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Aponte PM, Gutierrez-Reinoso MA, Garcia-Herreros M. Bridging the Gap: Animal Models in Next-Generation Reproductive Technologies for Male Fertility Preservation. Life (Basel) 2023; 14:17. [PMID: 38276265 PMCID: PMC10820126 DOI: 10.3390/life14010017] [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: 09/05/2023] [Revised: 12/12/2023] [Accepted: 12/19/2023] [Indexed: 01/27/2024] Open
Abstract
This review aims to explore advanced reproductive technologies for male fertility preservation, underscoring the essential role that animal models have played in shaping these techniques through historical contexts and into modern applications. Rising infertility concerns have become more prevalent in human populations recently. The surge in male fertility issues has prompted advanced reproductive technologies, with animal models playing a pivotal role in their evolution. Historically, animal models have aided our understanding in the field, from early reproductive basic research to developing techniques like artificial insemination, multiple ovulation, and in vitro fertilization. The contemporary landscape of male fertility preservation encompasses techniques such as sperm cryopreservation, testicular sperm extraction, and intracytoplasmic sperm injection, among others. The relevance of animal models will undoubtedly bridge the gap between traditional methods and revolutionary next-generation reproductive techniques, fortifying our collective efforts in enhancing male fertility preservation strategies. While we possess extensive knowledge about spermatogenesis and its regulation, largely thanks to insights from animal models that paved the way for human infertility treatments, a pressing need remains to further understand specific infertility issues unique to humans. The primary aim of this review is to provide a comprehensive analysis of how animal models have influenced the development and refinement of advanced reproductive technologies for male fertility preservation, and to assess their future potential in bridging the gap between current practices and cutting-edge fertility techniques, particularly in addressing unique human male factor infertility.
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Affiliation(s)
- Pedro M. Aponte
- Colegio de Ciencias Biológicas y Ambientales (COCIBA), Universidad San Francisco de Quito (USFQ), Quito 170901, Ecuador
- Instituto de Investigaciones en Biomedicina “One-Health”, Universidad San Francisco de Quito (USFQ), Campus Cumbayá, Quito 170901, Ecuador
| | - Miguel A. Gutierrez-Reinoso
- Facultad de Ciencias Agropecuarias y Recursos Naturales, Carrera de Medicina Veterinaria, Universidad Técnica de Cotopaxi (UTC), Latacunga 050150, Ecuador;
- Laboratorio de Biotecnología Animal, Departamento de Ciencia Animal, Facultad de Ciencias Veterinarias, Universidad de Concepción (UdeC), Chillán 3780000, Chile
| | - Manuel Garcia-Herreros
- Instituto Nacional de Investigação Agrária e Veterinária (INIAV), 2005-048 Santarém, Portugal
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Lin H, Cheng K, Kubota H, Lan Y, Riedel SS, Kakiuchi K, Sasaki K, Bernt KM, Bartolomei MS, Luo M, Wang PJ. Histone methyltransferase DOT1L is essential for self-renewal of germline stem cells. Genes Dev 2022; 36:752-763. [PMID: 35738678 PMCID: PMC9296001 DOI: 10.1101/gad.349550.122] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2022] [Accepted: 06/06/2022] [Indexed: 12/25/2022]
Abstract
Self-renewal of spermatogonial stem cells is vital to lifelong production of male gametes and thus fertility. However, the underlying mechanisms remain enigmatic. Here, we show that DOT1L, the sole H3K79 methyltransferase, is required for spermatogonial stem cell self-renewal. Mice lacking DOT1L fail to maintain spermatogonial stem cells, characterized by a sequential loss of germ cells from spermatogonia to spermatids and ultimately a Sertoli cell only syndrome. Inhibition of DOT1L reduces the stem cell activity after transplantation. DOT1L promotes expression of the fate-determining HoxC transcription factors in spermatogonial stem cells. Furthermore, H3K79me2 accumulates at HoxC9 and HoxC10 genes. Our findings identify an essential function for DOT1L in adult stem cells and provide an epigenetic paradigm for regulation of spermatogonial stem cells.
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Affiliation(s)
- Huijuan Lin
- School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei Province 430072, China;,Department of Biomedical Sciences, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104, USA
| | - Keren Cheng
- Department of Biomedical Sciences, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104, USA
| | - Hiroshi Kubota
- Laboratory of Cell and Molecular Biology, Department of Animal Science, School of Veterinary Medicine, Kitasato University, Towada, Aomori 034-8628, Japan
| | - Yemin Lan
- Epigenetics Institute, Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
| | - Simone S. Riedel
- Division of Pediatric Oncology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA;,Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA;,Abramson Cancer Center, Philadelphia, Pennsylvania 19104, USA
| | - Kazue Kakiuchi
- Laboratory of Cell and Molecular Biology, Department of Animal Science, School of Veterinary Medicine, Kitasato University, Towada, Aomori 034-8628, Japan
| | - Kotaro Sasaki
- Department of Biomedical Sciences, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104, USA
| | - Kathrin M. Bernt
- Division of Pediatric Oncology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA;,Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA;,Abramson Cancer Center, Philadelphia, Pennsylvania 19104, USA
| | - Marisa S. Bartolomei
- Epigenetics Institute, Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
| | - Mengcheng Luo
- School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei Province 430072, China
| | - P. Jeremy Wang
- Department of Biomedical Sciences, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104, USA
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Cellular Therapy via Spermatogonial Stem Cells for Treating Impaired Spermatogenesis, Non-Obstructive Azoospermia. Cells 2021; 10:cells10071779. [PMID: 34359947 PMCID: PMC8304133 DOI: 10.3390/cells10071779] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Revised: 07/04/2021] [Accepted: 07/12/2021] [Indexed: 12/18/2022] Open
Abstract
Male infertility is a major health problem affecting about 8–12% of couples worldwide. Spermatogenesis starts in the early fetus and completes after puberty, passing through different stages. Male infertility can result from primary or congenital, acquired, or idiopathic causes. The absence of sperm in semen, or azoospermia, results from non-obstructive causes (pretesticular and testicular), and post-testicular obstructive causes. Several medications such as antihypertensive drugs, antidepressants, chemotherapy, and radiotherapy could lead to impaired spermatogenesis and lead to a non-obstructive azoospermia. Spermatogonial stem cells (SSCs) are the basis for spermatogenesis and fertility in men. SSCs are characterized by their capacity to maintain the self-renewal process and differentiation into spermatozoa throughout the male reproductive life and transmit genetic information to the next generation. SSCs originate from gonocytes in the postnatal testis, which originate from long-lived primordial germ cells during embryonic development. The treatment of infertility in males has a poor prognosis. However, SSCs are viewed as a promising alternative for the regeneration of the impaired or damaged spermatogenesis. SSC transplantation is a promising technique for male infertility treatment and restoration of spermatogenesis in the case of degenerative diseases such as cancer, radiotherapy, and chemotherapy. The process involves isolation of SSCs and cryopreservation from a testicular biopsy before starting cancer treatment, followed by intra-testicular stem cell transplantation. In general, treatment for male infertility, even with SSC transplantation, still has several obstacles. The efficiency of cryopreservation, exclusion of malignant cells contamination in cancer patients, and socio-cultural attitudes remain major challenges to the wider application of SSCs as alternatives. Furthermore, there are limitations in experience and knowledge regarding cryopreservation of SSCs. However, the level of infrastructure or availability of regulatory approval to process and preserve testicular tissue makes them tangible and accurate therapy options for male infertility caused by non-obstructive azoospermia, though in their infancy, at least to date.
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Mao GP, Niu MH, Cui YH, Tang RL, Chen W, Liu B, He Z. Characterization, isolation, and culture of spermatogonial stem cells in Macaca fascicularis. Asian J Androl 2021; 23:240-248. [PMID: 33533740 PMCID: PMC8152426 DOI: 10.4103/aja.aja_95_20] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2019] [Accepted: 12/02/2020] [Indexed: 12/22/2022] Open
Abstract
Spermatogonial stem cells (SSCs) have great applications in both reproductive and regenerative medicine. Primates including monkeys are very similar to humans with regard to physiology and pathology. Nevertheless, little is known about the isolation, the characteristics, and the culture of primate SSCs. This study was designed to identify, isolate, and culture monkey SSCs. Immunocytochemistry was used to identify markers for monkey SSCs. Glial cell line-derived neurotrophic factor family receptor alpha-1 (GFRA1)-enriched spermatogonia were isolated from monkeys, namely Macaca fascicularis (M. fascicularis), by two-step enzymatic digestion and magnetic-activated cell sorting, and they were cultured on precoated plates in the conditioned medium. Reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and RNA sequencing were used to compare phenotype and transcriptomes in GFRA1-enriched spermatogonia between 0 day and 14 days of culture, and xenotransplantation was performed to evaluate the function of GFRA1-enriched spermatogonia. SSCs shared some phenotypes with rodent and human SSCs. GFRA1-enriched spermatogonia with high purity and viability were isolated from M. fascicularis testes. The freshly isolated cells expressed numerous markers for rodent SSCs, and they were cultured for 14 days. The expression of numerous SSC markers was maintained during the cultivation of GFRA1-enriched spermatogonia. RNA sequencing reflected a 97.3% similarity in global gene profiles between 0 day and 14 days of culture. The xenotransplantation assay indicated that the GFRA1-enriched spermatogonia formed colonies and proliferated in vivo in the recipient c-KitW/W (W) mutant mice. Collectively, GFRA1-enriched spermatogonia are monkey SSCs phenotypically both in vitro and in vivo. This study suggests that monkey might provide an alternative to human SSCs for basic research and application in human diseases.
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Affiliation(s)
- Guo-Ping Mao
- Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China
- The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200030, China
- Shanghai Key Laboratory of Embryo Original Diseases, Shanghai 200030, China
| | - Ming-Hui Niu
- Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China
| | - Ying-Hong Cui
- The Key Laboratory of Model Animals and Stem Cell Biology in Hunan Province, Hunan Normal University School of Medicine, Changsha 410013, China
| | - Rui-Ling Tang
- The Key Laboratory of Model Animals and Stem Cell Biology in Hunan Province, Hunan Normal University School of Medicine, Changsha 410013, China
| | - Wei Chen
- The Key Laboratory of Model Animals and Stem Cell Biology in Hunan Province, Hunan Normal University School of Medicine, Changsha 410013, China
| | - Bang Liu
- The Key Laboratory of Model Animals and Stem Cell Biology in Hunan Province, Hunan Normal University School of Medicine, Changsha 410013, China
| | - Zuping He
- The Key Laboratory of Model Animals and Stem Cell Biology in Hunan Province, Hunan Normal University School of Medicine, Changsha 410013, China
- Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China
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Shinohara T, Kanatsu-Shinohara M. Transgenesis and Genome Editing of Mouse Spermatogonial Stem Cells by Lentivirus Pseudotyped with Sendai Virus F Protein. Stem Cell Reports 2020; 14:447-461. [PMID: 32160520 PMCID: PMC7066332 DOI: 10.1016/j.stemcr.2020.02.001] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2019] [Revised: 02/05/2020] [Accepted: 02/10/2020] [Indexed: 12/31/2022] Open
Abstract
Spermatogonial stem cells (SSCs) serve as a resource for producing genetically modified animals. However, genetic manipulation of SSCs has met with limited success. Here, we show efficient gene transfer into SSCs via a lentivirus (FV-LV) using a fusion protein (F), a Sendai virus (SV) envelope protein involved in virion/cell membrane fusion. FV-LVs transduced cultured SSCs more efficiently than conventional LVs. Although SSCs infected with SV failed to produce offspring, those transduced with FV-LVs were fertile. In vivo microinjection showed that FV-LVs could penetrate not only the basement membrane of the seminiferous tubules but also the blood-testis barrier, which resulted in successful transduction of both spermatogenic cells and testicular somatic cells. Cultured SSCs transfected with FV-LVs that express drug-inducible CRISPR/Cas9 against Kit or Sycp3 showed impaired spermatogenesis upon transplantation and drug treatment in vivo. Thus, FV-LVs provide an efficient method for functional analysis of genes involved in SSCs and spermatogenesis.
Sendai virus-derived F protein enhances lentiviral infection of male germ cells Transfected spermatogonial stem cells undergo germline transmission Lentivirus pseudotyped with F protein penetrates the blood-testis barrier This method is compatible with in vivo conditional gene editing
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Affiliation(s)
- Takashi Shinohara
- Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo, Kyoto 606-8501, Japan.
| | - Mito Kanatsu-Shinohara
- Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo, Kyoto 606-8501, Japan
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Takashima S, Shinohara T. Culture and transplantation of spermatogonial stem cells. Stem Cell Res 2018; 29:46-55. [DOI: 10.1016/j.scr.2018.03.006] [Citation(s) in RCA: 37] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/29/2016] [Revised: 02/24/2018] [Accepted: 03/09/2018] [Indexed: 12/22/2022] Open
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BMP4 Cooperates with Retinoic Acid to Induce the Expression of Differentiation Markers in Cultured Mouse Spermatogonia. Stem Cells Int 2016; 2016:9536192. [PMID: 27795714 PMCID: PMC5067322 DOI: 10.1155/2016/9536192] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2016] [Revised: 08/28/2016] [Accepted: 09/08/2016] [Indexed: 02/08/2023] Open
Abstract
Spermatogenesis is sustained by the proliferation and differentiation of spermatogonial stem cells (SSCs). However, the molecules controlling these processes remain largely unknown. Here, we developed a simplified high concentration serum-containing system for the culture of mouse SSCs. Analysis of SSCs markers and transplantation results revealed that the cultured spermatogonia retained stem cell characteristics after long-term in vitro propagation. Using this culture system, the expression and function of bone morphogenetic protein 4 (BMP4) were explored. Immunostaining showed that BMP4 was predominantly expressed in germ cells and that its level increased as spermatogenesis progresses. BMP4 receptors BMPR1A and BMPRII were present in spermatogonia, spermatocytes, and round spermatids. Moreover, despite the mRNAs of these two genes being present in mouse Sertoli cells, only BMPRII was detected by using Western blotting assays. While exogenous BMP4 by itself did not induce the expression of Stra8 and c-Kit, two marker genes of differentiating spermatogonia, a significant cooperative effect of BMP4 and retinoic acid (RA) was observed. Moreover, pretreatment of cultured spermatogonia with the BMP4 antagonist Noggin could inhibit RA-induced expression of these two marker genes. In conclusion, BMP4 may exert autocrine effects and act cooperatively with RA to induce the differentiation of spermatogonia in vivo.
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González R, Dobrinski I. Beyond the mouse monopoly: studying the male germ line in domestic animal models. ILAR J 2016; 56:83-98. [PMID: 25991701 DOI: 10.1093/ilar/ilv004] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and essential to maintain the continuous production of spermatozoa after the onset of puberty in the male. The study of the male germ line is important for understanding the process of spermatogenesis, unravelling mechanisms of stemness maintenance, cell differentiation, and cell-to-cell interactions. The transplantation of SSCs can contribute to the preservation of the genome of valuable individuals in assisted reproduction programs. In addition to the importance of SSCs for male fertility, their study has recently stimulated interest in the generation of genetically modified animals because manipulations of the male germ line at the SSC stage will be maintained in the long term and transmitted to the offspring. Studies performed mainly in the mouse model have laid the groundwork for facilitating advancements in the field of male germ line biology, but more progress is needed in nonrodent species in order to translate the technology to the agricultural and biomedical fields. The lack of reliable markers for isolating germ cells from testicular somatic cells and the lack of knowledge of the requirements for germ cell maintenance have precluded their long-term maintenance in domestic animals. Nevertheless, some progress has been made. In this review, we will focus on the state of the art in the isolation, characterization, culture, and manipulation of SSCs and the use of germ cell transplantation in domestic animals.
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Affiliation(s)
- Raquel González
- Raquel González, DVM, PhD, is a postdoctoral research fellow at the Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada. Ina Dobrinski, DVM, MVSc, PhD, Dipl ACT, is a professor and the head of the Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada
| | - Ina Dobrinski
- Raquel González, DVM, PhD, is a postdoctoral research fellow at the Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada. Ina Dobrinski, DVM, MVSc, PhD, Dipl ACT, is a professor and the head of the Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada
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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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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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Liu S, Tang Z, Xiong T, Tang W. Isolation and characterization of human spermatogonial stem cells. Reprod Biol Endocrinol 2011; 9:141. [PMID: 22018465 PMCID: PMC3235066 DOI: 10.1186/1477-7827-9-141] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/10/2011] [Accepted: 10/24/2011] [Indexed: 03/30/2023] Open
Abstract
BACKGROUND To isolate and characterization of human spermatogonial stem cells from stem spermatogonium. METHODS The disassociation of spermatogonial stem cells (SSCs) were performed using enzymatic digestion of type I collagenase and trypsin. The SSCs were isolated by using Percoll density gradient centrifugation, followed by differential surface-attachment method. Octamer-4(OCT4)-positive SSC cells were further identified using immunofluorescence staining and flow cytometry technques. The purity of the human SSCs was also determined, and a co-culture system for SSCs and Sertoli cells was established. RESULTS The cell viability was 91.07% for the suspension of human spermatogonial stem cells dissociated using a two-step enzymatic digestion process. The cells isolated from Percoll density gradient coupled with differential surface-attachement purification were OCT4 positive, indicating the cells were human spermatogonial stem cells. The purity of isolated human spermatogonial stem cells was 86.7% as assessed by flow cytometry. The isolated SSCs were shown to form stable human spermatogonial stem cell colonies on the feeder layer of the Sertoli cells. CONCLUSIONS The two-step enzyme digestion (by type I collagenase and trypsin) process is an economical, simple and reproducible technique for isolating human spermatogonial stem cells. With little contamination and less cell damage, this method facilitates isolated human spermatogonial stem cells to form a stable cell colony on the supporting cell layer.
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Affiliation(s)
- Shixue Liu
- Department of Urology, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China
| | - Ziwei Tang
- West China Medical College, Sichuan University, Chengdu 610041, China
| | - Tao Xiong
- Department of Urology, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China
| | - Wei Tang
- Department of Urology, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China
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Parte S, Bhartiya D, Telang J, Daithankar V, Salvi V, Zaveri K, Hinduja I. Detection, characterization, and spontaneous differentiation in vitro of very small embryonic-like putative stem cells in adult mammalian ovary. Stem Cells Dev 2011; 20:1451-64. [PMID: 21291304 PMCID: PMC3148829 DOI: 10.1089/scd.2010.0461] [Citation(s) in RCA: 182] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
The present study was undertaken to detect, characterize, and study differentiation potential of stem cells in adult rabbit, sheep, monkey, and menopausal human ovarian surface epithelium (OSE). Two distinct populations of putative stem cells (PSCs) of variable size were detected in scraped OSE, one being smaller and other similar in size to the surrounding red blood cells in the scraped OSE. The smaller 1-3 μm very small embryonic-like PSCs were pluripotent in nature with nuclear Oct-4 and cell surface SSEA-4, whereas the bigger 4-7 μm cells with cytoplasmic localization of Oct-4 and minimal expression of SSEA-4 were possibly the tissue committed progenitor stem cells. Pluripotent gene transcripts of Oct-4, Oct-4A, Nanog, Sox-2, TERT, and Stat-3 in human and sheep OSE were detected by reverse transcriptase-polymerase chain reaction. The PSCs underwent spontaneous differentiation into oocyte-like structures, parthenote-like structures, embryoid body-like structures, cells with neuronal-like phenotype, and embryonic stem cell-like colonies, whereas the epithelial cells transformed into mesenchymal phenotype by epithelial-mesenchymal transition in 3 weeks of OSE culture. Germ cell markers like c-Kit, DAZL, GDF-9, VASA, and ZP4 were immuno-localized in oocyte-like structures. In conclusion, as opposed to the existing view of OSE being a bipotent source of oocytes and granulosa cells, mammalian ovaries harbor distinct very small embryonic-like PSCs and tissue committed progenitor stem cells population that have the potential to develop into oocyte-like structures in vitro, whereas mesenchymal fibroblasts appear to form supporting granulosa-like somatic cells. Research at the single-cell level, including complete gene expression profiling, is required to further confirm whether postnatal oogenesis is a conserved phenomenon in adult mammals.
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Affiliation(s)
- Seema Parte
- Department of Stem Cell Biology, National Institute for Research in Reproductive Health, Mumbai, India
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Bhartiya D, Kasiviswanathan S, Unni SK, Pethe P, Dhabalia JV, Patwardhan S, Tongaonkar HB. Newer insights into premeiotic development of germ cells in adult human testis using Oct-4 as a stem cell marker. J Histochem Cytochem 2010; 58:1093-1106. [PMID: 20805580 PMCID: PMC2989246 DOI: 10.1369/jhc.2010.956870] [Citation(s) in RCA: 83] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2010] [Accepted: 08/11/2010] [Indexed: 02/07/2023] Open
Abstract
The transcription factor octamer-binding transforming factor 4 (Oct-4) is central to the gene regulatory network responsible for self-renewal, pluripotency, and lineage commitment in embryonic stem (ES) cells and induced pluripotent stem cells (PSCs). This study was undertaken to evaluate differential localization and expression of two major transcripts of Oct-4, viz. Oct-4A and Oct-4B, in adult human testis. A novel population of 5- to 10-μm PSCs with nuclear Oct-4A was identified by ISH and immunolocalization studies. Besides Oct-4, other pluripotent markers like Nanog and TERT were also detected by RT-PCR. A(dark) spermatogonial stem cells (SSCs) were visualized in pairs and chains undergoing clonal expansion and stained positive for cytoplasmic Oct-4B. Quantitative PCR and Western blotting revealed both the transcripts, with higher expression of Oct-4B. It is proposed that PSCs undergo asymmetric cell division and give rise to A(dark) SSCs, which proliferate and initiate lineage-specific differentiation. The darkly stained nuclei in A(dark) SSCs may represent extensive nuclear reprogramming by epigenetic changes when a PSC becomes committed. Oct-4B eventually disappeared in mature germ cells, viz. spermatocytes, spermatids, and sperm. Besides maintaining normal testicular homeostasis, PSCs may also be implicated in germ cell tumors and ES-like colonies that have recently been derived from adult human testicular tissue.
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Affiliation(s)
- Deepa Bhartiya
- Dept. of Stem Cell Biology, National Institute for Research in Reproductive Health, JM Street, Parel, Mumbai 400012, India.
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Cheng CY, Mruk DD. The biology of spermatogenesis: the past, present and future. Philos Trans R Soc Lond B Biol Sci 2010; 365:1459-63. [PMID: 20403863 DOI: 10.1098/rstb.2010.0024] [Citation(s) in RCA: 66] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The physiological function of spermatogenesis in Caenorhabditis elegans, Drosophila melanogaster and mammals is to produce spermatozoa (1n, haploid) that contain only half of the genetic material of spermatogonia (2n, diploid). This half number of chromosomes from a spermatozoon will then be reconstituted to become a diploid cell upon fertilization with an egg, which is also haploid. Thus, genetic information from two parental individuals can be passed onto their offspring. Spermatogenesis takes place in the seminiferous epithelium of the seminiferous tubule, the functional unit of the mammalian testis. In mammals, particularly in rodents, the fascinating morphological changes that occur during spermatogenesis involving cellular differentiation and transformation, mitosis, meiosis, germ cell movement, spermiogenesis and spermiation have been well documented from the 1950s through the 1980s. During this time, however, the regulation of, as well as the biochemical and molecular mechanisms underlying these diverse cellular events occurring throughout spermatogenesis, have remained largely unexplored. In the past two decades, important advancements have been made using new biochemical, cell and molecular biology techniques to understand how different genes, proteins and signalling pathways regulate various aspects of spermatogenesis. These include studies on the differentiation of spermatogonia from gonocytes; regulation of spermatogonial stem cells; regulation of spermatogonial mitosis; regulation of meiosis, spermiogenesis and spermiation; role of hormones (e.g. oestrogens, androgens) in spermatogenesis; transcriptional regulation of spermatogenesis; regulation of apoptosis; cell-cell interactions; and the biology of junction dynamics during spermatogenesis. The impact of environmental toxicants on spermatogenesis has also become an urgent issue in the field in light of declining fertility levels in males. Many of these studies have helped investigators to understand important similarities, differences and evolutionary relationships between C. elegans, D. melanogaster and mammals relating to spermatogenesis. In this Special Issue of the Philosophical Transactions of the Royal Society B: Biological Sciences, we have covered many of these areas, and in this Introduction, we highlight the topic of spermatogenesis by examining its past, present and future.
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Affiliation(s)
- C Yan Cheng
- The Mary M. Wohlford Laboratory for Male Contraceptive Research, Population Council, 1230 York Avenue, New York, NY 10065, USA.
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Simon L, Hess RA, Cooke PS. Spermatogonial stem cells, in vivo transdifferentiation and human regenerative medicine. Expert Opin Biol Ther 2010; 10:519-30. [PMID: 20146635 DOI: 10.1517/14712591003614731] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
IMPORTANCE OF THE FIELD Embryonic stem (ES) cells have potential for use in regenerative medicine, but use of these cells is hindered by moral, legal and ethical issues. Induced pluripotent cells have promise in regenerative medicine. However, since generation of these cells involves genetic manipulation, it also faces significant hurdles before clinical use. This review discusses spermatogonial stem cells (SSCs) as a potential alternative source of pluripotent cells for use in human regenerative medicine. AREAS COVERED IN THE REVIEW The potential of SSCs to give rise to a wide range of other cell types either directly, when recombined with instructive inducers, or indirectly, after being converted to ES-like cells. Current understanding of the differentiation potential of murine SSCs and recent progress in isolating and culturing human SSCs and demonstrating their properties is also discussed. WHAT THE READER WILL GAIN Insight into the plasticity of SSCs and the unique properties of these cells for regenerative applications, the limitations of SSCs for stem-cell-based therapy and the potential alternatives available. TAKE HOME MESSAGE If methodologies for isolation and conversion of adult human SSCs directly into other cell types can be effectively developed, SSCs could represent an important alternate source of pluripotent cells that can be used in human tissue repair and/or regeneration.
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Affiliation(s)
- Liz Simon
- University of Illinois at Urbana Champaign, Veterinary Biosciences, VMBSB, 2001, S. Lincoln Avenue, Urbana, IL 61802, USA
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Saitou M, Yamaji M. Germ cell specification in mice: signaling, transcription regulation, and epigenetic consequences. Reproduction 2010; 139:931-42. [DOI: 10.1530/rep-10-0043] [Citation(s) in RCA: 105] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
The specification of germ cell fate in development initiates mechanisms essential for the perpetuation of genetic information across the generations. Recent studies in mice have shown that germ cell specification requires at least three key molecular/cellular events: repression of the somatic program, re-acquisition of potential pluripotency, and an ensuing genome-wide epigenetic reprogramming. Moreover, a signaling and transcriptional principle governing these processes has been identified, raising the possibility of inducing the germ cell fate precisely from pluripotent stem cells in culture. These advances will in turn serve as a basis to explore the mechanism of germ cell specification in other mammals, including humans. The recapitulation of germ cell development in humans in culture will provide unprecedented opportunities to understand the basis of the propagation of our genome, both under normal and diseased conditions.
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Takehashi M, Kanatsu-Shinohara M, Shinohara T. Generation of genetically modified animals using spermatogonial stem cells. Dev Growth Differ 2010; 52:303-10. [PMID: 20148923 DOI: 10.1111/j.1440-169x.2009.01167.x] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis, and are unique tissue-specific stem cells because of their ability to transmit genetic information to offspring. Generation of knockout mice using mouse SSCs became feasible after the successful establishment of protocols for the transplantation and long-term culture of these cells, called germline stem (GS) cells. Furthermore, SSCs can acquire pluripotentiality similar to that of embryonic stem (ES) cells, in addition to their highly differentiated spermatogenic potential. These ES-like cells, called multipotent GS (mGS) cells, are capable of generating knockout mice in a manner similar to that of ES cells. The use of GS and mGS cells for animal transgenesis has added a new dimension to gene-targeting technology using ES cells and somatic cell nuclear transfer, which has limited application. Furthermore, for regenerative medicine purposes, the use of mGS will settle problems such as ethics issues and immunological rejection associated with ES cells, as well as risks of insertional mutagenesis associated with integrated genes into induced pluripotent stem cells.
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Affiliation(s)
- Masanori Takehashi
- Faculty of Pharmacy, Osaka Ohtani University, Tondabayashi, Osaka, Japan.
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Kanatsu-Shinohara M, Shinohara T. Germline Modification Using Mouse Spermatogonial Stem Cells. Methods Enzymol 2010; 477:17-36. [DOI: 10.1016/s0076-6879(10)77002-6] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
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