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Segunda MN, Cortez J, Díaz C, Arancibia R, Torres CG, Parraguez VH, De Los Reyes M, Peralta OA. Potential of mesenchymal stromal/stem cells and spermatogonial stem cells for survival and colonization in bull recipient testes after allogenic transplantation. Theriogenology 2024; 230:192-202. [PMID: 39332379 DOI: 10.1016/j.theriogenology.2024.09.022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2024] [Revised: 07/18/2024] [Accepted: 09/23/2024] [Indexed: 09/29/2024]
Abstract
Stem cell transplantation into seminiferous tubules of recipient testis could become a tool for fertility restoration, genetic improvement, or conservation of endangered species. Spermatogonial stem cells (SSCs) are primary candidates for transplantation; however, limited abundance, complexity for isolation and culture, and lack of specific markers have limited their use. Mesenchymal stromal/stem cells (MSCs) are multipotent progenitors that are simple to isolate and culture and possess specific markers for identification, and immune evasive and migratory capacities. The objective of the present study was to evaluate the potential for survival and colonization in seminiferous tubules of two different concentrations of bovine fetal adipose tissue-derived MSCs (AT-MSCs), native of pre-induced, and to compare the fate of bovine adult peripheral blood-derived MSCs (PB-MSCs) and SSCs after allogenic transplantation in testis of recipient bulls. In experiment 1, AT-MSCs at two concentrations (1x107 and 2x107; n = 3) or pre-exposed to 2 μM testosterone and 1 μM retinoic acid (RA) for 14 days (n = 5) were evaluated. In experiment 2, adult PB-MSCs and SSCs (4x107 cells each) pre-exposed to Sertoli cell conditioned media (SCs/CM; n = 4) for 14 days were compared. Each cell type was separately labelled with PKH26 and then transplanted into testes of 8-month-old recipient bulls. Four weeks (Exp. 1) and two weeks (Exp. 2) after transplantation, testicular tissue was processed for confocal microscopy detection of PKH26-positive cells. Mean number of PKH26-positive cells were higher (P < 0.05) in testis transplanted with 2x107 AT-MSCs in the proximal (6.7 ± 3.7) and medial (6.6 ± 3.2) sections compared to testis transplanted with 1x107 AT-MSCs (proximal: 1.9 ± 1; medial: 1.9 ± 1) sections or pre-induced AT-MSCs (proximal: 4.7 ± 5.6; medial: 3.8 ± 4.1). In Exp. 2, mean number of PKH26-positive SSCs in medial testicular section (22.5 ± 1.3) were higher (P < 0.05) compared to respective section in PB-MSCs group (17 ± 4.2). Thus, in vivo data indicates that a higher number of transplanted AT-MSCs resulted in more cells surviving and colonizing seminiferous tubules; however, pre-induction with testosterone and RA did not improve these capacities. SSCs displayed a greater capacity for survival and colonization in recipient seminiferous tubules; however, PB-MSCs were observed in all sections of testis after two weeks of transplantation.
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Affiliation(s)
- Moisés N Segunda
- Faculty of Veterinary and Animal Sciences, University of Chile, Santa Rosa 11735, 8820808, Santiago, Chile; Doctorate Program of Forestry, Agriculture, and Veterinary Sciences (DCSAV), University of Chile, Santa Rosa 11315, 8820808, Santiago, Chile; Faculdade de Medicina Veterinária, Universidade José Eduardo Dos Santos, Bairro Santo António-Avenida Nuno Alvarez, 555, Huambo, Angola
| | - Jahaira Cortez
- Faculty of Veterinary and Animal Sciences, University of Chile, Santa Rosa 11735, 8820808, Santiago, Chile; Doctorate Program of Forestry, Agriculture, and Veterinary Sciences (DCSAV), University of Chile, Santa Rosa 11315, 8820808, Santiago, Chile
| | - Carlos Díaz
- Doctorate Program in Sciences, UNED, Bravo Murillo 38, 28015, Madrid, Spain
| | - Richard Arancibia
- Faculty of Veterinary and Animal Sciences, University of Chile, Santa Rosa 11735, 8820808, Santiago, Chile
| | - Cristian G Torres
- Faculty of Veterinary and Animal Sciences, University of Chile, Santa Rosa 11735, 8820808, Santiago, Chile
| | - Víctor H Parraguez
- Faculty of Veterinary and Animal Sciences, University of Chile, Santa Rosa 11735, 8820808, Santiago, Chile
| | - Mónica De Los Reyes
- Faculty of Veterinary and Animal Sciences, University of Chile, Santa Rosa 11735, 8820808, Santiago, Chile
| | - Oscar A Peralta
- Faculty of Veterinary and Animal Sciences, University of Chile, Santa Rosa 11735, 8820808, Santiago, Chile; Escuela de Medicina Veterinaria, Facultad de Agronomía e Ingeniería Forestal, Facultad de Ciencias Biológicas y Facultad de Medicina, Pontificia Universidad Católica de Chile, Vicuña Mackenna 4860, Macul, 7820436, Santiago, Chile.
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Damyanova KB, Nixon B, Johnston SD, Gambini A, Benitez PP, Lord T. Spermatogonial stem cell technologies: applications from human medicine to wildlife conservation†. Biol Reprod 2024; 111:757-779. [PMID: 38993049 PMCID: PMC11473898 DOI: 10.1093/biolre/ioae109] [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: 05/01/2024] [Revised: 07/05/2024] [Accepted: 07/10/2024] [Indexed: 07/13/2024] Open
Abstract
Spermatogonial stem cell (SSC) technologies that are currently under clinical development to reverse human infertility hold the potential to be adapted and applied for the conservation of endangered and vulnerable wildlife species. The biobanking of testis tissue containing SSCs from wildlife species, aligned with that occurring in pediatric human patients, could facilitate strategies to improve the genetic diversity and fitness of endangered populations. Approaches to utilize these SSCs could include spermatogonial transplantation or testis tissue grafting into a donor animal of the same or a closely related species, or in vitro spermatogenesis paired with assisted reproduction approaches. The primary roadblock to progress in this field is a lack of fundamental knowledge of SSC biology in non-model species. Herein, we review the current understanding of molecular mechanisms controlling SSC function in laboratory rodents and humans, and given our particular interest in the conservation of Australian marsupials, use a subset of these species as a case-study to demonstrate gaps-in-knowledge that are common to wildlife. Additionally, we review progress in the development and application of SSC technologies in fertility clinics and consider the translation potential of these techniques for species conservation pipelines.
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Affiliation(s)
- Katerina B Damyanova
- Discipline of Biological Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia
- Infertility and Reproduction Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia
| | - Brett Nixon
- Discipline of Biological Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia
- Infertility and Reproduction Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia
| | - Stephen D Johnston
- School of Environment, The University of Queensland, Gatton, QLD 4343, Australia
- School of Veterinary Science, The University of Queensland, Gatton, QLD 4343, Australia
| | - Andrés Gambini
- School of Veterinary Science, The University of Queensland, Gatton, QLD 4343, Australia
- School of Agriculture and Food Science, The University of Queensland, Gatton, QLD 4343, Australia
| | - Patricio P Benitez
- School of Agriculture and Food Science, The University of Queensland, Gatton, QLD 4343, Australia
| | - Tessa Lord
- Discipline of Biological Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia
- Infertility and Reproduction Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia
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Jung H, Yoon M. Transplantation of spermatogonial stem cells in stallions. JOURNAL OF ANIMAL SCIENCE AND TECHNOLOGY 2024; 66:635-644. [PMID: 39165739 PMCID: PMC11331362 DOI: 10.5187/jast.2024.e30] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 08/30/2023] [Revised: 02/07/2024] [Accepted: 02/29/2024] [Indexed: 08/22/2024]
Abstract
Spermatogonial stem cells originate from gonocytes and undergo self-renewal and differentiation to generate mature spermatozoa via spermatogenesis in the seminiferous tubules of the testis in male mammals. Owing to the unique capacity of these cells, the spermatogonial stem cell transplantation technique, which enables the restoration of male fertility by transfer of germlines between donor and recipient males, has been developed. Thus, spermatogonial stem cell transplantation can be used as an important next-generation reproductive and breeding tool in livestock production. However, in large animals, this approach is associated with many technical limitations and inefficiency. Furthermore, research regrading spermatogonial stem cell transplantation in stallions is limited. Therefore, this review article describes the history and current knowledge regarding spermatogonial stem cell transplantation in animals and challenges in establishing an experimental protocol for successful spermatogonial stem cell transplantation in stallions, which have been presented under the following heads: spermatogonial stem cell isolation, recipient preparation, and spermatogonial stem cell transplantation. Additionally, we suggest that further investigation based on previous unequivocal evidence regarding donor-derived spermatogenesis in large animals must be conducted. A detailed and better understanding of the physical and physiological aspects is required to discuss the current status of this technique field and develop future directions for the establishment of spermatogonial stem cell transplantation in stallions.
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Affiliation(s)
- Heejun Jung
- Research Center for Horse Industry,
Kyungpook National University, Sangju 37224, Korea
| | - Minjung Yoon
- Research Center for Horse Industry,
Kyungpook National University, Sangju 37224, Korea
- Department of Horse, Companion and Wild
Animal Science, Kyungpook National University, Sangju 37224,
Korea
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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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Reifarth L, Körber H, Packeiser EM, Goericke-Pesch S. Detection of spermatogonial stem cells in testicular tissue of dogs with chronic asymptomatic orchitis. Front Vet Sci 2023; 10:1205064. [PMID: 37396999 PMCID: PMC10311113 DOI: 10.3389/fvets.2023.1205064] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2023] [Accepted: 05/25/2023] [Indexed: 07/04/2023] Open
Abstract
Chronic asymptomatic idiopathic orchitis (CAO) is an important but neglected cause of acquired infertility due to non-obstructive azoospermia (NOA) in male dogs. The similarity of the pathophysiology in infertile dogs and men supports the dog's suitability as a possible animal model for studying human diseases causing disruption of spermatogenesis and evaluating the role of spermatogonial stem cells (SSCs) as a new therapeutic approach to restore or recover fertility in cases of CAO. To investigate the survival of resilient stem cells, the expression of the protein gene product (PGP9.5), deleted in azoospermia like (DAZL), foxo transcription factor 1 (FOXO1) and tyrosine-kinase receptor (C-Kit) were evaluated in healthy and CAO-affected canine testes. Our data confirmed the presence of all investigated germ cell markers at mRNA and protein levels. In addition, we postulate a specific expression pattern of FOXO1 and C-Kit in undifferentiated and differentiating spermatogonia, respectively, whereas DAZL and PGP9.5 expressions were confirmed in the entire spermatogonial population. Furthermore, this is the first study revealing a significant reduction of PGP9.5, DAZL, and FOXO1 in CAO at protein and/or gene expression level indicating a severe disruption of spermatogenesis. This means that chronic asymptomatic inflammatory changes in CAO testis are accompanied by a significant loss of SSCs. Notwithstanding, our data confirm the survival of putative stem cells with the potential of self-renewal and differentiation and lay the groundwork for further research into stem cell-based therapeutic options to reinitialize spermatogenesis in canine CAO-affected patients.
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Affiliation(s)
| | | | | | - Sandra Goericke-Pesch
- Reproductive Unit – Clinic for Small Animals, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany
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Pozor MA, Benson SM, Macpherson ML, Kelleman AA. Effects of therapeutic ultrasound and moderate heat on stallion testes. Theriogenology 2023; 203:21-32. [PMID: 36966582 DOI: 10.1016/j.theriogenology.2023.03.014] [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: 08/30/2022] [Revised: 02/19/2023] [Accepted: 03/13/2023] [Indexed: 04/12/2023]
Abstract
Transplantation of stem cells into dysfunctional testes is currently being investigated as a therapeutic option for men and stallions with advanced testicular degeneration. This series of "proof of concept" studies aimed to identify a safe and efficient method of inducing severe testicular degeneration to create an optimal equine recipient model for intratesticular stem cell transplantation (SCT). Two ex vivo and two in vivo experiments were conducted. At first, forty testes obtained from castrations were used to identify an effective therapeutic ultrasound (TUS) device and the protocol for increasing intratesticular temperature in stallions. Six min of treatment using the Vetrison Clinic Portable TUS machine raised the intratesticular temperature by 8°C-12.5 °C. This protocol was applied to treat three scrotal testes in three Miniature horse stallions, three times, every other day. Contralateral testes served as controls. There were signs of slight tubular degeneration in treated testes two and three weeks after TUS treatment. The number of seminiferous tubules (STs) with exfoliated germ cells (GCs) was increased in one testis only, three weeks after treatment. The degree of apoptosis of GCs was higher in each treated testis in comparison to the contralateral control testis. Next, the ability of various heating devices to increase intratesticular temperatures to at least 43 °C in stallion testes was tested, using twenty testes obtained from castrations. ThermaCare® Lower Back & Hip Pain Therapy Heatwrap (TC heat wrap) reliably increased intratesticular temperatures and kept them continuously between 43 °C and 48 °C for seven to 8 h. In the follow-up in vivo study, the left testes of three Miniature horse stallions were treated with TUS, after which both testes of each stallion were treated with moderate heat provided by the TC heat wrap (three times, every other day, for 5 h each time). There were signs of moderate tubular degeneration in the samples from all treated testes obtained three weeks after treatments (Heat only or Heat/TUS): areas with hypospermatogenesis, spermatogenic arrest, vacuolized Sertoli cells, numerous STs with exfoliated GCs, increased degree of GCs apoptosis, and changes in three histomorphometric numeric attributes of STs. We concluded that TUS or TC wraps increase intratesticular temperature of the isolated stallion testes. Further, treatment with TUS or moderate heat may induce mild to moderate degenerative changes in stallion testes. However, to achieve more robust result - severe testicular degeneration, our treatment protocol has to be modified.
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Affiliation(s)
- Malgorzata A Pozor
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA.
| | - Susanne M Benson
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA.
| | - Margo L Macpherson
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA.
| | - Audrey A Kelleman
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA.
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Mohamed Rasheed ZB, Nordin F, Wan Kamarul Zaman WS, Tan YF, Abd Aziz NH. Autologous Human Mesenchymal Stem Cell-Based Therapy in Infertility: New Strategies and Future Perspectives. BIOLOGY 2023; 12:108. [PMID: 36671799 PMCID: PMC9855776 DOI: 10.3390/biology12010108] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/08/2022] [Revised: 01/06/2023] [Accepted: 01/08/2023] [Indexed: 01/12/2023]
Abstract
Infertility could be associated with a few factors including problems with physical and mental health, hormonal imbalances, lifestyles, and genetic factors. Given that there is a concern about the rise of infertility globally, increased focus has been given to its treatment for the last several decades. Traditional assisted reproductive technology (ART) has been the prime option for many years in solving various cases of infertility; however, it contains significant risks and does not solve the fundamental problem of infertility such as genetic disorders. Attention toward the utilization of MSCs has been widely regarded as a promising option in the development of stem-cell-based infertility treatments. This narrative review briefly presents the challenges in the current ART treatment of infertility and the various potential applications of autologous MSCs in the treatment of these reproductive diseases.
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Affiliation(s)
- Zahirrah Begam Mohamed Rasheed
- UKM Medical Molecular Biology Institute (UMBI), Jalan Yaacob Latiff, Bandar Tun Razak, Cheras, Kuala Lumpur 56000, Malaysia
| | - Fazlina Nordin
- Centre for Tissue Engineering and Regenerative Medicine (CTERM), Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Yaacob Latiff, Bandar Tun Razak, Cheras, Kuala Lumpur 56000, Malaysia
| | | | - Yuen-Fen Tan
- PPUKM-MAKNA Cancer Center, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, WPKL, Kuala Lumpur 56000, Malaysia
- Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Sungai Long Campus, Bandar Sungai Long, Kajang 43000, Malaysia
| | - Nor Haslinda Abd Aziz
- Department of Obstetrics and Gynaecology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur 56000, Malaysia
- Research Laboratory of UKM Specialist Children’s Hospital, UKM Specialist Children’s Hospital, Universiti Kebangsaan Malaysia, Kuala Lumpur 56000, Malaysia
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Ciccarelli M, Oatley JM. Perspectives: Approaches for Studying Livestock Spermatogonia. Methods Mol Biol 2023; 2656:325-339. [PMID: 37249879 DOI: 10.1007/978-1-0716-3139-3_17] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/31/2023]
Abstract
At present, the knowledge base on characteristics and biology of spermatogonia in livestock is limited in comparison to rodents, yet the importance of studying these cells for comparative species analysis and enhancing reproductive capacity in food animals is high. Previous studies have established that although many core attributes of organ physiology and mechanisms governing essential cellular functions are conserved across eutherians, significant differences exist between mice and higher order mammals. In this chapter, we briefly discuss distinguishing aspects of testicular anatomy and the spermatogenic lineage in livestock and critical considerations for studying spermatogonial stem cell biology in these species.
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Affiliation(s)
- Michela Ciccarelli
- Center for Reproductive Biology, Washington State University, Pullman, WA, USA
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA, USA
| | - Jon M Oatley
- Department of Biology, University of Texas at San Antonio, San Antonio, TX, USA.
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Deebel NA, Soltanghoraee H, Bradshaw AW, Abdelaal O, Reynolds K, Howards S, Kogan S, Sadeghi MR, Atala A, Stogner-Underwood K, Sadri-Ardekani H. Morphometric and immunohistochemical analysis as a method to identify undifferentiated spermatogonial cells in adult subjects with Klinefelter syndrome: a cohort study. Fertil Steril 2022; 118:864-873. [PMID: 36116982 DOI: 10.1016/j.fertnstert.2022.07.015] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2022] [Revised: 07/08/2022] [Accepted: 07/14/2022] [Indexed: 01/13/2023]
Abstract
OBJECTIVE To study the prevalence of spermatogonia in adult subjects with Klinefelter syndrome (KS) using MAGE-A4 and UCHL1 (PGP9.5) immunohistochemistry as markers for undifferentiated spermatogonial cells. We aimed to compare this method to the gold standard of hematoxylin and eosin (H & E) staining with histologic analysis in the largest reported cohort of adult subjects with KS. DESIGN A retrospective cohort study. SETTING Infertility Clinic and Institute for Regenerative Medicine. PATIENT(S) This study consisted of 79 adult subjects with KS and 12 adult control subjects. INTERVENTION(S) The subjects with KS (n = 79) underwent bilateral testicular biopsy in an initial effort to recover spermatozoa for in vitro fertilization and intracytoplasmic sperm injection. The institutional review board approved the use of a portion of the archived diagnostic pathology paraffin blocks for the study. The samples were superimposed onto microscopic slides and labeled with the PGP9.5 and MAGE-A4 antibodies. Subjects (n = 12) who had previously consented to be organ donors via the National Disease Research Interchange were selected as controls. Dedicated genitourinary pathologists examined the H & E-, PGP9.5-, and MAGE-A4-stained tissue for presence of undifferentiated spermatogonia and spermatozoa with the use of a virtual microscopy software. MAIN OUTCOME MEASURE(S) The primary outcome was the presence of MAGE-A4-positive or UCHL1-positive tubules that indicate undifferentiated spermatogonia. Supportive outcomes include assessing the biopsy specimen for the following: total surface area; total seminiferous tubule surface area; total interstitium surface area; the total number of seminiferous tubules; and MAGE-A4- negative or UCHL1-negative tubules. Additionally, clinical information, such as age, karyotype, height, weight, mean testicle size, and hormonal panel (luteinizing hormone, follicle-stimulating hormone, and testosterone), was obtained and used in a single and multivariable analysis with linear regression to determine predictive factors for the number of UCHL1-positive tubules. RESULT(S) The mean age of the subjects in the KS group was 32.9 ± 0.7 years (range, 16-48). UCHL1 (PGP9.5) and MAGE-A4 staining showed that 74.7% (n = 59) and 40.5% (n = 32) of the subjects with KS, respectively, were positive for undifferentiated spermatogonia compared with 100% (n = 12) of the control subjects who were positive for both the markers. Hematoxylin and eosin with microscopic analysis showed that only 10.1% (n = 8) of the subjects were positive for spermatogonia. The mean number of positive tubules per subject with KS was 11.8 ± 1.8 for UCHL1 and 3.7 ± 1.0 for MAGE-A4. Secondary analysis showed 7 (8.9%) adult subjects with KS as positive for spermatozoa on biopsy. The population having negative testicular sperm extraction results (n = 72) showed a spermatogonia-positive rate of 1.4%, (n = 1), 72.2% (n = 52), and 34.7% (n = 25) using H & E, UCHL1, and MAGE-A4, respectively. Further analysis showed that 54 (75.0%) subjects were either positive for UCHL1 or MAGE-A4. Twenty (27.8%) subjects were positive for both UCHL1 and MAGE-A4. Multivariate analysis with linear regression showed no significant correlation between clinical variables and the number of UCHL1-positive tubules found on biopsy specimens. CONCLUSION(S) We report a cohort of adult subjects with KS undergoing analysis for the presence of undifferentiated spermatogonia. UCHL1 and MAGE-A4 immunostaining appear to be an effective way of identifying undifferentiated spermatogonia in testicular biopsy specimens of subjects with KS. Despite observing deterioration in the testicular architecture, many patients remain positive for undifferentiated spermatogonia, which could be harvested and potentially used for infertility therapy in a patient with KS who is azoospermic and has negative testicular sperm extraction results.
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Affiliation(s)
- Nicholas A Deebel
- Department of Urology, Wake Forest University School of Medicine, Winston-Salem, North Carolina; Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina
| | - Haleh Soltanghoraee
- Reproductive Biotechnology Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research, Tehran, Iran; Avicenna Infertility Clinic, Avicenna Research Institute, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Aaron William Bradshaw
- Department of Urology, Wake Forest University School of Medicine, Winston-Salem, North Carolina; Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina
| | - Omar Abdelaal
- Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina; Department of Urology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
| | - Karl Reynolds
- Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina
| | - Stuart Howards
- Department of Urology, Wake Forest University School of Medicine, Winston-Salem, North Carolina
| | - Stanley Kogan
- Department of Urology, Wake Forest University School of Medicine, Winston-Salem, North Carolina; Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina
| | - Mohammad Reza Sadeghi
- Reproductive Biotechnology Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research, Tehran, Iran; Avicenna Infertility Clinic, Avicenna Research Institute, Academic Center for Education, Culture and Research, Tehran, Iran
| | - Anthony Atala
- Department of Urology, Wake Forest University School of Medicine, Winston-Salem, North Carolina; Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina
| | - Kimberly Stogner-Underwood
- Department of Urology, Wake Forest University School of Medicine, Winston-Salem, North Carolina; Department of Urology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
| | - Hooman Sadri-Ardekani
- Department of Urology, Wake Forest University School of Medicine, Winston-Salem, North Carolina; Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina; Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, North Carolina.
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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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11
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Cheng H, Shang D, Zhou R. Germline stem cells in human. Signal Transduct Target Ther 2022; 7:345. [PMID: 36184610 PMCID: PMC9527259 DOI: 10.1038/s41392-022-01197-3] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2022] [Revised: 09/06/2022] [Accepted: 09/14/2022] [Indexed: 12/02/2022] Open
Abstract
The germline cells are essential for the propagation of human beings, thus essential for the survival of mankind. The germline stem cells, as a unique cell type, generate various states of germ stem cells and then differentiate into specialized cells, spermatozoa and ova, for producing offspring, while self-renew to generate more stem cells. Abnormal development of germline stem cells often causes severe diseases in humans, including infertility and cancer. Primordial germ cells (PGCs) first emerge during early embryonic development, migrate into the gentile ridge, and then join in the formation of gonads. In males, they differentiate into spermatogonial stem cells, which give rise to spermatozoa via meiosis from the onset of puberty, while in females, the female germline stem cells (FGSCs) retain stemness in the ovary and initiate meiosis to generate oocytes. Primordial germ cell-like cells (PGCLCs) can be induced in vitro from embryonic stem cells or induced pluripotent stem cells. In this review, we focus on current advances in these embryonic and adult germline stem cells, and the induced PGCLCs in humans, provide an overview of molecular mechanisms underlying the development and differentiation of the germline stem cells and outline their physiological functions, pathological implications, and clinical applications.
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Affiliation(s)
- Hanhua Cheng
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Renmin Hospital of Wuhan University, Wuhan University, 430072, Wuhan, China.
| | - Dantong Shang
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Renmin Hospital of Wuhan University, Wuhan University, 430072, Wuhan, China
| | - Rongjia Zhou
- Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Renmin Hospital of Wuhan University, Wuhan University, 430072, Wuhan, China.
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12
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Huanhuan Hu, Ji G, Shi X, Zhang J, Li M. Current Status of Male Fertility Preservation in Humans. Russ J Dev Biol 2022. [DOI: 10.1134/s1062360422020060] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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13
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Tran KTD, Valli-Pulaski H, Colvin A, Orwig KE. Male fertility preservation and restoration strategies for patients undergoing gonadotoxic therapies†. Biol Reprod 2022; 107:382-405. [PMID: 35403667 PMCID: PMC9382377 DOI: 10.1093/biolre/ioac072] [Citation(s) in RCA: 29] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2021] [Revised: 03/29/2022] [Accepted: 04/04/2022] [Indexed: 11/22/2022] Open
Abstract
Medical treatments for cancers or other conditions can lead to permanent infertility. Infertility is an insidious disease that impacts not only the ability to have a biological child but also the emotional well-being of the infertile individuals, relationships, finances, and overall health. Therefore, all patients should be educated about the effects of their medical treatments on future fertility and about fertility preservation options. The standard fertility preservation option for adolescent and adult men is sperm cryopreservation. Sperms can be frozen and stored for a long period, thawed at a later date, and used to achieve pregnancy with existing assisted reproductive technologies. However, sperm cryopreservation is not applicable for prepubertal patients who do not yet produce sperm. The only fertility preservation option available to prepubertal boys is testicular tissue cryopreservation. Next-generation technologies are being developed to mature those testicular cells or tissues to produce fertilization-competent sperms. When sperm and testicular tissues are not available for fertility preservation, inducing pluripotent stem cells derived from somatic cells, such as blood or skin, may provide an alternative path to produce sperms through a process call in vitro gametogenesis. This review describes standard and experimental options to preserve male fertility as well as the experimental options to produce functional spermatids or sperms from immature cryopreserved testicular tissues or somatic cells.
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Affiliation(s)
- Kien T D Tran
- Molecular Genetics and Developmental Biology Graduate Program, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA,Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA,Magee-Womens Research Institute, Pittsburgh, PA, USA
| | - Hanna Valli-Pulaski
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA,Magee-Womens Research Institute, Pittsburgh, PA, USA
| | - Amanda Colvin
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA,Magee-Womens Research Institute, Pittsburgh, PA, USA
| | - Kyle E Orwig
- Correspondence: Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, PA 15213, USA. Tel: 412-641-2460; E-mail:
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14
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Zhang W, Nie R, Cai Y, Xie W, Zou K. Progress in germline stem cell transplantation in mammals and the potential usage. Reprod Biol Endocrinol 2022; 20:59. [PMID: 35361229 PMCID: PMC8969385 DOI: 10.1186/s12958-022-00930-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/16/2021] [Accepted: 03/10/2022] [Indexed: 11/10/2022] Open
Abstract
Germline stem cells (GSCs) are germ cells with the capacities of self-renewal and differentiation into functional gametes, and are able to migrate to their niche and reconstitute the fertility of recipients after transplantation. Therefore, GSCs transplantation is a promising technique for fertility recovery in the clinic, protection of rare animals and livestock breeding. Though this novel technique faces tremendous challenges, numerous achievements have been made after several decades' endeavor. This review summarizes the current knowledge of GSCs transplantation and its utilization in mammals, and discusses the application prospect in reproductive medicine and animal science.
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Affiliation(s)
- Wen Zhang
- Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China
| | - Ruotian Nie
- College of Life Science, Nanjing Agricultural University, Nanjing, 210095, China
| | - Yihui Cai
- Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China
| | - Wenhai Xie
- School of Life Sciences, Shandong University of Technology, NO. 266 Xincun Road, Zibo, 255000, Shandong, China.
| | - Kang Zou
- Germline Stem Cells and Microenvironment Lab, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China.
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Nakami W, Kipyegon AN, Nguhiu-Mwangi J, Tiambo C, Kemp S. Culture of spermatogonial stem cells and use of surrogate sires as a breeding technology to propagate superior genetics in livestock production: A systematic review. Vet World 2021; 14:3235-3248. [PMID: 35153418 PMCID: PMC8829400 DOI: 10.14202/vetworld.2021.3235-3248] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2021] [Accepted: 11/15/2021] [Indexed: 11/16/2022] Open
Abstract
Background and Aim: Spermatogonial stem cells (SSCs) have previously been isolated from animals’ testes, cultured in vitro, and successfully transplanted into compatible recipients. The SSC unique characteristic has potential for exploitation as a reproductive tool and this can be achieved through SSC intratesticular transplantation to surrogate sires. Here, we aimed at comprehensively analyzing published data on in vitro maintenance of SSC isolated from the testes of livestock animals and their applications. Materials and Methods: The literature search was performed in PubMed, Science Direct, and Google Scholar electronic databases. Data screening was conducted using Rayyan Intelligent Systematic Review software (https://www.rayyan.ai/). Duplicate papers were excluded from the study. Abstracts were read and relevant full papers were reviewed for data extraction. Results: From a total of 4786 full papers screened, data were extracted from 93 relevant papers. Of these, eight papers reported on long-term culture conditions (>1 month) for SSC in different livestock species, 22 papers on short-term cultures (5-15 days), 10 papers on transfection protocols, 18 papers on transplantation using different methods of preparation of livestock recipients, and five papers on donor-derived spermatogenesis. Conclusion: Optimization of SSC long-term culture systems has renewed the possibilities of utilization of these cells in gene-editing technologies to develop transgenic animals. Further, the development of genetically deficient recipients in the endogenous germline layer lends to a future possibility for the utilization of germ cell transplantation in livestock systems.
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Affiliation(s)
- Wilkister Nakami
- Department of Clinical Studies, Faculty of Veterinary Medicine, University of Nairobi, 29053-00625 Nairobi, Kenya; Livestock Genetics Program International Livestock Research Institute, 30709-00100, Nairobi, Kenya; Centre for Tropical Livestock Genetics and Health (CTLGH)-ILRI, 30709-00100, Nairobi, Kenya
| | - Ambrose Ng'eno Kipyegon
- Department of Clinical Studies, Faculty of Veterinary Medicine, University of Nairobi, 29053-00625 Nairobi, Kenya
| | - James Nguhiu-Mwangi
- Department of Clinical Studies, Faculty of Veterinary Medicine, University of Nairobi, 29053-00625 Nairobi, Kenya
| | - Christian Tiambo
- Livestock Genetics Program International Livestock Research Institute, 30709-00100, Nairobi, Kenya; Centre for Tropical Livestock Genetics and Health (CTLGH)-ILRI, 30709-00100, Nairobi, Kenya
| | - Stephen Kemp
- Livestock Genetics Program International Livestock Research Institute, 30709-00100, Nairobi, Kenya; Centre for Tropical Livestock Genetics and Health (CTLGH)-ILRI, 30709-00100, Nairobi, Kenya
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Park JK, Song Y, Kim DW, Cho K, Yeo JM, Lee R, Lim YS, Lee WY, Park HJ. Helix-loop-helix protein ID4 expressed in bovine Sertoli cells. Acta Histochem 2021; 123:151800. [PMID: 34673438 DOI: 10.1016/j.acthis.2021.151800] [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: 07/14/2021] [Revised: 10/11/2021] [Accepted: 10/11/2021] [Indexed: 10/20/2022]
Abstract
Stage- and cell type-specific biomarkers are important for understanding spermatogenesis in mammalian testis. The present study identified several testicular cell marker proteins in 6- and 24-month old bovine testes. In 6-month old bovine testes, spermatogonia and spermatocytes were detected but complete spermatogenesis occurred in 24-month old testes. The diameters of the seminiferous tubules increased significantly in the 24-month old testes compared with those in the 6-month old testes. Protein Gene Product 9.5 (PGP9.5), also known as the undifferentiated spermatogonium marker, and GATA4 (GATA binding protein 4), vimentin, and SOX9 (SRY-Box Transcription Factor 9) were detected in the basement membrane region. Interestingly, ID4 (inhibitor of DNA binding protein 4; previously known as the undifferentiated cell marker) proteins were located in the basement membrane region but their expression patterns were different from those of PGP9.5. Co-immunohistochemistry results showed that ID4 was detected in the Sertoli cells expressing vimentin and SOX9 in 6- and 24-month old bovine testes. This result indicated that ID4 is a putative biomarker of Sertoli cell in the bovine system, which is different from the rodent models. Thus, these results will contribute in understanding the process of spermatogenesis that is different in bovines compared to other species.
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17
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Jung H, Yoon M. Germ Cell Transplantation in Stallion Testes. J Equine Vet Sci 2021; 106:103748. [PMID: 34670702 DOI: 10.1016/j.jevs.2021.103748] [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: 06/19/2021] [Revised: 08/18/2021] [Accepted: 08/18/2021] [Indexed: 10/20/2022]
Abstract
The production of donor-derived sperm using spermatogonial stem cell transplantation has been studied in various animals including mice, rats, goats, boar, dogs, sheep, and monkeys. However, germ cell transplantation has not been applied in stallions. The objective of this study was to produce donor germ cell-derived sperm using germ cell transplantation in stallions. Donor germ cells were transplanted into the parenchyma of 3 recipient stallions that had been treated with busulfan IV injections of 15 mg/kg body weight. For the preparation of donor single germ cells, tissue (20 g) from each testis was subjected to a 2-enzyme digestion procedure. Donor testicular germ cells in minimum essential medium α supplemented with 10% fetal bovine serum were transplanted in the testis of recipient stallions at a rate of 2 ml/min. The semen of each recipient stallion was collected using an artificial vagina at 8 weeks after germ cell transplantation. General sperm evaluation and libido tests were performed. Microsatellite fingerprinting with 17 markers was performed to identify the presence of donor-derived sperm in the semen of the recipient stallions. Sperm were observed to have total and progressive motility exceeding 50% throughout the experimental period. The libido of the recipient stallions was unchanged. No donor-derived sperm could be detected in the semen of the recipient stallions by genotyping. In conclusion, the transplantation of donor germ cells into the testicular parenchyma of stallions was not an optimal transplantation technique for producing donor-derived sperm.
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Affiliation(s)
- Heejun Jung
- Department of Animal Science and Biotechnology, Kyungpook National University, Sangju, Republic of Korea
| | - Minjung Yoon
- Department of Animal Science and Biotechnology, Kyungpook National University, Sangju, Republic of Korea; Department of Horse, Companion and Wild Animal Science, Kyungpook National University, Sangju, Republic of Korea.
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18
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Kawahara T, Kanouchi M, Naniwa Y, Koyago M, Numabe T, Mizutani K, Tanemura K, Hara K. Persistence of undifferentiated spermatogonia in aged Japanese Black cattle. Anim Sci J 2021; 92:e13572. [PMID: 34254411 PMCID: PMC8365669 DOI: 10.1111/asj.13572] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2021] [Revised: 05/09/2021] [Accepted: 05/14/2021] [Indexed: 12/26/2022]
Abstract
Aging is a major risk factor for spermatogenesis deterioration. However, the influence of age on spermatogenic stem cells and their progenitors in bulls is largely unknown. Here, we report age‐related changes in undifferentiated and differentiating spermatogonia in Japanese Black cattle with nearly constant sperm output, by using spermatogonial markers. The numbers of differentiating spermatogonia and more differentiated spermatogenic cells were significantly decreased in aged bovine testes compared with those in young testes. In contrast, the number of undifferentiated spermatogonia was maintained, and their proliferative activity did not differ significantly between young and aged bovine testes. Although severe calcification was only observed to a small extent in aged testes, fewer Sertoli cells and interstitial fibrosis were observed in noncalcified testicular regions. These results suggest that, even in old bulls with nearly constant sperm output, testicular spermatogenic activity declined whereas undifferentiated spermatogonia numbers were maintained. Thus, we propose that undifferentiated spermatogonia may be resistant to age‐related changes in bovine testes. Because undifferentiated spermatogonia may contain stem cell activity, our findings highlight the potential utility of undifferentiated spermatogonia as an agricultural resource to produce spermatozoa beyond the natural bovine lifetime through transplantation and in vitro spermatogenesis in future animal production.
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Affiliation(s)
- Terumichi Kawahara
- Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan
| | - Miki Kanouchi
- Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan
| | - Yousuke Naniwa
- Maebashi Institute of Animal Science, Livestock Improvement Association of Japan, Inc., Maebashi, Gunma, Japan
| | - Masanori Koyago
- Maebashi Institute of Animal Science, Livestock Improvement Association of Japan, Inc., Maebashi, Gunma, Japan
| | - Takashi Numabe
- Miyagi Agricultural Development Corporation, Sendai, Miyagi, Japan
| | - Keishi Mizutani
- Morioka AI Center, Livestock Improvement Association of Japan, Inc., Morioka, Iwate, Japan
| | - Kentaro Tanemura
- Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan
| | - Kenshiro Hara
- Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japan
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Brannigan RE, Fantus RJ, Halpern JA. Fertility preservation in men: a contemporary overview and a look toward emerging technologies. Fertil Steril 2021; 115:1126-1139. [PMID: 33933174 DOI: 10.1016/j.fertnstert.2021.03.026] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2021] [Revised: 03/15/2021] [Accepted: 03/17/2021] [Indexed: 12/18/2022]
Abstract
Cancer and oncologic therapies can have significant adverse effects on male reproductive potential, leaving many men permanently infertile. Fertility preservation has emerged as a key survivorship issue over the past 20 years, and numerous professional societies have published guidelines calling for fertility preservation to become a routine component of oncologic care. Most males with cancer are able to produce a semen specimen for fertility preservation, but numerous other methods of sperm procurement are available for patients who cannot provide a sufficient sample. Despite these options, fertility preservation will remain a challenge for prepubertal boys and men without sperm production. For these patients, experimental and investigational approaches offer the hope that one day they will translate to the clinical arena, offering additional pathways for successful fertility preservation care.
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Affiliation(s)
- Robert E Brannigan
- Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, Illinois.
| | - Richard J Fantus
- Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, Illinois
| | - Joshua A Halpern
- Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, Illinois
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20
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Cai H, Jiang Y, Zhang S, Cai NN, Zhu WQ, Yang R, Tang B, Li ZY, Zhang XM. Culture bovine prospermatogonia with 2i medium. Andrologia 2021; 53:e14056. [PMID: 33763906 DOI: 10.1111/and.14056] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2020] [Revised: 01/28/2021] [Accepted: 03/09/2021] [Indexed: 12/31/2022] Open
Abstract
Germplasm cryopreservation and expansion of gonocytes/prospermatogonia or spermatogonial stem cells (SSCs) are important; however, it's difficult in cattle. Since inhibitors of Mek1/2 and Gsk3β (2i) can enhance pluripotency maintenance, effects of 2i-based medium on the cultivation of bovine prospermatogonia from the cryopreserved tissues were examined. The testicular tissues of newborn bulls were well cryopreserved. High mRNA levels of prospermatogonium/SSC markers (PLZF, GFRα-1) and pluripotency markers (Oct4/Pouf5, Sox2, Nanog) were detected and the PLZF+ /GFRα-1+ prospermatogonia were consistently identified immunohistochemically in the seminiferous cords. Using differential plating and Percoll-based centrifugation, 41.59% prospermatogonia were enriched and they proliferated robustly in 2i medium. The 2i medium boosted mRNA abundances of Pouf5, Sox2, Nanog, GFRα-1, PLZF, anti-apoptosis gene Bcl2, LIF receptor gene LIFR and enhanced PLZF protein expression, but suppressed mRNA expressions of spermatogonial differentiation marker c-kit and pro-apoptotic gene Bax, in the cultured prospermatogonia. It also alleviated H2 O2 -induced apoptosis of the enriched cells and decreased histone H3 lysine (K9) trimethylation (H3K9me3) and its methylase Suv39h1/2 mRNA level in the cultured seminiferous cords. Overall, 2i medium improves the cultivation of bovine prospermatogonia isolated from the cryopreserved testes, by inhibiting Suv39h1/2-mediated H3K9me3 through Mek1/2 and Gsk3β signalling, evidencing successful cryopreservation and expansion of bovine germplasm.
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Affiliation(s)
- Huan Cai
- College of Veterinary Medicine, Jilin University, Changchun, China
| | - Yu Jiang
- College of Veterinary Medicine, Jilin University, Changchun, China
| | - Sheng Zhang
- First Bethune Hospital, Jilin University, Changchun, China
| | - Ning-Ning Cai
- College of Veterinary Medicine, Jilin University, Changchun, China
| | - Wen-Qian Zhu
- College of Veterinary Medicine, Jilin University, Changchun, China
| | - Rui Yang
- College of Veterinary Medicine, Jilin University, Changchun, China
| | - Bo Tang
- College of Veterinary Medicine, Jilin University, Changchun, China
| | - Zi-Yi Li
- First Bethune Hospital, Jilin University, Changchun, China
| | - Xue-Ming Zhang
- College of Veterinary Medicine, Jilin University, Changchun, China
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21
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Spermatogonial Stem Cell Transplantation in Large Animals. Animals (Basel) 2021; 11:ani11040918. [PMID: 33805058 PMCID: PMC8064064 DOI: 10.3390/ani11040918] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2020] [Revised: 03/16/2021] [Accepted: 03/22/2021] [Indexed: 12/22/2022] Open
Abstract
Simple Summary The spermatogonial stem cell (SSC) is the only adult stem cell in males to transmit genetic information to offspring. SSC transplantation (SSCT) is a laboratory technique to regenerate spermatogenesis in recipient males, thus can be used as a novel breeding tool to benefit animal production. Although successful SSCT in rodent models has been established, progress in realizing SSCT in large animals has been limited. Here we discuss what we learned in this area from past experiments and highlight possible directions in developing effective SSCT protocol in large animals. Abstract Spermatogonial stem cell transplantation (SSCT) can restore male fertility through transfer of germline between donor and recipient males. From an agricultural perspective, SSCT could be an important next-generation reproductive and breeding tool in livestock production. Current SSCT approaches in large animals remain inefficient and many technical details need further investigation. This paper reviews the current knowledge on SSCT in large animals, addressing (1) donor spermatogonial stem cell (SSC) preparation, (2) recipient male treatment, and (3) SSC injection, homing, and detection. The major studies showing unequivocal evidence of donor SSC-derived spermatogenesis in large animals (mainly in livestock for breeding purpose) are summarized to discuss the current status of the field and future directions.
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22
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Kazemzadeh S, Rastegar T, Zangi BM, Malekzadeh M, Khanehzad M, Khanlari P, Madadi S, Bashghareh A, Hedayatpour A. Effect of a Freezing Medium Containing Melatonin on Markers of Pre-meiotic and Post-meiotic Spermatogonial Stem Cells (SSCs) After Transplantation in an Azoospermia Mouse Model Due to Testicular Torsion. Reprod Sci 2021; 28:1508-1522. [PMID: 33481217 DOI: 10.1007/s43032-020-00447-5] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2020] [Accepted: 12/27/2020] [Indexed: 01/07/2023]
Abstract
Spermatogonial stem cells (SSCs) are essential to the initiation of spermatogenesis. Cryopreservation, long-term maintenance, and auto-transplantation of SSCs could be a new treatment for infertility. The aim of this study was to add melatonin to the basic freezing medium and to evaluate its effect on the efficiency of the thawed SSCs after transplantation into the testicles of azoospermic mice. SSCs were isolated from newborn NMRI mice, and the cells were enriched to assess morphological features. The thawed SSCs were evaluated for survival, apoptosis, and ROS level before transplantation, and the proliferation (MVH and ID4) and differentiation (c-Kit, SCP3, TP1, TP2, and Prm1) markers of SSCs were examined using immunofluorescence, western blot, and quantitative real-time polymerase chain reaction (PCR) after transplantation. It was found that the survival rate of SSCs after thawing was significantly higher in the melatonin group compared with the cryopreservation group containing basic freezing medium, and the rate of apoptosis and level of ROS production also decreased significantly in the cryopreservation group with melatonin (p < 0.05). The expression of proliferation and differentiation markers after transplantation was significantly higher in the cryopreservation group with melatonin compared to the cryopreservation group (p < 0.05). The results suggest that adding melatonin to the basic freezing medium can effectively protect the SSCs by increasing the viability and reducing the ROS production and apoptosis and improve the transplantation efficiency of SSCs after cryopreservation, which will provide a significant suggestion for fertility protection in the clinic.
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Affiliation(s)
- Shokoofeh Kazemzadeh
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Tayebeh Rastegar
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Bagher Minaei Zangi
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Mehrnoush Malekzadeh
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Maryam Khanehzad
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Parastoo Khanlari
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Soheila Madadi
- Department of Anatomy, School of Medicine, Arak University of Medical Sciences, Arak, Iran
| | - Alieh Bashghareh
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
| | - Azim Hedayatpour
- Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
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McLean ZL, Appleby SJ, Wei J, Snell RG, Oback B. Testes of DAZL null neonatal sheep lack prospermatogonia but maintain normal somatic cell morphology and marker expression. Mol Reprod Dev 2020; 88:3-14. [PMID: 33251684 DOI: 10.1002/mrd.23443] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2020] [Accepted: 11/17/2020] [Indexed: 01/21/2023]
Abstract
Multiplying the germline would increase the number of offspring that can be produced from selected animals, accelerating genetic improvement for livestock breeding. This could be achieved by producing multiple chimaeric animals, each carrying a mix of donor and host germ cells in their gonads. However, such chimaeric germlines would produce offspring from both donor and host genotypes, limiting the rate of genetic improvement. To resolve this problem, we disrupted the RNA-binding protein DAZL and generated germ cell-deficient host animals. Using Cas9-mediated homology-directed repair (HDR), we introduced a DAZL loss-of-function mutation in male ovine fetal fibroblasts. Following manual single cell isolation, 4/48 (8.3%) of donor cell strains were homozygously HDR-edited. Sequence-validated strains were used as nuclear donors for somatic cell cloning to generate three lambs, which died at birth. All DAZL null male neonatal sheep lacked germ cells on histological sections and showed greatly reduced germ cell markers. Somatic cells within their testes were morphologically intact and expressed normal levels of lineage-specific markers, suggesting that the germ cell niche remained intact. This extends the DAZL mutant phenotype beyond mice into agriculturally relevant ruminants, providing a pathway for using absolute germline transmitters in rapid livestock improvement.
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Affiliation(s)
- Zachariah L McLean
- Reproduction, AgResearch, Ruakura Research Centre, Hamilton.,Applied Translational Research Group and Centre for Brain Research, School of Biological Sciences, University of Auckland, Auckland, New Zealand
| | - Sarah J Appleby
- Reproduction, AgResearch, Ruakura Research Centre, Hamilton.,Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand
| | - Jingwei Wei
- Reproduction, AgResearch, Ruakura Research Centre, Hamilton
| | - Russell G Snell
- Applied Translational Research Group and Centre for Brain Research, School of Biological Sciences, University of Auckland, Auckland, New Zealand
| | - Björn Oback
- Reproduction, AgResearch, Ruakura Research Centre, Hamilton.,Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand
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Gul M, Hildorf S, Dong L, Thorup J, Hoffmann ER, Jensen CFS, Sønksen J, Cortes D, Fedder J, Andersen CY, Goossens E. Review of injection techniques for spermatogonial stem cell transplantation. Hum Reprod Update 2020; 26:368-391. [PMID: 32163572 DOI: 10.1093/humupd/dmaa003] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2019] [Revised: 01/07/2020] [Indexed: 12/22/2022] Open
Abstract
BACKGROUND Although the prognosis of childhood cancer survivors has increased dramatically during recent years, chemotherapy and radiation treatments for cancer and other conditions may lead to permanent infertility in prepubertal boys. Recent developments have shown that spermatogonial stem cell (SSC) transplantation may be a hope for restoring fertility in adult survivors of childhood cancers. For this reason, several centres around the world are collecting and cryopreserving testicular tissue or cells anticipating that, in the near future, some patients will return for SSC transplantation. This review summarizes the current knowledge and utility of SSC transplantation techniques. OBJECTIVE AND RATIONALE The aim of this narrative review is to provide an overview of the currently used experimental injection techniques for SSC transplantation in animal and human testes. This is crucial in understanding and determining the role of the different techniques necessary for successful transplantation. SEARCH METHODS A comprehensive review of peer-reviewed publications on this topic was performed using the PubMed and Google Scholar databases. The search was limited to English language work and studies between 1994 (from the first study on SSC transplantation) and April 2019. Key search terms included mouse, rat, boar, ram, dog, sheep, goat, cattle, monkey, human, cadaver, testes, SSC transplantation, injection and technique. OUTCOMES This review provides an extensive clinical overview of the current research in the field of human SSC transplantation. Rete testis injection with ultrasonography guidance currently seems the most promising injection technique thus far; however, the ability to draw clear conclusions is limited due to long ischemia time of cadaver testis, the relatively decreased volume of the testis, the diminishing size of seminiferous tubules, a lack of intratesticular pressure and leakage into the interstitium during the injection on human cadaver testis. Current evidence does not support improved outcomes from multiple infusions through the rete testes. Overall, further optimization is required to increase the efficiency and safety of the infusion method. WIDER IMPLICATIONS Identifying a favourable injection method for SSC transplantation will provide insight into the mechanisms of successful assisted human reproduction. Future research could focus on reducing leakage and establishing the optimal infusion cell concentrations and pressure.
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Affiliation(s)
- Murat Gul
- Laboratory of Reproductive Biology, Copenhagen University Hospital Rigshospitalet, 2100 Copenhagen, Denmark.,Department of Urology, Selcuk University School of Medicine, 42250 Konya, Turkey
| | - Simone Hildorf
- Department of Pediatric Surgery, Copenhagen University Hospital Rigshospitalet, 2100 Copenhagen, Denmark
| | - Lihua Dong
- Laboratory of Reproductive Biology, Copenhagen University Hospital Rigshospitalet, 2100 Copenhagen, Denmark
| | - Jorgen Thorup
- Department of Pediatric Surgery, Copenhagen University Hospital Rigshospitalet, 2100 Copenhagen, Denmark
| | - Eva R Hoffmann
- DNRF Center for Chromosome Stability, Department of Molecular and Cellular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark
| | | | - Jens Sønksen
- Department of Urology, Herlev and Gentofte University Hospital, 2930 Herlev, Denmark.,Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark
| | - Dina Cortes
- Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.,Department of Pediatrics, Copenhagen University Hospital Hvidovre, 2650 Hvidovre, Denmark
| | - Jens Fedder
- Centre of Andrology & Fertility Clinic, Department D, Odense University Hospital, 5000 Odense, Denmark.,Research Unit of Human Reproduction, Institute of Clinical Research, University of Southern Denmark, 5230 Odense, Denmark
| | - Claus Yding Andersen
- Laboratory of Reproductive Biology, Copenhagen University Hospital Rigshospitalet, 2100 Copenhagen, Denmark.,Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark
| | - Ellen Goossens
- Biology of the Testis, Research Laboratory for Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), 1090 Brussels, Belgium
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Liu HC, Xie Y, Deng CH, Liu GH. Stem cell-based therapies for fertility preservation in males: Current status and future prospects. World J Stem Cells 2020; 12:1097-1112. [PMID: 33178394 PMCID: PMC7596443 DOI: 10.4252/wjsc.v12.i10.1097] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/26/2020] [Revised: 05/13/2020] [Accepted: 08/26/2020] [Indexed: 02/06/2023] Open
Abstract
With the decline in male fertility in recent years, strategies for male fertility preservation have received increasing attention. In this study, by reviewing current treatments and recent publications, we describe research progress in and the future directions of stem cell-based therapies for male fertility preservation, focusing on the use of spermatogonial stem cells (SSCs), SSC niches, SSC-based testicular organoids, other stem cell types such as mesenchymal stem cells, and stem cell-derived extracellular vesicles. In conclusion, a more comprehensive understanding of the germ cell microenvironment, stem cell-derived extracellular vesicles, and testicular organoids will play an important role in achieving male fertility preservation.
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Affiliation(s)
- Han-Chao Liu
- Department of Andrology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
| | - Yun Xie
- Department of Andrology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
| | - Chun-Hua Deng
- Department of Andrology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
| | - Gui-Hua Liu
- Reproductive Medicine Research Center, The Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou 510655, Guangdong Province, China
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SHARMA A, KUMARESAN A, NALA N, TIWARI M, ROSHAN M, SINGH MK, PALTA P, SINGLA SK, MANIK RS, CHAUHAN MS. Homologous transplantation of fluorescently labelled enriched buffalo (Bubalus bubalis) spermatogonial stem cells to prepubertal recipients. THE INDIAN JOURNAL OF ANIMAL SCIENCES 2020. [DOI: 10.56093/ijans.v90i5.104608] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/03/2022]
Abstract
Spermatogonial stem cell transplantation provides a unique opportunity to study the biology of spermatogenesis and also offers an alternative approach for genetic modification in large animals. The present study aimed to extend this technique to the water buffalo. Spermatogonial stem cells (SSCs) were isolated from prepubertal buffalo testes (3-6 months of age) using two-step enzymatic digestion method and enriched by differential plating and Percoll density gradient centrifugation. The enriched SSCs expressed numerous spermatogonial transcriptional markers, viz. ID4, THY1, BCL6B, UCHL1, ETV5 and REX1 which confirmed their bonafide SSC identity. Subsequently, the enriched SSCs were labelled with a fluorescent dye PKH26 and transplanted into buffalo calves under ultrasound guidance. The recipient testes were recovered after 7-8 weeks by castration and their fluorescence microscopebased examination exhibited the persistence and localization of the fluorescent donor cells within the recipient seminiferous tubules. Further validation was done by the flow cytometric evaluation of PKH26 labeled donor cells among those isolated by two-step enzymatic digestion of recipient testicular parenchyma. In conclusion, we demonstrated the feasibility of SSC transplantation technique in the water buffalo.
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Deebel NA, Galdon G, Zarandi NP, Stogner-Underwood K, Howards S, Lovato J, Kogan S, Atala A, Lue Y, Sadri-Ardekani H. Age-related presence of spermatogonia in patients with Klinefelter syndrome: a systematic review and meta-analysis. Hum Reprod Update 2020; 26:58-72. [PMID: 31822886 DOI: 10.1093/humupd/dmz038] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2019] [Revised: 09/15/2019] [Accepted: 09/30/2019] [Indexed: 12/12/2022] Open
Abstract
BACKGROUND Klinefelter syndrome (KS) has been defined by sex chromosome aneuploidies (classically 47, XXY) in the male patient. The peripubertal timeframe in KS patients has been associated with the initiation of progressive testicular fibrosis, loss of spermatogonial stem cells (SSC), hypogonadism and impaired fertility. Less than half of KS patients are positive for spermatozoa in the ejaculate or testis via semen analysis or testicular sperm extraction, respectively. However, the chance of finding spermatogonia including a sub-population of SSCs in KS testes has not been well defined. Given the recent demonstration of successful cell culture for mouse and human SSCs, it could be feasible to isolate and propagate SSCs and transplant the cells back to the patient or to differentiate them in vitro to haploid cells. OBJECTIVE AND RATIONALE The main objective of this study was to meta-analyse the currently available data from KS patients to identify the prevalence of KS patients with spermatogonia on testicular biopsy across four age groups (year): fetal/infantile (age ≤ 1), prepubertal (age 1 ≤ x ≤ 10), peripubertal/adolescent (age 10 < x < 18) and adult (age ≥ 18) ages. Additionally, the association of endocrine parameters with presence or absence of spermatogonia was tested to obtain a more powered analysis of whether FSH, LH, testosterone and inhibin B can serve as predictive markers for successful spermatogonia retrieval. SEARCH METHODS A thorough Medline/PubMed search was conducted using the following search terms: 'Klinefelter, germ cells, spermatogenesis and spermatogonia', yielding results from 1 October 1965 to 3 February 2019. Relevant articles were added from the bibliographies of selected articles. Exclusion criteria included non-English language, abstracts only, non-human data and review papers. OUTCOMES A total of 751 papers were identified with independent review returning 36 papers with relevant information for meta-analysis on 386 patients. For the most part, articles were case reports, case-controlled series and cohort studies (level IV-VI evidence). Spermatogonial cells were present in all of the fetal/infantile and 83% of the prepubertal patients' testes, and in 42.7% and 48.5% of the peripubertal and adult groups, respectively were positive for spermatogonia. Additionally, 26 of the 56 (46.4%) peripubertal/adolescent and 37 of the 152 (24.3%) adult patients negative for spermatozoa were positive for spermatogonia (P < 0.05). In peripubertal/adolescent patients, the mean ± SEM level for FSH was 12.88 ± 3.13 IU/L for spermatogonia positive patients and 30.42 ± 4.05 IU/L for spermatogonia negative patients (P = 0.001); the mean ± SEM level LH levels were 4.36 ± 1.31 and 11.43 ± 1.68 IU/L for spermatogonia positive and negative, respectively (P < 0.01); the mean ± SEM level for testosterone levels were 5.04 ± 1.37 and 9.05 ± 0.94 nmol/L (equal to 145 ± 40 and 261 ± 27 and ng/dl) for the spermatogonia positive and negative groups, respectively (P < 0.05), while the difference in means for inhibin B was not statistically significant (P > 0.05). A similar analysis in the adult group showed the FSH levels in spermatogonia positive and negative patients to be 25.77 ± 2.78 and 36.12 ± 2.90 IU/L, respectively (mean ± SEM level, P < 0.05). All other hormone measurements were not statistically significantly different between groups. WIDER IMPLICATIONS While azoospermia is a common finding in the KS patient population, many patients are positive for spermatogonia. Recent advances in SSC in vitro propagation, transplantation and differentiation open new avenues for these patients for fertility preservation. This would offer a new subset of KS patients a chance of biological paternity. Data surrounding the hormonal profiles of KS patients and their relation to fertility should be interpreted with caution as a paucity of adequately powered data exists. Future work is needed to clarify the utility of FSH, LH, testosterone and inhibin B as biomarkers for successful retrieval of spermatogonia.
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Affiliation(s)
- Nicholas A Deebel
- Department of Urology, Wake Forest University School of Medicine, Winston-Salem, NC, USA.,Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA
| | - Guillermo Galdon
- Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA
| | - Nima Pourhabibi Zarandi
- Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA
| | | | - Stuart Howards
- Department of Urology, Wake Forest University School of Medicine, Winston-Salem, NC, USA
| | - James Lovato
- Department of Biostatistics and Data Science, Wake Forest University School of Medicine, Winston-Salem, NC, USA
| | - Stanley Kogan
- Department of Urology, Wake Forest University School of Medicine, Winston-Salem, NC, USA.,Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA
| | - Anthony Atala
- Department of Urology, Wake Forest University School of Medicine, Winston-Salem, NC, USA.,Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA
| | - Yanhe Lue
- Division of Endocrinology, Department of Medicine, Los Angeles Biomedical Research Institute and Harbor-UCLA Medical Center, Torrance, CA, USA
| | - Hooman Sadri-Ardekani
- Department of Urology, Wake Forest University School of Medicine, Winston-Salem, NC, USA.,Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA
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Rasouli MH, Zandi M, Sadeghi AA, Emamjomeh-Kashan N. Spermatogonial stem cell survival in ram lambs following busulfan treatment. Anim Reprod 2020; 17:e20200001. [PMID: 32714457 PMCID: PMC7375864 DOI: 10.1590/1984-3143-ar2020-0001] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
To clarify the effect of busulfan on the depletion of spermatogonial stem cells (SSCs) from shal rams testis, in the first experiment, lambs were treated by intraperitoneal injection with 4 mg/kg busulfan. In the second experiment, different concentrations of busulfan (1, 2 and 4 mg/kg) were injected directly into both sides of the left testis. The testes of 8 lambs were collected by standard castration procedure for histological analysis five weeks after the treatments and the left testis of remaining lambs were collected after eight weeks and a two-time enzymatic digestion process was used to isolate SSCs. The results showed that all rams that had received intraperitoneal injections of busulfan died. But by testicular injecting of same dose of the drug, 40% of the animals died. The testicular injection of rams with 1, 2 and 4 mg/kg of busulfan resulted in a dose dependent decrease in testis size and also spermatocytes population after 5 weeks of treatments. From the results of colony formation 8 weeks after treatment with busulfan, it can be concluded that only in 1 and 2 mg/kg of busulfan, recovery of endogenous germ cells was performed. In conclusion, the results demonstrated that intra-testicular injections of busulfan (2 mg/kg) reduced spermatocytes population in ram testis within 5 weeks of treatments, and this effect was reversible within 8 weeks of injection. However, it was not recommended to inject 4 mg/kg busulfan into the peritoneal cavity or testis of lambs based on its side effects.
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Affiliation(s)
- Mohammad Hadi Rasouli
- Department of Animal Science, Faculty of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University, Tehran, Iran
| | - Mohammad Zandi
- Department of Agriculture, Iranian Research Organization for Science and Technology, Tehran, Iran
| | - Ali Asghar Sadeghi
- Department of Animal Science, Faculty of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University, Tehran, Iran
| | - Naser Emamjomeh-Kashan
- Department of Animal Science, Faculty of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University, Tehran, Iran
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Live-cell imaging and ultrastructural analysis reveal remarkable features of cultured porcine gonocytes. Cell Tissue Res 2020; 381:361-377. [PMID: 32388763 DOI: 10.1007/s00441-020-03218-5] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2019] [Accepted: 04/13/2020] [Indexed: 12/31/2022]
Abstract
Gonocytes in the neonatal testis have male germline stem cell potential. The objective of the present study was to examine the behavior and ultrastructure of gonocytes in culture. Neonatal porcine testis cells were cultured for 4 weeks and underwent live-cell imaging to explore real-time interactions among cultured cells. This included imaging every 1 h from day 0 to day 3, every 2 h from day 4 to day 7, and every 1 h for 24 h at days 14, 21, and 28. Samples also underwent scanning electron microscopy, transmission electron microscopy, morphometric evaluations, immunofluorescence, and RT-PCR. Live-cell imaging revealed an active amoeboid-like movement of gonocytes, assisted by the formation of extensive cytoplasmic projections, which, using scanning electron microscopy, were categorized into spike-like filopodia, leaf-like lamellipodia, membrane ruffles, and cytoplasmic blebs. In the first week of culture, gonocytes formed loose attachments on top of a somatic cell monolayer and, in week 2, formed grape-like clusters, which, over time, grew in cell number. Starting at week 3 of culture, some of the gonocyte clusters transformed into large multinucleated embryoid body-like colonies (EBLCs) that expressed both gonocyte- and pluripotent-specific markers. The number and diameter of individual gonocytes, the number and density of organelles within gonocytes, as well as the number and diameter of the EBLCs increased over time (P < 0.05). In conclusion, cultured porcine gonocytes displayed extensive migratory behavior facilitated by their various cytoplasmic projections, propagated, and transformed into EBLCs that increased in size and complexity over time.
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Abstract
Infertility caused by chemotherapy or radiation treatments negatively impacts patient-survivor quality of life. The only fertility preservation option available to prepubertal boys who are not making sperm is cryopreservation of testicular tissues that contain spermatogonial stem cells (SSCs) with potential to produce sperm and/or restore fertility. SSC transplantation to regenerate spermatogenesis in infertile adult survivors of childhood cancers is a mature technology. However, the number of SSCs obtained in a biopsy of a prepubertal testis may be small. Therefore, methods to expand SSC numbers in culture before transplantation are needed. Here we review progress with human SSC culture.
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Affiliation(s)
- Sherin David
- Department of Obstetrics, Gynecology and Reproductive Sciences, Molecular Genetics and Developmental Biology Graduate Program, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, 204 Craft Avenue, Pittsburgh, PA 15213, USA
| | - Kyle E Orwig
- Department of Obstetrics, Gynecology and Reproductive Sciences, Molecular Genetics and Developmental Biology Graduate Program, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, 204 Craft Avenue, Pittsburgh, PA 15213, USA.
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31
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The study and manipulation of spermatogonial stem cells using animal models. Cell Tissue Res 2020; 380:393-414. [PMID: 32337615 DOI: 10.1007/s00441-020-03212-x] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2020] [Accepted: 03/30/2020] [Indexed: 02/08/2023]
Abstract
Spermatogonial stem cells (SSCs) are a rare group of cells in the testis that undergo self-renewal and complex sequences of differentiation to initiate and sustain spermatogenesis, to ensure the continuity of sperm production throughout adulthood. The difficulty of unequivocal identification of SSCs and complexity of replicating their differentiation properties in vitro have prompted the introduction of novel in vivo models such as germ cell transplantation (GCT), testis tissue xenografting (TTX), and testis cell aggregate implantation (TCAI). Owing to these unique animal models, our ability to study and manipulate SSCs has dramatically increased, which complements the availability of other advanced assisted reproductive technologies and various genome editing tools. These animal models can advance our knowledge of SSCs, testis tissue morphogenesis and development, germ-somatic cell interactions, and mechanisms that control spermatogenesis. Equally important, these animal models can have a wide range of experimental and potential clinical applications in fertility preservation of prepubertal cancer patients, and genetic conservation of endangered species. Moreover, these models allow experimentations that are otherwise difficult or impossible to be performed directly in the target species. Examples include proof-of-principle manipulation of germ cells for correction of genetic disorders or investigation of potential toxicants or new drugs on human testis formation or function. The primary focus of this review is to highlight the importance, methodology, current and potential future applications, as well as limitations of using these novel animal models in the study and manipulation of male germline stem cells.
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Three-dimensional decellularized amnion membrane scaffold promotes the efficiency of male germ cells generation from human induced pluripotent stem cells. Exp Cell Res 2019; 384:111544. [DOI: 10.1016/j.yexcr.2019.111544] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2019] [Revised: 07/21/2019] [Accepted: 08/01/2019] [Indexed: 12/30/2022]
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Dong L, Gul M, Hildorf S, Pors SE, Kristensen SG, Hoffmann ER, Cortes D, Thorup J, Andersen CY. Xeno-Free Propagation of Spermatogonial Stem Cells from Infant Boys. Int J Mol Sci 2019; 20:ijms20215390. [PMID: 31671863 PMCID: PMC6862004 DOI: 10.3390/ijms20215390] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2019] [Revised: 10/03/2019] [Accepted: 10/28/2019] [Indexed: 12/13/2022] Open
Abstract
Spermatogonial stem cell (SSC) transplantation therapy is a promising strategy to renew spermatogenesis for prepubertal boys whose fertility is compromised. However, propagation of SSCs is required due to a limited number of SSCs in cryopreserved testicular tissue. This propagation must be done under xeno-free conditions for clinical application. SSCs were propagated from infant testicular tissue (7 mg and 10 mg) from two boys under xeno-free conditions using human platelet lysate and nutrient source. We verified SSC-like cell clusters (SSCLCs) by quantitative real-time polymerase chain reaction (PCR) and immune-reaction assay using the SSC markers undifferentiated embryonic cell transcription factor 1 (UTF1), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), GDNF receptor alpha-1 (GFRα-1) Fα and promyelocytic leukaemia zinc finger protein (PLZF). The functionality of the propagated SSCs was investigated by pre-labelling using green fluorescent Cell Linker PKH67 and xeno-transplantation of the SSCLCs into busulfan-treated, therefore sterile, immunodeficient mice. SSC-like cell clusters (SSCLCs) appeared after 2 weeks in primary passage. The SSCLCs were SSC-like as the UTF1, UCHL1, GFRα1 and PLZF were all positive. After 2.5 months’ culture period, a total of 13 million cells from one sample were harvested for xenotransplantation. Labelled human propagated SSCs were identified and verified in mouse seminiferous tubules at 3–6 weeks, confirming that the transplanted cells contain SSCLCs. The present xeno-free clinical culture protocol allows propagation of SSCs from infant boys.
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Affiliation(s)
- Lihua Dong
- Laboratory of Reproductive Biology, Rigshospitalet, University Hospital of Copenhagen, 2100 Copenhagen, Denmark.
| | - Murat Gul
- Laboratory of Reproductive Biology, Rigshospitalet, University Hospital of Copenhagen, 2100 Copenhagen, Denmark.
- Department of Urology, Aksaray University School of Medicine, Aksaray 68100, Turkey.
| | - Simone Hildorf
- Department of Pediatric Surgery, Rigshospitalet, Copenhagen University Hospital, 2100 Copenhagen, Denmark.
| | - Susanne Elisabeth Pors
- Laboratory of Reproductive Biology, Rigshospitalet, University Hospital of Copenhagen, 2100 Copenhagen, Denmark.
| | - Stine Gry Kristensen
- Laboratory of Reproductive Biology, Rigshospitalet, University Hospital of Copenhagen, 2100 Copenhagen, Denmark.
| | - Eva R Hoffmann
- Center for Chromosome Stability, Institute of Molecular and Cellular Medicine, 2200 Copenhagen, Denmark.
- Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
| | - Dina Cortes
- Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
- Department of Pediatrics, Hvidovre, Copenhagen University Hospital, 2650 Copenhagen, Denmark.
| | - Jorgen Thorup
- Department of Pediatric Surgery, Rigshospitalet, Copenhagen University Hospital, 2100 Copenhagen, Denmark.
- Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
| | - Claus Yding Andersen
- Laboratory of Reproductive Biology, Rigshospitalet, University Hospital of Copenhagen, 2100 Copenhagen, Denmark.
- Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.
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Kadam P, Ntemou E, Onofre J, Van Saen D, Goossens E. Does co-transplantation of mesenchymal and spermatogonial stem cells improve reproductive efficiency and safety in mice? Stem Cell Res Ther 2019; 10:310. [PMID: 31640769 PMCID: PMC6805426 DOI: 10.1186/s13287-019-1420-9] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2019] [Revised: 09/03/2019] [Accepted: 09/16/2019] [Indexed: 12/12/2022] Open
Abstract
Background Spermatogonial stem cell transplantation (SSCT) is a promising therapy in restoring the fertility of childhood cancer survivors. However, the low efficiency of SSCT is a significant concern. SSCT could be improved by co-transplanting transforming growth factor beta 1 (TGFβ1)-induced mesenchymal stem cells (MSCs). In this study, we investigated the reproductive efficiency and safety of co-transplanting spermatogonial stem cells (SSCs) and TGFβ1-induced MSCs. Methods A mouse model for long-term infertility was used to transplant SSCs (SSCT, n = 10) and a combination of SSCs and TGFβ1-treated MSCs (MSi-SSCT, n = 10). Both transplanted groups and a fertile control group (n = 7) were allowed to mate naturally to check the reproductive efficiency after transplantation. Furthermore, the testes from transplanted males and donor-derived male offspring were analyzed for the epigenetic markers DNA methyltransferase 3A (DNMT3A) and histone 4 lysine 5 acetylation (H4K5ac). Results The overall tubular fertility index (TFI) after SSCT (76 ± 12) was similar to that after MSi-SSCT (73 ± 14). However, the donor-derived TFI after MSi-SSCT (26 ± 14) was higher compared to the one after SSCT (9 ± 5; P = 0.002), even after injecting half of the number of SSCs in MSi-SSCT. The litter sizes after SSCT (3.7 ± 3.7) and MSi-SSCT (3.7 ± 3.6) were similar but differed significantly with the control group (7.6 ± 1.0; P < 0.001). The number of GFP+ offspring per litter obtained after SSCT (1.6 ± 0.5) and MSi-SSCT (2.0 ± 1.0) was also similar. The expression of DNMT3A and H4K5ac in germ cells of transplanted males was found to be significantly reduced compared to the control group. However, in donor-derived offspring, DNMT3A and H4K5ac followed the normal pattern. Conclusion Co-transplanting SSCs and TGFβ1-treated MSCs results in reproductive efficiency as good as SSCT, even after transplanting half the number of SSCs. Although transplanted males showed lower expression of DNMT3A and H4K5ac in donor-derived germ cells, the expression was restored to normal levels in germ cells of donor-derived offspring. This procedure could become an efficient method to restore fertility in a clinical setup, but more studies are needed to ensure safety in the long term.
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Affiliation(s)
- Prashant Kadam
- Biology of the Testis (BITE) Laboratory, Department of Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium
| | - Elissavet Ntemou
- Biology of the Testis (BITE) Laboratory, Department of Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium
| | - Jaime Onofre
- Biology of the Testis (BITE) Laboratory, Department of Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium
| | - Dorien Van Saen
- Biology of the Testis (BITE) Laboratory, Department of Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium
| | - Ellen Goossens
- Biology of the Testis (BITE) Laboratory, Department of Reproduction, Genetics and Regenerative Medicine, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium.
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Kubota H, Brinster RL. Spermatogonial stem cells. Biol Reprod 2019; 99:52-74. [PMID: 29617903 DOI: 10.1093/biolre/ioy077] [Citation(s) in RCA: 146] [Impact Index Per Article: 24.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2017] [Accepted: 03/29/2018] [Indexed: 12/19/2022] Open
Abstract
Spermatogonial stem cells (SSCs) are the most primitive spermatogonia in the testis and have an essential role to maintain highly productive spermatogenesis by self-renewal and continuous generation of daughter spermatogonia that differentiate into spermatozoa, transmitting genetic information to the next generation. Since the 1950s, many experimental methods, including histology, immunostaining, whole-mount analyses, and pulse-chase labeling, had been used in attempts to identify SSCs, but without success. In 1994, a spermatogonial transplantation method was reported that established a quantitative functional assay to identify SSCs by evaluating their ability to both self-renew and differentiate to spermatozoa. The system was originally developed using mice and subsequently extended to nonrodents, including domestic animals and humans. Availability of the functional assay for SSCs has made it possible to develop culture systems for their ex vivo expansion, which dramatically advanced germ cell biology and allowed medical and agricultural applications. In coming years, SSCs will be increasingly used to understand their regulation, as well as in germline modification, including gene correction, enhancement of male fertility, and conversion of somatic cells to biologically competent male germline cells.
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Affiliation(s)
- Hiroshi Kubota
- Laboratory of Cell and Molecular Biology, Department of Animal Science, School of Veterinary Medicine, Kitasato University, Towada, Aomori, Japan
| | - Ralph L Brinster
- Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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Giassetti MI, Ciccarelli M, Oatley JM. Spermatogonial Stem Cell Transplantation: Insights and Outlook for Domestic Animals. Annu Rev Anim Biosci 2019; 7:385-401. [PMID: 30762440 DOI: 10.1146/annurev-animal-020518-115239] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The demand for food will increase to an unprecedented level over the next 30 years owing to human population expansion, thus necessitating an evolution that improves the efficiency of livestock production. Genetic gain to improve production traits of domestic animal populations is most effectively achieved via selective use of gametes from animals deemed to be elite, and this principle has been the basis of selective breeding strategies employed by humans for thousands of years. In modern-day animal agriculture, artificial insemination (AI) has been the staple of selective breeding programs, but it has inherent limitations for applications in beef cattle and pig production systems. In this review, we discuss the potential and current state of development for a concept termed Surrogate Sires as a next-generation breeding tool in livestock production. The scheme capitalizes on the capacity of spermatogonial stem cells to regenerate sperm production after isolation from donor testicular tissue and transfer into the testes of a recipient male that lacks endogenous germline, thereby allowing the surrogate male to produce offspring with the donor haplotype via natural mating. This concept provides an effective selective breeding tool to achieve genetic gain that is conducive for livestock production systems in which AI is difficult to implement.
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Affiliation(s)
- Mariana I Giassetti
- School of Molecular Biosciences, Center for Reproductive Biology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164, USA;
| | - Michela Ciccarelli
- School of Molecular Biosciences, Center for Reproductive Biology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164, USA;
| | - Jon M Oatley
- School of Molecular Biosciences, Center for Reproductive Biology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164, USA;
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Ma W, Wang J, Gao W, Jia H. The Safe Recipient of SSC Transplantation Prepared by Heat Shock With Busulfan Treatment in Mice. Cell Transplant 2018; 27:1451-1458. [PMID: 30187774 PMCID: PMC6180719 DOI: 10.1177/0963689718794126] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Safety is the chief consideration in recipient preparation of spermatogonial stem cell (SSC) transplantation in mammals, especially humans. In this study, we compared the safety of the SSC transplantation recipients that were prepared both by testes heat shock plus testes busulfan injection (heat shock+busulfan(t)) and by busulfan intraperitoneal injection (busulfan i.p.) only. Our results showed that heat shock+busulfan(t) treatment significantly (p < 0.05) reduced mortality in mice and did not produce bone marrow cell toxicity. Furthermore, heat shock+busulfan(t) treatment directly damaged SSCs and exhausted almost all of the germ cells in the testis; the exhaustion of these cells is considered a key factor in the successful preparation of the recipients. Therefore, we used heat shock+busulfan(t) treatment to prepare recipients of SSC transplantation. Two months after SSC transplantation, the number and length of donor SSC-derived colonies in the testis of recipient in heat shock+busulfan(t) group was closed to that in busulfan i.p. group. Therefore, compared with busulfan i.p. treatment, heat shock+busulfan(t) treatment improved the safety of recipient preparation without reducing the efficiency of SSC transplantation. Two GFP-positive offspring were produced from 1 of the 20 recipients that had mated with female mice 72 days after SSC transplantation. In conclusion, heat shock with busulfan treatment is a safe method to prepare the recipient of SSC transplantation in mice.
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Affiliation(s)
- Wenzhi Ma
- 1 Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education,Key Laboratory of Reproduction and Genetic of Ningxia Hui Autonomous Region, and Department of Anatomy, Histology and Embryology, School of Basic Medical Science, Ningxia Medical University, Yinchuan, China.,Wenzhi Ma and Jia Wang contributed equally to this work
| | - Jia Wang
- 1 Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education,Key Laboratory of Reproduction and Genetic of Ningxia Hui Autonomous Region, and Department of Anatomy, Histology and Embryology, School of Basic Medical Science, Ningxia Medical University, Yinchuan, China.,Wenzhi Ma and Jia Wang contributed equally to this work
| | - Weijun Gao
- 1 Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education,Key Laboratory of Reproduction and Genetic of Ningxia Hui Autonomous Region, and Department of Anatomy, Histology and Embryology, School of Basic Medical Science, Ningxia Medical University, Yinchuan, China
| | - Hua Jia
- 1 Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education,Key Laboratory of Reproduction and Genetic of Ningxia Hui Autonomous Region, and Department of Anatomy, Histology and Embryology, School of Basic Medical Science, Ningxia Medical University, Yinchuan, China
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Olejnik J, Suchowerska N, Herrid M, Jackson M, Hinch G, Hill J. Spermatogonia survival in young ram lambs following irradiation, Busulfan or thermal treatment. Small Rumin Res 2018. [DOI: 10.1016/j.smallrumres.2018.07.017] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
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Olejnik J, Suchowerska N, Herrid M, Jackson A, Jackson M, Andronicos NM, Hinch GN, Hill JR. Sensitivity of spermatogonia to irradiation varies with age in pre-pubertal ram lambs. Anim Reprod Sci 2018; 193:58-67. [PMID: 29636209 DOI: 10.1016/j.anireprosci.2018.03.037] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2018] [Revised: 03/23/2018] [Accepted: 03/29/2018] [Indexed: 01/15/2023]
Abstract
Although germ cells from donor rams transplanted into irradiated recipient testes have produced donor derived offspring, efficiency is low. Further optimization of recipient irradiation protocols will add precision to the depletion of recipient spermatogonia prior to germ cell transplant. Three irradiation doses (9,12,15 Gy) were administered to ram lambs aged 14 weeks (Group 1) and 20 weeks (Group 2), then testicular biopsies were collected 1, 2 and 3 months after irradiation. At 1 month after irradiation of Group 1, only the largest dose (15 Gy) reduced spermatogonia numbers below 10% of non-irradiated controls, whereas in Group 2 lambs, each irradiation dose reduced spermatogonia below 10% of controls. In both Groups, fewer differentiated germ cells were present in seminiferous tubules compared to controls. At 2 months after irradiation, spermatogonia numbers in both Groups increased more than sixfold to be similar to controls, whereas fewer differentiated germ cells were present in the tubules of both Groups. At 3 months in Group 1, each irradiation dose reduced spermatogonia numbers to <30% of controls and fewer tubules contained differentiated germ cells. Lesser expression of spermatogonial genes, VASA and UCHL-1, was observed in the 15 Gy group. In Group 2, only 12 Gy treated tubules contained fewer spermatogonia. Knowledge of these subtle differences between age groups in the effect of irradiation doses on spermatogonia or differentiated germ cell numbers and the duration of recovery of spermatogonia numbers after irradiation will aid the timing of germ cell transplants into prepubertal recipient lambs.
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Affiliation(s)
- J Olejnik
- CSIRO Food Futures National Research Flagship, Australia; CSIRO Animal, Food and Health Sciences, F. D. McMaster Laboratory, Armidale, NSW, 2350 Australia; University of New England, Armidale, NSW, 2350, Australia
| | - N Suchowerska
- School of Physics, University of Sydney, NSW, Australia; Faculty of Medicine, University of Sydney, Sydney, NSW, Australia
| | - M Herrid
- CSIRO Food Futures National Research Flagship, Australia
| | - A Jackson
- CSIRO Food Futures National Research Flagship, Australia
| | - M Jackson
- Faculty of Medicine, University of Sydney, Sydney, NSW, Australia
| | - N M Andronicos
- CSIRO Animal, Food and Health Sciences, F. D. McMaster Laboratory, Armidale, NSW, 2350 Australia; University of New England, Armidale, NSW, 2350, Australia
| | - G N Hinch
- University of New England, Armidale, NSW, 2350, Australia
| | - J R Hill
- CSIRO Food Futures National Research Flagship, Australia; University of Queensland, School of Veterinary Science, Gatton, QLD 4343, Queensland, Australia.
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Sahare MG, Suyatno, Imai H. Recent advances of in vitro culture systems for spermatogonial stem cells in mammals. Reprod Med Biol 2018; 17:134-142. [PMID: 29692670 PMCID: PMC5902468 DOI: 10.1002/rmb2.12087] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2017] [Accepted: 12/23/2017] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND Spermatogonial stem cells (SSCs) in the mammalian testis are unipotent stem cells for spermatozoa. They show unique cell characteristics as stem cells and germ cells after being isolated from the testis and cultured in vitro. This review introduces recent progress in the development of culture systems for the establishment of SSC lines in mammalian species, including humans. METHODS Based on the published reports, the isolation and purification of SSCs, identification and characteristics of SSCs, and culture system for mice, humans, and domestic animals have been summarized. RESULTS In mice, cell lines from SSCs are established and can be reprogrammed to show pluripotent stem cell potency that is similar to embryonic stem cells. However, it is difficult to establish cell lines for animals other than mice because of the dearth of understanding about species-specific requirements for growth factors and mechanisms supporting the self-renewal of cultured SSCs. Among the factors that are associated with the development of culture systems, the enrichment of SSCs that are isolated from the testis and the combination of growth factors are essential. CONCLUSION Providing an example of SSC culture in cattle, a rational consideration was made about how it can be possible to establish cell lines from neonatal and immature testes.
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Affiliation(s)
- Mahesh G Sahare
- National Facility for Gene Function in Health and Disease Indian Institute of Science, Education and Research Pune India
| | - Suyatno
- Indonesian Agency for Agricultural Research and Development Jakarta Indonesia
- Laboratory of Reproductive Biology Graduate School of Agriculture Kyoto University Kyoto Japan
| | - Hiroshi Imai
- Laboratory of Reproductive Biology Graduate School of Agriculture Kyoto University Kyoto Japan
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Tang L, Bondareva A, González R, Rodriguez-Sosa JR, Carlson DF, Webster D, Fahrenkrug S, Dobrinski I. TALEN-mediated gene targeting in porcine spermatogonia. Mol Reprod Dev 2018; 85:250-261. [PMID: 29393557 DOI: 10.1002/mrd.22961] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2017] [Revised: 01/22/2018] [Accepted: 01/25/2018] [Indexed: 01/05/2023]
Abstract
Spermatogonia represent a diploid germ cell population that includes spermatogonial stem cells. In this report, we describe new methods for isolation of highly enriched porcine spermatogonia based on light scatter properties, and for targeted mutagenesis in porcine spermatogonia using nucleofection and TALENs. We optimized a nucleofection protocol to deliver TALENs specifically targeting the DMD locus in porcine spermatogonia. We also validated specific sorting of porcine spermatogonia based on light scatter properties. We were able to obtain a highly enriched germ cell population with over 90% of cells being UCH-L1 positive undifferentiated spermatogonia. After gene targeting in porcine spermatogonia, indel (insertion or deletion) mutations as a result of non-homologous end joining (NHEJ) were detected in up to 18% of transfected cells. Our report demonstrates for the first time an approach to obtain a live cell population highly enriched in undifferentiated spermatogonia from immature porcine testes, and that gene targeting can be achieved in porcine spermatogonia which will enable germ line modification.
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Affiliation(s)
- Lin Tang
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada
| | - Alla Bondareva
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada
| | - Raquel González
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada
| | - Jose R Rodriguez-Sosa
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada
| | | | | | | | - Ina Dobrinski
- Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada
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Awang-Junaidi AH, Honaramooz A. Optimization of culture conditions for short-term maintenance, proliferation, and colony formation of porcine gonocytes. J Anim Sci Biotechnol 2018; 9:8. [PMID: 29372053 PMCID: PMC5771198 DOI: 10.1186/s40104-017-0222-0] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2017] [Accepted: 12/19/2017] [Indexed: 01/14/2023] Open
Abstract
Background Gonocytes give rise to spermatogonial stem cells, and thereby play an essential role in establishing spermatogenesis. Optimized culture conditions for gonocytes provide an opportunity for their study and in vitro manipulation for potential application in reproductive technologies. Using six experiments in a step-wise design, we examined the effects of several culture conditions on the maintenance, proliferation, and colony formation of porcine gonocytes. Testis cells from neonatal piglets were cultured for 7 d in DMEM supplemented with 10% fetal bovine serum. The examined culture conditions included using different cell seeding densities, gonocyte proportions, incubation temperatures, sampling strategies, and medium changing regimens. Results Confluency of cells was optimal (>90% by ~6 d) when 3.0 × 104 testis cells/cm2 containing ~40% gonocytes were used. Incubating the cells at 35 °C or 37 °C resulted in similar cell number and viability at confluency, but incubation at 35 °C resulted in a delayed confluency. In the first 2 d of culture, gonocytes remained mostly floating in the medium and gradually settled over the next 5 d. Consequently, not changing the medium for 7 d (as opposed to changing it every 2 d) led to a significant increase in the number of gonocyte colonies by reducing the loss of “floating gonocytes”. Conclusion We found that gonocytes require the presence of a critical minimum number of somatic cells for settlement, and can proliferate and form growing colonies even in a basic medium. Large numbers of viable gonocytes remain floating in the medium for several days. The optimized culture conditions in the present study included seeding with 3.0 × 104 testis cells/cm2 containing ~40% gonocytes, incubating at 37 °C, and without changing the medium in the first week, which can result in improved colony formation of porcine gonocytes.
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Affiliation(s)
- Awang Hazmi Awang-Junaidi
- Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK S7N 5B4 Canada
| | - Ali Honaramooz
- Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK S7N 5B4 Canada
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Mulder CL, Catsburg LAE, Zheng Y, de Winter-Korver CM, van Daalen SKM, van Wely M, Pals S, Repping S, van Pelt AMM. Long-term health in recipients of transplanted in vitro propagated spermatogonial stem cells. Hum Reprod 2018; 33:81-90. [PMID: 29165614 PMCID: PMC5850721 DOI: 10.1093/humrep/dex348] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2017] [Revised: 10/26/2017] [Accepted: 11/01/2017] [Indexed: 12/25/2022] Open
Abstract
STUDY QUESTION Is testicular transplantation of in vitro propagated spermatogonial stem cells associated with increased cancer incidence and decreased survival rates in recipient mice? SUMMARY ANSWER Cancer incidence was not increased and long-term survival rate was not altered after transplantation of in vitro propagated murine spermatogonial stem cells (SSCs) in busulfan-treated recipients as compared to non-transplanted busulfan-treated controls. WHAT IS KNOWN ALREADY Spermatogonial stem cell autotransplantation (SSCT) is a promising experimental reproductive technique currently under development to restore fertility in male childhood cancer survivors. Most preclinical studies have focused on the proof-of-principle of the functionality and efficiency of this technique. The long-term health of recipients of SSCT has not been studied systematically. STUDY DESIGN, SIZE, DURATION This study was designed as a murine equivalent of a clinical prospective study design. Long-term follow-up was performed for mice who received a busulfan treatment followed by either an intratesticular transplantation of in vitro propagated enhanced green fluorescent protein (eGFP) positive SSCs (cases, n = 34) or no transplantation (control, n = 37). Using a power calculation, we estimated that 36 animals per group would be sufficient to provide an 80% power and with a 5% level of significance to demonstrate a 25% increase in cancer incidence in the transplanted group. The survival rate and cancer incidence was investigated until the age of 18 months. PARTICIPANTS/MATERIALS, SETTING, METHODS Neonatal male B6D2F1 actin-eGFP transgenic mouse testis were used to initiate eGFP positive germline stem (GS) cell culture, which harbor SSCs. Six-week old male C57BL/6 J mice received a single dose busulfan treatment to deplete the testis from endogenous spermatogenesis. Half of these mice received a testicular transplantation of cultured eGFP positive GS cells, while the remainder of mice served as a control group. Mice were followed up until the age of 18 months (497-517 days post-busulfan) or sacrificed earlier due to severe discomfort or illness. Survival data were collected. To evaluate cancer incidence a necropsy was performed and tissues were collected. eGFP signal in transplanted testis and in benign and malignant lesions was assessed by standard PCR. MAIN RESULTS AND THE ROLE OF CHANCE We found 9% (95% CI: 2-25%) malignancies in the transplanted busulfan-treated animals compared to 26% (95% CI: 14-45%) in the busulfan-treated control group, indicating no statistically significant difference in incidence of malignant lesions in transplanted and control mice (OR: 0.3, 95% CI: 0.1-1.1). Furthermore, none of the malignancies that arose in the transplanted animals contained eGFP signal, suggesting that they are not derived from the in vitro propagated transplanted SSCs. Mean survival time after busulfan treatment was found to be equal, with a mean survival time for transplanted animals of 478 days and 437 days for control animals (P = 0.076). LARGE SCALE DATA NA. LIMITATIONS, REASONS FOR CAUTION Although we attempted to mimic the future clinical application of SSCT in humans as close as possible, the mouse model that we used might not reflect all aspects of the future clinical setting. WIDER IMPLICATIONS OF THE FINDINGS The absence of an increase in cancer incidence and a decrease in survival of mice that received a testicular transplantation of in vitro propagated SSCs is reassuring in light of the future clinical application of SSCT in humans. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by KiKa (Kika86) and ZonMw (TAS 116003002). The authors report no financial or other conflict of interest relevant to the subject of this article.
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Affiliation(s)
- Callista L Mulder
- Center for Reproductive Medicine, Amsterdam Research Institute Reproduction and Development, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
| | - Lisa A E Catsburg
- Center for Reproductive Medicine, Amsterdam Research Institute Reproduction and Development, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
| | - Yi Zheng
- Center for Reproductive Medicine, Amsterdam Research Institute Reproduction and Development, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
| | - Cindy M de Winter-Korver
- Center for Reproductive Medicine, Amsterdam Research Institute Reproduction and Development, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
| | - Saskia K M van Daalen
- Center for Reproductive Medicine, Amsterdam Research Institute Reproduction and Development, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
| | - Madelon van Wely
- Center for Reproductive Medicine, Amsterdam Research Institute Reproduction and Development, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
| | - Steven Pals
- Department of Pathology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
| | - Sjoerd Repping
- Center for Reproductive Medicine, Amsterdam Research Institute Reproduction and Development, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
| | - Ans M M van Pelt
- Center for Reproductive Medicine, Amsterdam Research Institute Reproduction and Development, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
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Thorup J, Clasen-Linde E, Dong L, Hildorf S, Kristensen SG, Andersen CY, Cortes D. Selecting Infants With Cryptorchidism and High Risk of Infertility for Optional Adjuvant Hormonal Therapy and Cryopreservation of Germ Cells: Experience From a Pilot Study. Front Endocrinol (Lausanne) 2018; 9:299. [PMID: 29922233 PMCID: PMC5996032 DOI: 10.3389/fendo.2018.00299] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/13/2018] [Accepted: 05/18/2018] [Indexed: 01/15/2023] Open
Abstract
INTRODUCTION Orchiopexy for congenital cryptorchid testes is recommended between ½ and 1 year of age to preserve testicular germ cell maturation. Early operation is not enough to preserve fertility in 22 and 36% of cases. Aim of this study was to set up a protocol for optional adjuvant hormonal therapy after orchiopexy and thereafter cryopreservation of testicular biopsies from infants with bilateral cryptorchidism and high infertility risk. MATERIALS AND METHODS We included 17 boys with bilateral cryptorchidism, normal FSH, and impaired germ cell number per tubular transverse section (G/T) in testicular biopsies at orchiopexy, 7 months to 3½ years old. Postoperatively, optional adjuvant LHRH (kryptocur®) 0.2 mg/0.1 mL 2× every second day in 16 weeks were offered. Ten boys were applicable for age matching according to parent's choice of treatment regime and G/T. Five of them had kryptocur®, and five were controls. Repeat bilateral testicular biopsy evaluation and cryopreservation were offered to all boys 12 months after primary orchiopexy. For cryopreservation, tissue pieces were incubated with a cryoprotectant with a slow program freezing. RESULTS Two out of five kryptorcur®-treated boys normalized both the average G/T and the number of adult dark spermatogonia (Ad-S). Another kryptocur®-treated boy with initial low G/T and no Ad-S increased the G/T and achieved normal number of Ad-S at time of cryopreservation. In the control group, two patients reached only normal lower range regarding the G/T and the number of Ad-S. None of boys with less than average 0.2 G/T improved significantly, whether they were kryptocur®-treated or not. CONCLUSION Based on literature and the present results, we recommend adjuvant LHRH treatment to boys with cryptorchidism and insufficient genuine gonadotropin stimulation at time of surgery, as these patients have high infertility risk. Cryopreservation should be an option in case of treatment failure of adjuvant LHRH. However, to avoid repeat surgery with biopsy, some parents may choose biopsy for cryopreservation at time of the initial bilateral orchiopexy, well informed that the procedure may only be truly indicated in 22 and 36% of the cases.
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Affiliation(s)
- Jorgen Thorup
- The Department of Pediatric Surgery, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark
- Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- *Correspondence: Jorgen Thorup, ; Dina Cortes,
| | - Erik Clasen-Linde
- The Department of Pathology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark
| | - Lihua Dong
- Laboratory of Reproductive Biology, Section 5712, Juliane Marie Centre for Women, Children and Reproduction, Rigshospitalet, Copenhagen, Denmark
| | - Simone Hildorf
- The Department of Pediatric Surgery, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark
| | - Stine Gry Kristensen
- Laboratory of Reproductive Biology, Section 5712, Juliane Marie Centre for Women, Children and Reproduction, Rigshospitalet, Copenhagen, Denmark
| | - Claus Yding Andersen
- Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- Laboratory of Reproductive Biology, Section 5712, Juliane Marie Centre for Women, Children and Reproduction, Rigshospitalet, Copenhagen, Denmark
| | - Dina Cortes
- Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- Section of Endocrinology, Department of Pediatrics, Copenhagen University Hospital Hvidovre, Hvidovre, Denmark
- *Correspondence: Jorgen Thorup, ; Dina Cortes,
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Spermatogonial stem cell transplantation and male infertility: Current status and future directions. Arab J Urol 2017; 16:171-180. [PMID: 29713548 PMCID: PMC5922182 DOI: 10.1016/j.aju.2017.11.015] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2017] [Revised: 11/25/2017] [Accepted: 11/26/2017] [Indexed: 01/07/2023] Open
Abstract
Objective To summarise the current state of research into spermatogonial stem cell (SSC) therapies with a focus on future directions, as SSCs show promise as a source for preserving or initiating fertility in otherwise infertile men. Materials and methods We performed a search for publications addressing spermatogonial stem cell transplantation in the treatment of male infertility. The search engines PubMed and Google Scholar were used from 1990 to 2017. Search terms were relevant for spermatogonial stem cell therapies. Titles of publications were screened for relevance; abstracts were read, if related and full papers were reviewed for directly pertinent original research. Results In all, 58 papers were found to be relevant to this review, and were included in appropriate subheadings. This review discusses the various techniques that SSCs are being investigated to treat forms of male infertility. Conclusions Evidence does not yet support clinical application of SSCs in humans. However, significant progress in the in vitro and in vivo development of SSCs, including differentiation into functional germ cells, gives reason for cautious optimism for future research.
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Key Words
- ART, assisted reproductive technologies
- Allograft
- BMP4, bone morphogenetic protein 4
- Bcl6b, B-Cell CLL/Lymphoma 6B
- CD(24)(34), cluster of differentiation (24)(34)
- FGF2, Fibroblast growth factor 2
- FISH, fluorescence in situ hybridisation
- Fertility preservation
- GDNF, glial cell line-derived neurotrophic factor
- ICSI, intracytoplasmic sperm injection
- ID4, inhibitor of differentiation 4
- KS, Klinefelter syndrome
- Male infertility
- Non-obstructive azoospermia
- Onco-fertility
- PGC, primordial germ cells
- PLZF, promyelocytic leukaemia zinc finger
- PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses
- RA(R), retinoic acid (receptor)
- SPG, spermatogonia
- SSC, spermatogonial stem cell
- Stem cell therapy
- Stra8, stimulated by RA 8
- ZBTB, zinc finger and broad complex/Tramtrack/bric-a-brac
- c-Kit, KIT Proto-oncogene receptor tyrosine kinase
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Jabarpour M, Department of Theriogenology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.. Evaluation of the effect of follicular stimulating hormone on the in vitro bovine spermatogonial stem cells self-renewal: An experimental study. Int J Reprod Biomed 2017. [DOI: 10.29252/ijrm.15.12.795] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
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Ghadimi F, Shakeri M, Zhandi M, Zaghari M, Piryaei A, Moslehifar P, Rajabinejad A. Different approaches to establish infertile rooster. Anim Reprod Sci 2017; 186:31-36. [PMID: 28919176 DOI: 10.1016/j.anireprosci.2017.08.019] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2017] [Revised: 08/13/2017] [Accepted: 08/21/2017] [Indexed: 01/13/2023]
Abstract
Several methods have been developed to suppress spermatogenesis in recipient males before spermatogonial stem cells (SSCs) transplantation. The aim of this study was to compare two different methods of depleting endogenous spermatogenesis in recipient ROSS 308 strain adult roosters. Gamma-radiation and alkylating agent busulfan were utilized to infertilize adult roosters (ROSS 308 strain). Two radiation therapy regimes (based on 60co isotope) were conducted locally to testes using 40Gy (5×8Gy with three-day intervals) and 30Gy (3×10Gy with three-day intervals). And two different levels of busulfan 60mg(40+20) and 50mg(30+20) with 10-day intervals were injected intraperitoneally. The results showed that both radiation therapy regimes and both busulfan levels reduced sperm motility and sperm concentration significantly compared with control group. Moreover, there were no significant differences between gamma radiation and busulfan treatments in progressive and total motility of sperm reduction. Sperm concentration reached to zero at the end of the 4th week of experiment in all treatment groups. Also histological examinations revealed that both treatments could significantly reduce the diameter of seminiferous tubules and thickness of epithelium. None of the treatments had significant effect on body weight in comparison with control group and the health status of experimental roosters remained good throughout the study. Given that, the risk probability of high doses of radiation exposure and busulfan, it can be concluded that the 30Gy (3×10Gy) and 50mg (30+20) are appropriate for suppression of endogenous spermatogenesis in mature roosters.
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Affiliation(s)
- Fereshteh Ghadimi
- Department of Animal Science, Faculty of Agricultural Science and Engineering, University of Tehran, Karaj, Iran
| | - Malak Shakeri
- Department of Animal Science, Faculty of Agricultural Science and Engineering, University of Tehran, Karaj, Iran.
| | - Mahdi Zhandi
- Department of Animal Science, Faculty of Agricultural Science and Engineering, University of Tehran, Karaj, Iran
| | - Mojtaba Zaghari
- Department of Animal Science, Faculty of Agricultural Science and Engineering, University of Tehran, Karaj, Iran
| | - Abbas Piryaei
- Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences
| | - Parham Moslehifar
- Department of Animal Science, Faculty of Agricultural Science and Engineering, University of Tehran, Karaj, Iran
| | - Alireza Rajabinejad
- Department of Animal Science, Faculty of Agricultural Science and Engineering, University of Tehran, Karaj, Iran
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Ibtisham F, Wu J, Xiao M, An L, Banker Z, Nawab A, Zhao Y, Li G. Progress and future prospect of in vitro spermatogenesis. Oncotarget 2017; 8:66709-66727. [PMID: 29029549 PMCID: PMC5630449 DOI: 10.18632/oncotarget.19640] [Citation(s) in RCA: 44] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2017] [Accepted: 07/12/2017] [Indexed: 12/25/2022] Open
Abstract
Infertility has become a major health issue in the world. It affects the social life of couples and of all infertility cases; approximately 40–50% is due to “male factor” infertility. Male infertility could be due to genetic factors, environment or due to gonadotoxic treatment. Developments in reproductive biotechnology have made it possible to rescue fertility and uphold biological fatherhood. In vitro production of haploid male germ cell is a powerful tool, not only for the treatment of infertility including oligozoospermic or azoospermic patient, but also for the fertility preservation in pre-pubertal boys whose gonadal function is threatened by gonadotoxic therapies. Genomic editing of in-vitro cultured germ cells could also potentially cure flaws in spermatogenesis due to genomic mutation. Furthermore, this ex-vivo maturation technique with genomic editing may be used to prevent paternal transmission of genomic diseases. Here, we summarize the historical progress of in vitro spermatogenesis research by using organ and cell culture techniques and the future clinical application of in vitro spermatogenesis.
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Affiliation(s)
- Fahar Ibtisham
- Agricultural College, Guangdong Ocean University, Zhanjiang, Guangdong, China
| | - Jiang Wu
- Agricultural College, Guangdong Ocean University, Zhanjiang, Guangdong, China
| | - Mei Xiao
- Agricultural College, Guangdong Ocean University, Zhanjiang, Guangdong, China
| | - Lilong An
- Agricultural College, Guangdong Ocean University, Zhanjiang, Guangdong, China
| | - Zachary Banker
- Foreign Language College, Guangdong Ocean University, Zhanjiang, Guangdong, China
| | - Aamir Nawab
- Agricultural College, Guangdong Ocean University, Zhanjiang, Guangdong, China
| | - Yi Zhao
- Agricultural College, Guangdong Ocean University, Zhanjiang, Guangdong, China
| | - Guanghui Li
- Agricultural College, Guangdong Ocean University, Zhanjiang, Guangdong, China
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Enrichment and In Vitro Culture of Spermatogonial Stem Cells from Pre-Pubertal Monkey Testes. Tissue Eng Regen Med 2017; 14:557-566. [PMID: 30603509 DOI: 10.1007/s13770-017-0058-x] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2017] [Revised: 04/12/2017] [Accepted: 04/16/2017] [Indexed: 12/22/2022] Open
Abstract
Spermatogonial stem cells (SSCs) are essential for spermatogenesis throughout the lifespan of the male. However, the rarity of SSCs has raised the need for an efficient selection method, but little is known about culture conditions that stimulate monkey SSC proliferation in vitro. In this study, we report the development of effective enrichment techniques and in vitro culturing of germ cells from pre-pubertal monkey testes. Testis cells were analyzed by fluorescence-activated cell sorting techniques and were transplanted into the testes of nude mice to characterize SSCs. Thy-1-positive cells showed a higher number of colonies than the unselected control after xenotransplantation. Extensive colonization of monkey cells in the mouse testes indicated the presence of highly enriched populations of SSCs in the Thy-1-positive sorted cells. Furthermore, monkey testis cells were enriched by differential plating using extracellular matrix, laminin, and gelatin, and then cultured under various conditions. Isolation of monkey testicular germ cells by differential plating increased germ cell purity by 2.7-fold, following the combinational isolation method using gelatin and laminin. These enriched germ cells actively proliferated under culture conditions involving StemPro medium supplemented with bFGF, GDNF, LIF, and EGF at 37 °C. These results suggest that the enrichment and in vitro culture method proposed in the present study for harvesting a large number of functionally active monkey SSCs can be applied as the basis for efficient in vitro expansion of human SSCs.
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