glial-derived neurotrophic factor family receptor α-1 ( Gfra1). Consistent with these data, we observed selective down-regulation of genes associated with germ cell differentiation in Cep55-overexpressing testes at postnatal day 10, including early growth response-4 ( Egr4) and spermatogenesis and oogenesis specific basic helix-loop-helix-1 ( Sohlh1). Thus, Cep55 amplification leads to a shift toward the initial maintenance of undifferentiated spermatogonia and ultimately results in progressive germ cell loss. Collectively, our findings demonstrate that Cep55 overexpression causes change in germ cell proportions and manifests as a Sertoli cell only tubule phenotype, similar to that seen in many azoospermic men.-Sinha, D., Kalimutho, M., Bowles, J., Chan, A.-L., Merriner, D. J., Bain, A. L., Simmons, J. L., Freire, R., Lopez, J. A., Hobbs, R. M., O'Bryan, M. K., Khanna, K. K. Cep55 overexpression causes male-specific sterility in mice by suppressing Foxo1 nuclear retention through sustained activation of PI3K/Akt signaling.
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Affiliation(s)
- Debottam Sinha
- Queensland Institute of Medical Research (QIMR) Berghofer Medical Research Institute, Herston, Queensland, Australia.,School of Natural Sciences, Griffith University, Nathan, Queensland, Australia
| | - Murugan Kalimutho
- Queensland Institute of Medical Research (QIMR) Berghofer Medical Research Institute, Herston, Queensland, Australia.,School of Natural Sciences, Griffith University, Nathan, Queensland, Australia
| | - Josephine Bowles
- School of Biomedical Sciences, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, Australia
| | - Ai-Leen Chan
- Germline Stem Cell Laboratory, Australian Regenerative Medicine Institute and Monash Biomedicine Discovery Institute, Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia
| | - D Jo Merriner
- Male Infertility and Germ Cell Biology Laboratory, the School of Biological Sciences, Monash University, Clayton, Victoria, Australia; and
| | - Amanda L Bain
- Queensland Institute of Medical Research (QIMR) Berghofer Medical Research Institute, Herston, Queensland, Australia
| | - Jacinta L Simmons
- Queensland Institute of Medical Research (QIMR) Berghofer Medical Research Institute, Herston, Queensland, Australia
| | - Raimundo Freire
- Unidad de Investigación, Hospital Universitario de Canarias, Instituto de Tecnologías Biomédicas, Tenerife, Spain
| | - J Alejandro Lopez
- Queensland Institute of Medical Research (QIMR) Berghofer Medical Research Institute, Herston, Queensland, Australia.,School of Natural Sciences, Griffith University, Nathan, Queensland, Australia
| | - Robin M Hobbs
- Germline Stem Cell Laboratory, Australian Regenerative Medicine Institute and Monash Biomedicine Discovery Institute, Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria, Australia
| | - Moira K O'Bryan
- Male Infertility and Germ Cell Biology Laboratory, the School of Biological Sciences, Monash University, Clayton, Victoria, Australia; and
| | - Kum Kum Khanna
- Queensland Institute of Medical Research (QIMR) Berghofer Medical Research Institute, Herston, Queensland, Australia
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Expression patterns and role of SDF-1/CXCR4 axis in boar spermatogonial stem cells. Theriogenology 2018; 113:221-228. [PMID: 29573661 DOI: 10.1016/j.theriogenology.2018.03.008] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2017] [Revised: 03/09/2018] [Accepted: 03/10/2018] [Indexed: 01/01/2023]
Abstract
The signaling of chemokine stromal cell-derived factor (SDF)-1 and its receptor C-X-C motif chemokine receptor 4 (CXCR4) is involved in the cellular proliferation, survival, and migration of various cell types. Although SDF-1/CXCR4 has been implicated in the maintenance of the spermatogonial population during mouse testis development, their expression patterns and functions in boar testis remain unclear. In the present study, the expression pattern of SDF-1 and CXCR4 was determined during pre-pubertal and post-pubertal stage boar testes and in vitro cultured porcine spermatogonial stem cells (pSSCs). The role of these proteins in colony formation in cultured pSSCs was also investigated. Interestingly, SDF-1 expression was observed in PGP 9.5-positve spermatogonia in all developing stages of boar testis; however, CXCR4 expression was only detected in spermatogonia from 5-day-old boar testis. In addition, SDF-1 and CXCR4 expression was observed in cultured pSSCs from 5-day-old boar testes, and inhibition of the CXCR4 receptor signaling pathway by AMD3100 significantly decreased the colony formation of pSSCs. These results suggest that SDF-1 and CXCR4 are useful markers for detecting stage-specific spermatogonia in boar testis. Our results reveal the role of the SDF-1/CXCR4 axis in pSSC in vitro culture.
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Park JE, Park MH, Kim MS, Park YR, Yun JI, Cheong HT, Kim M, Choi JH, Lee E, Lee ST. Porcine spermatogonial stem cells self-renew effectively in a three dimensional culture microenvironment. Cell Biol Int 2017; 41:1316-1324. [DOI: 10.1002/cbin.10844] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2017] [Accepted: 08/12/2017] [Indexed: 01/15/2023]
Affiliation(s)
- Ji Eun Park
- Department of Animal Life Science; Kangwon National University; Chuncheon 24341 Korea
| | - Min Hee Park
- Department of Animal Life Science; Kangwon National University; Chuncheon 24341 Korea
| | - Min Seong Kim
- Department of Animal Life Science; Kangwon National University; Chuncheon 24341 Korea
| | - Yeo Reum Park
- College of Veterinary Medicine; Kangwon National University; Chuncheon 24341 Korea
| | - Jung Im Yun
- Division of Animal Resource Science; Kangwon National University; Chuncheon 24341 Korea
| | - Hee Tae Cheong
- College of Veterinary Medicine; Kangwon National University; Chuncheon 24341 Korea
| | - Minseok Kim
- Animal Nutrition and Physiology Team; National Institute of Animal Science, RDA; Wanju 55365 Korea
| | - Jung Hoon Choi
- College of Veterinary Medicine; Kangwon National University; Chuncheon 24341 Korea
| | - Eunsong Lee
- College of Veterinary Medicine; Kangwon National University; Chuncheon 24341 Korea
| | - Seung Tae Lee
- Department of Animal Life Science; Kangwon National University; Chuncheon 24341 Korea
- Division of Applied Animal Science, Department of Animal Life Science, Laboratory of Stem Cell Biomodulation; Kangwon National University; Chuncheon 24341 Korea
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Lee WY, Lee R, Park HJ, Do JT, Park C, Kim JH, Jhun H, Lee JH, Hur T, Song H. Characterization of male germ cell markers in canine testis. Anim Reprod Sci 2017; 182:1-8. [DOI: 10.1016/j.anireprosci.2017.01.002] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2016] [Revised: 01/06/2017] [Accepted: 01/07/2017] [Indexed: 12/27/2022]
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Komsky-Elbaz A, Roth Z. Effect of the herbicide atrazine and its metabolite DACT on bovine sperm quality. Reprod Toxicol 2017; 67:15-25. [DOI: 10.1016/j.reprotox.2016.11.001] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2016] [Revised: 10/30/2016] [Accepted: 11/01/2016] [Indexed: 01/07/2023]
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Pirnia A, Parivar K, Hemadi M, Yaghmaei P, Gholami M. Stemness of spermatogonial stem cells encapsulated in alginate hydrogel during cryopreservation. Andrologia 2016; 49. [DOI: 10.1111/and.12650] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/28/2016] [Indexed: 01/15/2023] Open
Affiliation(s)
- A. Pirnia
- Department of Biology; Science and Research Branch; Islamic Azad University; Tehran Iran
| | - K. Parivar
- Department of Biology; Science and Research Branch; Islamic Azad University; Tehran Iran
| | - M. Hemadi
- Fertility and Infertility Research Center; Ahvaz Jundishapur University of Medical Sciences; Ahvaz Iran
| | - P. Yaghmaei
- Department of Biology; Science and Research Branch; Islamic Azad University; Tehran Iran
| | - M. Gholami
- Razi Herbal Medicine Research center and department of Anatomical sciences; Lorestan University of Medical Sciences; Khorramabad Iran
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Giassetti MI, Goissis MD, Moreira PV, de Barros FRO, Assumpção MEOD, Visintin JA. Effect of age on expression of spermatogonial markers in bovine testis and isolated cells. Anim Reprod Sci 2016; 170:68-74. [DOI: 10.1016/j.anireprosci.2016.04.004] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2015] [Revised: 03/23/2016] [Accepted: 04/04/2016] [Indexed: 01/15/2023]
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Lanza DG, Dawson EP, Rao P, Heaney JD. Misexpression of cyclin D1 in embryonic germ cells promotes testicular teratoma initiation. Cell Cycle 2016; 15:919-30. [PMID: 26901436 DOI: 10.1080/15384101.2016.1149272] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Testicular teratomas result from anomalies in embryonic germ cell development. In the 129 family of inbred mouse strains, teratomas arise during the same developmental period that male germ cells normally enter G1/G0 mitotic arrest and female germ cells initiate meiosis (the mitotic:meiotic switch). Dysregulation of this switch associates with teratoma susceptibility and involves three germ cell developmental abnormalities seemingly critical for tumor initiation: delayed G1/G0 mitotic arrest, retention of pluripotency, and misexpression of genes normally restricted to embryonic female and adult male germ cells. One misexpressed gene, cyclin D1 (Ccnd1), is a known regulator of cell cycle progression and an oncogene in many tissues. Here, we investigated whether Ccnd1 misexpression in embryonic germ cells is a determinant of teratoma susceptibility in mice. We found that CCND1 localizes to teratoma-susceptible germ cells that fail to enter G1/G0 arrest during the mitotic:meiotic switch and is the only D-type cyclin misexpressed during this critical developmental time frame. We discovered that Ccnd1 deficiency in teratoma-susceptible mice significantly reduced teratoma incidence and suppressed the germ cell proliferation and pluripotency abnormalities associated with tumor initiation. Importantly, Ccnd1 expression was dispensable for somatic cell development and male germ cell specification and maturation in tumor-susceptible mice, implying that the mechanisms by which Ccnd1 deficiency reduced teratoma incidence were germ cell autonomous and specific to tumorigenesis. We conclude that misexpression of Ccnd1 in male germ cells is a key component of a larger pro-proliferative program that disrupts the mitotic:meiotic switch and predisposes 129 inbred mice to testicular teratocarcinogenesis.
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Affiliation(s)
- Denise G Lanza
- a Department of Molecular and Human Genetics , Baylor College of Medicine , Houston , TX , USA
| | - Emily P Dawson
- a Department of Molecular and Human Genetics , Baylor College of Medicine , Houston , TX , USA
| | - Priya Rao
- b Department of Pathology , MD Anderson Cancer Center, The University of Texas , Houston , TX , USA
| | - Jason D Heaney
- a Department of Molecular and Human Genetics , Baylor College of Medicine , Houston , TX , USA.,c Dan L Duncan Cancer Center, Baylor College of Medicine , Houston , TX , USA.,d Center For Reproductive Medicine, Baylor College of Medicine , Houston , TX , USA
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Park MH, Park JE, Kim MS, Lee KY, Hwang JY, Yun JI, Choi JH, Lee E, Lee ST. Effects of Extracellular Matrix Protein-derived Signaling on the Maintenance of the Undifferentiated State of Spermatogonial Stem Cells from Porcine Neonatal Testis. ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES 2016; 29:1398-406. [PMID: 26954208 PMCID: PMC5003964 DOI: 10.5713/ajas.15.0856] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/20/2015] [Revised: 12/17/2015] [Accepted: 01/25/2016] [Indexed: 12/24/2022]
Abstract
In general, the seminiferous tubule basement membrane (STBM), comprising laminin, collagen IV, perlecan, and entactin, plays an important role in self-renewal and spermatogenesis of spermatogonial stem cells (SSCs) in the testis. However, among the diverse extracellular matrix (ECM) proteins constituting the STBM, the mechanism by which each regulates SSC fate has yet to be revealed. Accordingly, we investigated the effects of various ECM proteins on the maintenance of the undifferentiated state of SSCs in pigs. First, an extracellular signaling-free culture system was optimized, and alkaline phosphatase (AP) activity and transcriptional regulation of SSC-specific genes were analyzed in porcine SSCs (pSSCs) cultured for 1, 3, and 5 days on non-, laminin- and collagen IV-coated Petri dishes in the optimized culture system. The microenvironment consisting of glial cell-derived neurotrophic factor (GDNF)-supplemented mouse embryonic stem cell culture medium (mESCCM) (GDNF-mESCCM) demonstrated the highest efficiency in the maintenance of AP activity. Moreover, under the established extracellular signaling-free microenvironment, effective maintenance of AP activity and SSC-specific gene expression was detected in pSSCs experiencing laminin-derived signaling. From these results, we believe that laminin can serve as an extracellular niche factor required for the in vitro maintenance of undifferentiated pSSCs in the establishment of the pSSC culture system.
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Affiliation(s)
- Min Hee Park
- Department of Animal Life Science, Kangwon National University, Chuncheon 200-701, Korea
| | - Ji Eun Park
- Department of Animal Life Science, Kangwon National University, Chuncheon 200-701, Korea
| | - Min Seong Kim
- Department of Animal Life Science, Kangwon National University, Chuncheon 200-701, Korea
| | - Kwon Young Lee
- College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon 200-701, Korea
| | - Jae Yeon Hwang
- Division of Applied Animal Science, Kangwon National University, Chuncheon 200-701, Korea
| | - Jung Im Yun
- Division of Animal Resource Science, Kangwon National University, Chuncheon 200-701, Korea
| | - Jung Hoon Choi
- College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon 200-701, Korea
| | - Eunsong Lee
- College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon 200-701, Korea
| | - Seung Tae Lee
- Department of Animal Life Science, Kangwon National University, Chuncheon 200-701, Korea.,Division of Applied Animal Science, Kangwon National University, Chuncheon 200-701, Korea
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Advances in cryopreservation of spermatogonial stem cells and restoration of male fertility. Microsc Res Tech 2015; 79:122-9. [DOI: 10.1002/jemt.22605] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2015] [Accepted: 11/07/2015] [Indexed: 11/07/2022]
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Huleihel M, Nourashrafeddin S, Plant TM. Application of three-dimensional culture systems to study mammalian spermatogenesis, with an emphasis on the rhesus monkey (Macaca mulatta). Asian J Androl 2015; 17:972-80. [PMID: 26067870 PMCID: PMC4814948 DOI: 10.4103/1008-682x.154994] [Citation(s) in RCA: 74] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2014] [Revised: 11/26/2014] [Accepted: 03/04/2015] [Indexed: 12/19/2022] Open
Abstract
In vitro culture of spermatogonial stem cells (SSCs) has generally been performed using two-dimensional (2D) culture systems; however, such cultures have not led to the development of complete spermatogenesis. It seems that 2D systems do not replicate optimal conditions of the seminiferous tubules (including those generated by the SSC niche) and necessary for spermatogenesis. Recently, one of our laboratories has been able to induce proliferation and differentiation of mouse testicular germ cells to meiotic and postmeiotic stages including generation of sperm in a 3D soft agar culture system (SACS) and a 3D methylcellulose culture system (MCS). It was suggested that SACS and MCS form a special 3D microenvironment that mimics germ cell niche formation in the seminiferous tubules, and thus permits mouse spermatogenesis in vitro. In this review, we (1) provide a brief overview of the differences in spermatogenesis in rodents and primates, (2) summarize data related to attempts to generate sperm in vitro, (3) report for the first time formation of colonies/clusters of cells and differentiation of meiotic (expression of CREM-1) and postmeiotic (expression of acrosin) germ cells from undifferentiated spermatogonia isolated from the testis of prepubertal rhesus monkeys and cultured in SACS and MCS, and (4) indicate research needed to optimize 3D systems for in vitro primate spermatogenesis and for possible future application to man.
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Affiliation(s)
- Mahmoud Huleihel
- The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel
| | - Seyedmehdi Nourashrafeddin
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Magee-Womens Research Institute, Pittsburgh, PA 15213, USA
| | - Tony M Plant
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Magee-Womens Research Institute, Pittsburgh, PA 15213, USA
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Kang HR, Lee YA, Kim YH, Lee DG, Kim BJ, Kim KJ, Kim BG, Oh MG, Han CK, Lee S, Ryu BY. Petasites japonicus Stimulates the Proliferation of Mouse Spermatogonial Stem Cells. PLoS One 2015. [PMID: 26207817 PMCID: PMC4514868 DOI: 10.1371/journal.pone.0133077] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Oriental natural plants have been used as medical herbs for the treatment of various diseases for over 2,000 years. In this study, we evaluated the effect of several natural plants on the preservation of male fertility by assessing the ability of plant extracts to stimulate spermatogonial stem cell (SSC) proliferation by using a serum-free culture method. In vitro assays showed that Petasites japonicus extracts, especially the butanol fraction, have a significant effect on germ cells proliferation including SSCs. The activity of SSCs cultured in the presence of the Petasites japonicus butanol fraction was confirmed by normal colony formation and spermatogenesis following germ cell transplantation of the treated SSCs. Our findings could lead to the discovery of novel factors that activate SSCs and could be useful for the development of technologies for the prevention of male infertility.
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Affiliation(s)
- Hye-Ryun Kang
- Department of Animal Science and Technology, Chung-Ang University, Anseong, Republic of Korea
| | - Yong-An Lee
- Department of Animal Science and Technology, Chung-Ang University, Anseong, Republic of Korea
| | - Yong-Hee Kim
- Department of Animal Science and Technology, Chung-Ang University, Anseong, Republic of Korea
| | - Dong Gu Lee
- Department of Integrative Plant Science, Chung-Ang University, Anseong, Republic of Korea
| | - Bang-Jin Kim
- Department of Animal Science and Technology, Chung-Ang University, Anseong, Republic of Korea
| | - Ki-Jung Kim
- Department of Animal Science and Technology, Chung-Ang University, Anseong, Republic of Korea
| | - Byung-Gak Kim
- Department of Obstetrics, Gynecology & Reproductive Biology, Van Andel Institute, Michigan State University, Grand Rapids, Michigan, United States of America
| | - Myeong-Geun Oh
- Department of Animal Science and Technology, Chung-Ang University, Anseong, Republic of Korea
| | - Chan Kyu Han
- Korea Food Research Institute, Sungnam, Republic of Korea
| | - Sanghyun Lee
- Department of Integrative Plant Science, Chung-Ang University, Anseong, Republic of Korea
| | - Buom-Yong Ryu
- Department of Animal Science and Technology, Chung-Ang University, Anseong, Republic of Korea
- * E-mail:
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Komsky-Elbaz A, Raziel A, Ben-Ami I, Bern O, Maslansky B, Gidoni YS, Ron-El R, Strassburger D. Ploidy of spermatogenic cells of men with non-mosaic Klinefelter's syndrome as measured by a computerized cell scanning system. J Assist Reprod Genet 2015; 32:1113-21. [PMID: 26081126 DOI: 10.1007/s10815-015-0508-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2015] [Accepted: 06/02/2015] [Indexed: 12/14/2022] Open
Abstract
PURPOSE This study aims to characterize the origin of testicular post-meiotic cells in non-mosaic Klinefelter's syndrome (KS). METHODS The study included testicular tissue specimens from 11 non-mosaic KS patients, with (6 positive) and without (5 negative) spermatozoa presence. The obtained testicular cells were affixed and stained for morphology followed by fluorescence in situ hybridization (FISH) for centromeric probes X, Y, and 18. We used a computerized automated cell scanning system that enables simultaneous viewing of morphology and FISH in the same cell. RESULTS A total of 12,387 cells from the positive cases, 11,991 cells from the negative cases, and 1,711 cells from the controls were analyzed. The majority of spermatogonia were 47, XXY in both the positive and negative KS cases (88.9 ± 4.76 % and 90.6 ± 4.58 %) as were primary spermatocytes (76.8 ± 8.14 % and 79.6 ± 7.30 %). The respective rates of secondary spermatocytes and post-meiotic cells (round, elongating spermatids and sperm cells) were 1.1 ± 1.39 % in the positive cases, 2.9 ± 3.33 % in the negative cases, compared to 67.6 ± 6.22 % in the controls (P < 0.02). Pairing of both 18 and XY homologous chromosomes in 46,XY primary spermatocytes was 2.5 ± 2.31 % and 3.4 ± 2.39 %, respectively, compared to 19.8 ± 8.95 % in the control group (P < 0.02) and in 47,XXY primary spermatocytes in 2.4 ± 3.8 % in the positive group and 3.2 ± 2.26 % in the negative group. CONCLUSIONS This study presents data to indicate that the majority of primary spermatocytes in the testes of non-mosaic KS patients are 47,XXY and could possibly develop into post-meiotic cells.
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Affiliation(s)
- Alisa Komsky-Elbaz
- Infertility and IVF Unit, Assaf Harofeh Medical Center, Zerifin, 703000, Israel
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Aponte PM. Spermatogonial stem cells: Current biotechnological advances in reproduction and regenerative medicine. World J Stem Cells 2015; 7:669-680. [PMID: 26029339 PMCID: PMC4444608 DOI: 10.4252/wjsc.v7.i4.669] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/28/2015] [Revised: 03/13/2015] [Accepted: 04/14/2015] [Indexed: 02/06/2023] Open
Abstract
Spermatogonial stem cells (SSCs) are the germ stem cells of the seminiferous epithelium in the testis. Through the process of spermatogenesis, they produce sperm while concomitantly keeping their cellular pool constant through self-renewal. SSC biology offers important applications for animal reproduction and overcoming human disease through regenerative therapies. To this end, several techniques involving SSCs have been developed and will be covered in this article. SSCs convey genetic information to the next generation, a property that can be exploited for gene targeting. Additionally, SSCs can be induced to become embryonic stem cell-like pluripotent cells in vitro. Updates on SSC transplantation techniques with related applications, such as fertility restoration and preservation of endangered species, are also covered on this article. SSC suspensions can be transplanted to the testis of an animal and this has given the basis for SSC functional assays. This procedure has proven technically demanding in large animals and men. In parallel, testis tissue xenografting, another transplantation technique, was developed and resulted in sperm production in testis explants grafted into ectopical locations in foreign species. Since SSC culture holds a pivotal role in SSC biotechnologies, current advances are overviewed. Finally, spermatogenesis in vitro, already demonstrated in mice, offers great promises to cope with reproductive issues in the farm animal industry and human clinical applications.
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Kim KJ, Lee YA, Kim BJ, Kim YH, Kim BG, Kang HG, Jung SE, Choi SH, Schmidt JA, Ryu BY. Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing. Cryobiology 2015; 70:175-83. [DOI: 10.1016/j.cryobiol.2015.02.007] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2014] [Revised: 02/21/2015] [Accepted: 02/23/2015] [Indexed: 01/15/2023]
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Chakraborty P, Buaas FW, Sharma M, Snyder E, de Rooij DG, Braun RE. LIN28A marks the spermatogonial progenitor population and regulates its cyclic expansion. Stem Cells 2015; 32:860-73. [PMID: 24715688 DOI: 10.1002/stem.1584] [Citation(s) in RCA: 60] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2013] [Revised: 09/02/2013] [Accepted: 09/05/2013] [Indexed: 12/22/2022]
Abstract
One of the hallmarks of highly proliferative adult tissues is the presence of a stem cell population that produces progenitor cells bound for differentiation. Progenitor cells undergo multiple transit amplifying (TA) divisions before initiating terminal differentiation. In the adult male germline, daughter cells arising from the spermatogonial stem cells undergo multiple rounds of TA divisions to produce undifferentiated clones of interconnected 2, 4, 8, and 16 cells, collectively termed A(undifferentiated) (A(undiff)) spermatogonia, before entering a stereotypic differentiation cascade. Although the number of TA divisions markedly affects the tissue output both at steady state and during regeneration, mechanisms regulating the expansion of the TA cell population are poorly understood in mammals. Here, we show that mice with a conditional deletion of Lin28a in the adult male germline, display impaired clonal expansion of the progenitor TA A(undiff) spermatogonia. The in vivo proliferative activity of Au(ndiff) spermatogonial cells as indicated by BrdU incorporation during S-phase was reduced in the absence of LIN28A. Thus, contrary to the role of LIN28A as a key determinant of cell fate signals in multiple stem cell lineages, in the adult male germline it functions as an intrinsic regulator of proliferation in the population of A(undiff) TA spermatogonia. In addition, neither precocious differentiation nor diminished capacity for self-renewal potential as assessed by transplantation was observed, suggesting that neither LIN28A itself nor the pool of Aal progenitor cells substantially contribute to the functional stem cell compartment.
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Koster R, Mitra N, D'Andrea K, Vardhanabhuti S, Chung CC, Wang Z, Loren Erickson R, Vaughn DJ, Litchfield K, Rahman N, Greene MH, McGlynn KA, Turnbull C, Chanock SJ, Nathanson KL, Kanetsky PA. Pathway-based analysis of GWAs data identifies association of sex determination genes with susceptibility to testicular germ cell tumors. Hum Mol Genet 2014; 23:6061-8. [PMID: 24943593 PMCID: PMC4204765 DOI: 10.1093/hmg/ddu305] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2013] [Revised: 05/28/2014] [Accepted: 06/12/2014] [Indexed: 02/06/2023] Open
Abstract
Genome-wide association (GWA) studies of testicular germ cell tumor (TGCT) have identified 18 susceptibility loci, some containing genes encoding proteins important in male germ cell development. Deletions of one of these genes, DMRT1, lead to male-to-female sex reversal and are associated with development of gonadoblastoma. To further explore genetic association with TGCT, we undertook a pathway-based analysis of SNP marker associations in the Penn GWAs (349 TGCT cases and 919 controls). We analyzed a custom-built sex determination gene set consisting of 32 genes using three different methods of pathway-based analysis. The sex determination gene set ranked highly compared with canonical gene sets, and it was associated with TGCT (FDRG = 2.28 × 10(-5), FDRM = 0.014 and FDRI = 0.008 for Gene Set Analysis-SNP (GSA-SNP), Meta-Analysis Gene Set Enrichment of Variant Associations (MAGENTA) and Improved Gene Set Enrichment Analysis for Genome-wide Association Study (i-GSEA4GWAS) analysis, respectively). The association remained after removal of DMRT1 from the gene set (FDRG = 0.0002, FDRM = 0.055 and FDRI = 0.009). Using data from the NCI GWA scan (582 TGCT cases and 1056 controls) and UK scan (986 TGCT cases and 4946 controls), we replicated these findings (NCI: FDRG = 0.006, FDRM = 0.014, FDRI = 0.033, and UK: FDRG = 1.04 × 10(-6), FDRM = 0.016, FDRI = 0.025). After removal of DMRT1 from the gene set, the sex determination gene set remains associated with TGCT in the NCI (FDRG = 0.039, FDRM = 0.050 and FDRI = 0.055) and UK scans (FDRG = 3.00 × 10(-5), FDRM = 0.056 and FDRI = 0.044). With the exception of DMRT1, genes in the sex determination gene set have not previously been identified as TGCT susceptibility loci in these GWA scans, demonstrating the complementary nature of a pathway-based approach for genome-wide analysis of TGCT.
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Affiliation(s)
- Roelof Koster
- Translational Medicine and Human Genetics, Department of Medicine
| | | | - Kurt D'Andrea
- Translational Medicine and Human Genetics, Department of Medicine
| | | | - Charles C Chung
- Division of Cancer Epidemiology and Genetics, Department of Health and Human Services,National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
| | - Zhaoming Wang
- Division of Cancer Epidemiology and Genetics, Department of Health and Human Services,National Cancer Institute, National Institutes of Health, Bethesda, MD, USA, Cancer Genome Research Laboratory, Division of Cancer Epidemiology and Genetics, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD, USA
| | - R Loren Erickson
- Walter Reed Army Institute of Research, Silver Spring, MD, USA and
| | - David J Vaughn
- Division of Hematology-Oncology, Department of Medicine and, Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
| | - Kevin Litchfield
- Division of Genetics and Epidemiology, Institute of Cancer Research, Sutton, Surrey, UK
| | - Nazneen Rahman
- Division of Genetics and Epidemiology, Institute of Cancer Research, Sutton, Surrey, UK
| | - Mark H Greene
- Division of Cancer Epidemiology and Genetics, Department of Health and Human Services,National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
| | - Katherine A McGlynn
- Division of Cancer Epidemiology and Genetics, Department of Health and Human Services,National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
| | - Clare Turnbull
- Division of Genetics and Epidemiology, Institute of Cancer Research, Sutton, Surrey, UK
| | - Stephen J Chanock
- Division of Cancer Epidemiology and Genetics, Department of Health and Human Services,National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
| | - Katherine L Nathanson
- Translational Medicine and Human Genetics, Department of Medicine, Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
| | - Peter A Kanetsky
- Department of Biostatistics and Epidemiology, Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA,
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Lee WY, Lee KH, Heo YT, Kim NH, Kim JH, Kim JH, Moon SH, Chung HJ, Yoon MJ, Song H. Transcriptional coactivator undifferentiated embryonic cell transcription factor 1 expressed in spermatogonial stem cells: A putative marker of boar spermatogonia. Anim Reprod Sci 2014; 150:115-24. [DOI: 10.1016/j.anireprosci.2014.09.010] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2014] [Revised: 09/10/2014] [Accepted: 09/17/2014] [Indexed: 12/12/2022]
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Identification of Niche Conditions Supporting Short-term Culture of Spermatogonial Stem Cells Derived from Porcine Neonatal Testis. JOURNAL OF ANIMAL REPRODUCTION AND BIOTECHNOLOGY 2014. [DOI: 10.12750/jet.2014.29.3.221] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
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Abstract
Male germline or spermatogonial stem cells (SSCs) are conserved across many species and essential for uninterrupted production of sperm over long periods of reproductive life span. A better understanding of SSC biology provides limitless opportunities in male reproductive health, fertility preservation, and regenerative medicine. Although several potential markers define SSCs, not many definitive markers exist that are specific for a rare subset of SSCs that self-renew and have the ability to give rise to other progenitors, eventually contributing to all stages of spermatogenesis. In the September 2014 issue of the JCI, Aloisio and colleagues report that PAX7 is a new marker expressed uniquely in a rare subset of SSCs in mouse testes. PAX7+ cells fulfill all the criteria required for bona fide SSCs. Surprisingly, male germline-specific deletion of Pax7 indicates that it is dispensable for spermatogenesis.
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Spermatogonial stem cell enrichment using simple grafting of testis and in vitro cultivation. Sci Rep 2014; 4:5923. [PMID: 25080919 PMCID: PMC4118148 DOI: 10.1038/srep05923] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2013] [Accepted: 07/15/2014] [Indexed: 01/15/2023] Open
Abstract
Enrichment of spermatogonial stem cells (SSCs) from the mammalian adult testis faces several limitations owing to their relatively low numbers among many types of advanced germ cells and somatic cells. The aim of the present study was to improve the isolation efficiency of SSCs using a simple tissue grafting method to eliminate the existing advanced germ cells. Sliced testis parenchyma obtained from adult ICR or EGFP-expressing transgenic mice were grafted heterotropically under the dorsal skin of nude mice. The most advanced germ cells disappeared in the grafted tissues after 2–4 weeks. Grafted tissues were dissociated enzymatically and plated in culture dishes. During in vitro culture, significantly more SSCs were obtained from the grafted testes than from non-grafted controls, and the isolated SSCs had proliferative potential and were successfully maintained. Additionally, EGFP-expressing SSCs derived from graft parenchyma were transplanted into bulsufan-treated recipient mice testes. Finally, we obtained EGFP-expressing pups after in vitro fertilization using spermatozoa derived from transplanted SSCs. These results suggest that subcutaneous grafting of testis parenchyma and the subsequent culture methods provide a simple and efficient isolation method to enrich for SSCs in adult testis without specific cell sorting methods and may be useful tools for clinical applications.
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Park MH, Park JE, Kim MS, Lee KY, Park HJ, Yun JI, Choi JH, Lee ES, Lee ST. Development of a high-yield technique to isolate spermatogonial stem cells from porcine testes. J Assist Reprod Genet 2014; 31:983-91. [PMID: 24938360 DOI: 10.1007/s10815-014-0271-7] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2014] [Accepted: 06/01/2014] [Indexed: 01/15/2023] Open
Abstract
PURPOSE To date, the methods available for isolating spermatogonial stem cells (SSCs) from porcine testicular cells have a low efficiency of cell separating. Therefore, we tried to develop a novel isolation technique with a high-yield cell separating ability to isolate SSCs from porcine testes. METHODS We confirmed the presence of SSCs by measuring alkaline phosphatase (AP) activity and SSC-specific gene expression in neonatal porcine testis-derived testicular cells. Subsequently, the isolation of SSCs from testicular cells was performed using different techniques as follows: differential plating (DP), double DP, Petri dish plating post-DP, magnetic-activated cell sorting (MACS), and MACS post-DP. Positive AP staining was used to assess and compare the isolation efficiency of each method. RESULTS Petri dish plating post-DP resulted in the highest isolation efficiency. The putative SSCs isolated using this method was then further characterized by analyzing the expression of SSC-specific genes and -related proteins, and germ cell-specific genes. OCT4, NANOG, EPCAM, THY1, and UCHL1 were expressed transcriptionally, and OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81, and PLZF were expressed translationally in 86 % of the isolated SSCs. In contrast, no difference was observed in the percentage of cells expressing luteinizing hormone receptor (LHR), a Leydig cell-specific protein, or GATA4, a Sertoli cell-specific protein, between SSCs and negative control cells. In addition, transcriptional expression of VASA, a primordial germ cell-specific marker, and DAZL, a premeiotic germ cell-specific marker, wasn't and was detected, respectively. CONCLUSIONS We successfully developed a novel high-yield technique to isolate SSCs from porcine testes to facilitate future porcine SSC-related research.
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Affiliation(s)
- Min Hee Park
- Department of Animal Life Science, Kangwon National University, Chuncheon, 200-701, South Korea
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Sadri-Ardekani H, Atala A. Testicular tissue cryopreservation and spermatogonial stem cell transplantation to restore fertility: from bench to bedside. Stem Cell Res Ther 2014; 5:68. [PMID: 25157677 PMCID: PMC4056749 DOI: 10.1186/scrt457] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Male infertility management has made significant progress during the past three decades, especially after the introduction of intracytoplasmic sperm injection in 1992. However, many boys and men still suffer from primary testicular failure due to acquired or genetic causes. New and novel treatments are needed to address these issues. Spermatogenesis originates from spermatogonial stem cells (SSCs) that reside in the testis. Many of these men lack SSCs or have lost SSCs over time as a result of specific medical conditions or toxic exposures. Loss of SSCs is critical in prepubertal boys who suffer from cancer and are going through gonadotoxic cancer treatments, as there is no option of sperm cryopresrvation due to sexual immaturity. The development of SSC transplantation in a mouse model to repopulate spermatozoa in depleted testes has opened new avenues of research in other animal models, including non-human primates. Recent advances in cryopreservation and in vitro propagation of human SSCs offer promise for human SSC autotransplantation in the near future. Ongoing research is focusing on safety and technical issues of human SSC autotransplantation. This is the time to counsel parents and boys at risk of infertility on the possibility of cryopreserving and banking a small amount of testis tissue for potential future use in SSC transplantation.
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Herrid M, McFarlane JR. Application of testis germ cell transplantation in breeding systems of food producing species: a review. Anim Biotechnol 2014; 24:293-306. [PMID: 23947666 DOI: 10.1080/10495398.2013.785431] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
A major benefit of advanced reproduction technologies (ART) in animal breeding is the ability to produce more progeny per individual parent. This is particularly useful with animals of high genetic merit. Testis germ cell transplantation (TGCT) is emerging as a novel reproductive technology with application in animal breeding systems, including the potential for use as an alternative to artificial insemination (AI), an alternative to transgenesis, part of an approach to reducing generation intervals, or an approach toward development of interspecies hybrids. There is one major difference in TGCT between rodents and some other species associated with immunotolerance in heterologous transplantation. In particular, livestock and aquatic species do not require an immunesuppression procedure to allow donor cell survival in recipient testis. Testicular stem cells from a genetically elite individual transplanted into others can develop and produce a surrogate male-an animal that produces the functional sperm of the original individual. Spermatozoa produced from testis stem cells are the only cells in the body of males that can transmit genetic information to the offspring. The isolation and genetic manipulation of testis stem cells prior to transplantation has been shown to create transgenic animals. However, the current success rate of the transplantation procedure in livestock and aquatic species is low, with a corresponding small proportion of donor spermatozoa in the recipient's semen. The propagation of donor cells in culture and preparation of recipient animals are the two main factors that limit the commercial application of this technique. The current paper reviews and compares recent progress and examines the difficulties of TGCT in both livestock and aquatic species, thereby providing new insights into the application of TGCT in food producing animals.
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Affiliation(s)
- Muren Herrid
- a Center for Bioactive Discovery in Health and Aging, University of New England , Armidale , Australia
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48
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Sadri-Ardekani H, Homburg CH, van Capel TMM, van den Berg H, van der Veen F, van der Schoot CE, van Pelt AMM, Repping S. Eliminating acute lymphoblastic leukemia cells from human testicular cell cultures: a pilot study. Fertil Steril 2014; 101:1072-1078.e1. [PMID: 24581582 DOI: 10.1016/j.fertnstert.2014.01.014] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2013] [Revised: 01/10/2014] [Accepted: 01/10/2014] [Indexed: 01/21/2023]
Abstract
OBJECTIVE To study whether acute lymphoblastic leukemia (ALL) cells survive in a human testicular cell culture system. DESIGN Experimental laboratory study. SETTING Reproductive biology laboratory, academic medical center. PATIENT(S) Acute lymphoblastic leukemia cells from three patients and testicular cells from three other patients. INTERVENTION(S) Acute lymphoblastic leukemia cells were cultured alone or in combination with testicular cells, at various concentrations, in a system that has recently been developed to propagate human spermatogonial stem cells. MAIN OUTCOME MEASURE(S) Viability of ALL and testicular cells during culture was evaluated by flow cytometry using markers for live/dead cells. Furthermore, the presence of ALL cells among testicular cells was determined by highly sensitive (1:10,000 to 1:100,000 cells) patient-specific antigen-receptor minimal residual disease polymerase chain reaction. The presence of spermatogonia at the end of culture was determined by reverse transcription-polymerase chain reaction for ZBTB16, UCHL1, and GPR125. RESULT(S) The ALL cells cultured separately did not survive beyond 14 days of culture. When cultured together with testicular cells, even at extremely high initial concentrations (40% ALL cells), ALL cells were undetectable beyond 26 days of culture. Reverse transcription-polymerase chain reaction confirmed the presence of spermatogonia at the end of the culture period. CONCLUSION(S) Our pilot study shows that the described testicular cell culture system not only allows for efficient propagation of spermatogonial stem cells but also eliminates contaminating ALL cells.
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Affiliation(s)
- Hooman Sadri-Ardekani
- Center for Reproductive Medicine, Women's and Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands; Reproductive Biotechnology Research Center, Avicenna Research Institute, The Academic Center for Education, Culture and Research (ACECR), Tehran, Iran.
| | - Christa H Homburg
- Experimental Immunohematology, Sanquin Research at the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB), Amsterdam, the Netherlands
| | - Toni M M van Capel
- Departments of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
| | - Henk van den Berg
- Department of Pediatric Oncology, Women's and Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
| | - Fulco van der Veen
- Center for Reproductive Medicine, Women's and Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
| | - C Ellen van der Schoot
- Experimental Immunohematology, Sanquin Research at the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB), Amsterdam, the Netherlands
| | - Ans M M van Pelt
- Center for Reproductive Medicine, Women's and Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
| | - Sjoerd Repping
- Center for Reproductive Medicine, Women's and Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
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Li Y, Zhang Y, Zhang X, Sun J, Hao J. BMP4/Smad Signaling Pathway Induces the Differentiation of Mouse Spermatogonial Stem Cells via Upregulation of Sohlh2. Anat Rec (Hoboken) 2014; 297:749-57. [DOI: 10.1002/ar.22891] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2013] [Accepted: 01/10/2014] [Indexed: 01/17/2023]
Affiliation(s)
- Yi Li
- Department of Histology and Embryology; School of Medicine, Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University; Jinan 250012 People's Republic of China
- Obstetric Genetic Disease Laboratory; Maternal and Child Health Hospital of Zibo City; Zibo 255029 People's Republic of China
| | - Yuecun Zhang
- Department of Gynaecology and Obstetrics; Qilu Hospital, Shandong University; Jinan 250012 People's Republic of China
| | - Xiaoli Zhang
- Department of Histology and Embryology; School of Medicine, Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University; Jinan 250012 People's Republic of China
| | - Jinhao Sun
- Department of Human Anatomy; School of Medicine; Shandong University; Jinan 250012 People's Republic of China
| | - Jing Hao
- Department of Histology and Embryology; School of Medicine, Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University; Jinan 250012 People's Republic of China
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Mukherjee A, Koli S, Reddy KVR. Regulatory non-coding transcripts in spermatogenesis: shedding light on ‘dark matter’. Andrology 2014; 2:360-9. [DOI: 10.1111/j.2047-2927.2014.00183.x] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2013] [Revised: 12/26/2013] [Accepted: 12/26/2013] [Indexed: 11/29/2022]
Affiliation(s)
- A. Mukherjee
- Division of Molecular Immunology and Microbiology; National Institute for Research in Reproductive Health; Indian Council of Medical Research; Mumbai India
| | - S. Koli
- Division of Molecular Immunology and Microbiology; National Institute for Research in Reproductive Health; Indian Council of Medical Research; Mumbai India
| | - K. V. R. Reddy
- Division of Molecular Immunology and Microbiology; National Institute for Research in Reproductive Health; Indian Council of Medical Research; Mumbai India
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