ABSTRACT
Testicular aging is mainly characterized by a gradual decline in the capability of testosterone synthesis and spermatogenesis, which not only affects male fertility, but also correlates with aging-related chronic diseases intimately. Therefore, delaying testicular aging plays a significant role in improving the health and quality of life of middle-aged and elderly men. Stem cells are a cell group with potent self-renewal capability and multi-directional differentiation potential. In recent years, the research of stem cells in basic and clinical application has been carried out in-depth, which has accelerated the development of cell therapy. Currently, stem cell transplantation has been employed to treat multiple diseases, which has captivated widespread attention in the field of aging and regenerative medicine. Stem cell transplantation has demonstrated promising prospects in the treatment of testicular aging. In this article, research profile and progress of stem cell transplantation in the treatment of testicular aging were reviewed, and bottleneck issues encountered in clinical translation and strategies for optimizing clinical efficacy were discussed, aiming to provide novel ideas for the research and development and clinical translation of stem cell therapy for testicular aging.
ABSTRACT
Although somatic cells can be reprogrammed to pluripotent stem cells (PSCs) with pure chemicals, authentic pluripotency of chemically induced pluripotent stem cells (CiPSCs) has never been achieved through tetraploid complementation assay. Spontaneous reprogramming of spermatogonial stem cells (SSCs) was another non-transgenic way to obtain PSCs, but this process lacks mechanistic explanation. Here, we reconstructed the trajectory of mouse SSC reprogramming and developed a five-chemical combination, boosting the reprogramming efficiency by nearly 80- to 100-folds. More importantly, chemical induced germline-derived PSCs (5C-gPSCs), but not gPSCs and chemical induced pluripotent stem cells, had authentic pluripotency, as determined by tetraploid complementation. Mechanistically, SSCs traversed through an inverted pathway of in vivo germ cell development, exhibiting the expression signatures and DNA methylation dynamics from spermatogonia to primordial germ cells and further to epiblasts. Besides, SSC-specific imprinting control regions switched from biallelic methylated states to monoallelic methylated states by imprinting demethylation and then re-methylation on one of the two alleles in 5C-gPSCs, which was apparently distinct with the imprinting reprogramming in vivo as DNA methylation simultaneously occurred on both alleles. Our work sheds light on the unique regulatory network underpinning SSC reprogramming, providing insights to understand generic mechanisms for cell-fate decision and epigenetic-related disorders in regenerative medicine.
Subject(s)
Male , Mice , Animals , Cellular Reprogramming/genetics , Tetraploidy , Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/metabolism , DNA Methylation , Spermatogonia/metabolism , Germ Cells/metabolismABSTRACT
Continuous spermatogenesis depends on the self-renewal and differentiation of spermatogonial stem cells (SSCs). SSCs, the only male reproductive stem cells that transmit genetic material to subsequent generations, possess an inherent self-renewal ability, which allows the maintenance of a steady stem cell pool. SSCs eventually differentiate to produce sperm. However, in an in vitro culture system, SSCs can be induced to differentiate into various types of germ cells. Rodent SSCs are well defined, and a culture system has been successfully established for them. In contrast, available information on the biomolecular markers and a culture system for livestock SSCs is limited. This review summarizes the existing knowledge and research progress regarding mammalian SSCs to determine the mammalian spermatogenic process, the biology and niche of SSCs, the isolation and culture systems of SSCs, and the biomolecular markers and identification of SSCs. This information can be used for the effective utilization of SSCs in reproductive technologies for large livestock animals, enhancement of human male fertility, reproductive medicine, and protection of endangered species.
Subject(s)
Animals , Male , Adult Germline Stem Cells , Cell Differentiation , Spermatogenesis , Spermatogonia , Stem CellsABSTRACT
An ideal animal model of azoospermia would be a powerful tool for the evaluation of spermatogonial stem cell (SSC) transplantation. Busulfan has been commonly used to develop such a model, but 30%-87% of mice die when administered an intraperitoneal injection of 40 mg kg-1. In the present study, hematoxylin and eosin staining, Western blot, immunofluorescence, and quantitative real-time polymerase chain reaction were used to test the effects of busulfan exposure in a mouse model that received two intraperitoneal injections of busulfan at a 3-h interval at different doses (20, 30, and 40 mg kg-1) on day 36 or a dose of 40 mg kg-1 at different time points (0, 9, 18, 27, 36, and 63 days). The survival rate of the mice was 100%. When the mice were treated with 40 mg kg-1 busulfan, dramatic SSC depletion occurred 18 days later and all of the germ cells were cleared by day 36. In addition, the gene expressions of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), chemokine (C-X-C Motif) ligand 12 (CXCL12), and colony-stimulating factor 1 (CSF1) were moderately increased by day 36. A 63-day, long-term observation showed the rare restoration of endogenous germ cells in the testes, suggesting that the potential period for SSC transplantation was between day 36 and day 63. Our results demonstrate that the administration of two intraperitoneal injections of busulfan (40 mg kg-1 in total) at a 3-h interval to mice provided a nonlethal and efficient method for recipient preparation in SSC transplantation and could improve treatments for infertility and the understanding of chemotherapy-induced gonadotoxicity.
ABSTRACT
An ideal animal model of azoospermia would be a powerful tool for the evaluation of spermatogonial stem cell (SSC) transplantation. Busulfan has been commonly used to develop such a model, but 30%-87% of mice die when administered an intraperitoneal injection of 40 mg kg-1. In the present study, hematoxylin and eosin staining, Western blot, immunofluorescence, and quantitative real-time polymerase chain reaction were used to test the effects of busulfan exposure in a mouse model that received two intraperitoneal injections of busulfan at a 3-h interval at different doses (20, 30, and 40 mg kg-1) on day 36 or a dose of 40 mg kg-1 at different time points (0, 9, 18, 27, 36, and 63 days). The survival rate of the mice was 100%. When the mice were treated with 40 mg kg-1 busulfan, dramatic SSC depletion occurred 18 days later and all of the germ cells were cleared by day 36. In addition, the gene expressions of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), chemokine (C-X-C Motif) ligand 12 (CXCL12), and colony-stimulating factor 1 (CSF1) were moderately increased by day 36. A 63-day, long-term observation showed the rare restoration of endogenous germ cells in the testes, suggesting that the potential period for SSC transplantation was between day 36 and day 63. Our results demonstrate that the administration of two intraperitoneal injections of busulfan (40 mg kg-1 in total) at a 3-h interval to mice provided a nonlethal and efficient method for recipient preparation in SSC transplantation and could improve treatments for infertility and the understanding of chemotherapy-induced gonadotoxicity.
Subject(s)
Animals , Male , Mice , Adult Germline Stem Cells/transplantation , Azoospermia/chemically induced , Busulfan/toxicity , Disease Models, Animal , Infertility, Male/chemically induced , Injections, Intraperitoneal , Spermatogenesis/drug effects , Spermatogonia/drug effects , Stem Cell Transplantation/methodsABSTRACT
Spermatogonial stem cells (SSCs) transmit genetic information to the next progeny in males. Thus, SSCs are a potential target for germline modifications to generate transgenic animals. In this study, we report a technique for the generation of transgenic rats by in vivo manipulation of SSCs with a high success rate. SSCs in juvenile rats were transduced in vivo with high titers of lentivirus harboring enhanced green fluorescent protein and mated with wild-type females to create founder rats. These founder rats expressed the transgene and passed on the transgene with an overall success rate of 50.0%. Subsequent generations of progeny from the founder rats both expressed and passed on the transgene. Thus, direct modification of SSCs in juvenile rats is an effective means of generating transgenic rats through the male germline. This technology could be adapted to larger animals, in which existing methods for gene modification are inadequate or inapplicable, resulting in the generation of transgenic animals in a variety of species.
ABSTRACT
Spermatogonial stem cells (SSCs) transmit genetic information to the next progeny in males. Thus, SSCs are a potential target for germline modifications to generate transgenic animals. In this study, we report a technique for the generation of transgenic rats by in vivo manipulation of SSCs with a high success rate. SSCs in juvenile rats were transduced in vivo with high titers of lentivirus harboring enhanced green fluorescent protein and mated with wild-type females to create founder rats. These founder rats expressed the transgene and passed on the transgene with an overall success rate of 50.0%. Subsequent generations of progeny from the founder rats both expressed and passed on the transgene. Thus, direct modification of SSCs in juvenile rats is an effective means of generating transgenic rats through the male germline. This technology could be adapted to larger animals, in which existing methods for gene modification are inadequate or inapplicable, resulting in the generation of transgenic animals in a variety of species.
Subject(s)
Animals , Male , Rats , Green Fluorescent Proteins , Lentivirus , Rats, Transgenic , Spermatogonia/metabolismABSTRACT
Spermatogenesis originates from spermatogonial stem cells (SSCs). SSCs can continually renew and eventually differentiate into spermatozoa under control of various growth factors, microenvironments and self-signaling. The molecules involved in SSCs self-renewal include glial cell-derived nerve growth factor, fibroblast growth factor and downstream signaling pathways, as well as the transcription factors and the epigenetic regulators. Molecules related to SSCs differentiation include retinoic acid, a variety of transcription factors and the eptigenetic regulatory factors. The research on the mechanism of SSCs self-renewal and differentiation is of great significance for the understanding of spermatogenesis and the diagnosis and treatment of male infertility. This review summarized the exogenous factors, transcription factors, and epigenetic regulators that are involved in the regulation of SSCs self-renewal and differentiation in recent years.
ABSTRACT
Spermatogenesis originates from spermatogonial stem cells (SSCs).SSCs can continually renew and eventually differentiate into spermatozoa under control of various growth factors,microenvironments and self-signaling.The molecules involved in SSCs self-renewal include glial cell-derived nerve growth factor,fibroblast growth factor and downstream signaling pathways,as well as the transcription factors and the epigenetic regulators.Molecules related to SSCs differentiation include retinoic acid,a variety of transcription factors and the eptigenetic regulatory factors.The research on the mechanism of SSCs self-renewal and differentiation is of great significance for the understanding of spermatogenesis and the diagnosis and treatment of male infertility.This review summarized the exogenous factors,transcription factors,and epigenetic regulators that are involved in the regulation of SSCs self-renewal and differentiation in recent years.
ABSTRACT
<p><b>Objective</b>To study the expression of the gene of myosin regulatory light chain-2 (MYL2) in the development of rat testis tissue.</p><p><b>METHODS</b>Using real-time PCR and immunohistochemistry, we determined the mRNA transcription level and protein expression of MYL2 in the rat testis.</p><p><b>RESULTS</b>The mRNA expression of the MYL2 gene changed in an age-dependent manner, reaching the highest value on postnatal day (PND) 2, then dropped rapidly till PND 8, increased slowly on PNDs 10 and 12, decreased on PND 14, rose slightly from PND 15 and rapidly on PNDs 20 and 25, and declined slowly from PND 65. Immunohistochemistry showed that the MYL2 protein was mainly expressed in testicular sperm cells.</p><p><b>CONCLUSIONS</b>The MYL2 gene may be involved in the proliferation of spermatogonial stem cells and the process of sperm cells developing into mature sperm.</p>
Subject(s)
Animals , Male , Rats , Gene Expression Regulation , RNA, Messenger , Metabolism , Real-Time Polymerase Chain Reaction , Spermatozoa , Metabolism , Testis , Metabolism , Time FactorsABSTRACT
Nanos2, a member of the Nanos2 gene family, is a specific gene in male germ cells and encodes an evolutionarily conserved RNA binding protein expressed in male primordial germ cells (PGCs) during the embryonic period as well as in the spermatogonial stem cells (SSCs) of the testis. In the embryonic period, Nanos2 promotes the development of male PGCs and inhibits them from meiosis. In the process of spermatogenesis, Nanos2 suppresses the differentiation of SSCs in the testis and maintains the stability of the SSC pool. The knockout of Nanos2 may cause the disappearance of germ cells and sterility in male mice while its overexpression in the testis may lead to accumulation of SSCs in seminiferous tubules. Besides, Nanos2 is involved in the degradation of specific RNAs and possibly associated with some diseases of the male reproductive system. This review focuses on the recent progress in the studies of Nanos2 in the male reproductive system.
Subject(s)
Animals , Male , Mice , Cell Differentiation , Gene Knockout Techniques , Meiosis , RNA , Metabolism , RNA-Binding Proteins , Genetics , Metabolism , Spermatogenesis , Physiology , Spermatogonia , Spermatozoa , Testis , Cell BiologyABSTRACT
Spermatogonial stem cells (SSCs) have the ability of self-renewal and differentiate into sperm throughout the life of male animals. They are the unique adult stem cells that transmit genetic information to subsequent generations. Recently, accumulating evidences have demonstrated that SSCs can be reprogrammed to become ES-like cells that differentiate into all cell lineages of the three germ layers. These studies were mainly focused on non-primate mammals. Therefore, this review summarized the characterization, isolation, purification, cultivation, identification and transplantation of SSCs in non-primate mammals, and discussed the unlimited pluripotency and plasticity of SSCs. We also provided valuable insights of SSCs in the treatment of male infertility and application potential of human regenerative medicine.
ABSTRACT
Transplantation of spermatogonial stem cells (SSCs) in experimental animal models has been used to study germ line stem cell biology and to produce transgenic animals. The species-specific recipient model preparation is important for the characterization of SSCs and the production of offspring. Here, we investigated the effects of surgically induced cryptorchidism in dog as a new recipient model for spermatogonial stem cell transplantation. Artificially unilateral or bilateral cryptorchidism was induced in ten mature male dogs by surgically returning the testis and epididymis to the abdominal cavity. The testes and epididymides were collected every week after the induction of artificial cryptorchidism (surgery) for one month. To determine the effect of surgical cryptorchidism, the seminiferous tubule diameter was measured and immunohistochemistry using PGP9.5 and GATA4 antibodies was analyzed. The diameters of the seminiferous tubules of abdominal testes were significantly reduced compared to those of the scrotal testes. Immunohistochemistry results showed that PGP9.5 positive undifferentiated spermatogonia were significantly reduced after surgical cryptorchidism induction, but there were no significant changes in GATA-4 positive sertoli cells. To evaluate the testis function recovery rate, orchiopexy was performed on two dogs after 30 days of bilateral cryptorchidism. In the orchiopexy group, SCP3 positive spermatocytes were detected, and spermatogenesis was recovered 8 weeks after orchiopexy. In this study, we provided optimum experimental conditions and time for surgical preparation of a recipient canine model for SSC transplantation. Additionally, our data will contribute to recipient preparation by using surgically induced cryptorchidism in non-rodent species.
Subject(s)
Animals , Dogs , Humans , Male , Abdominal Cavity , Animals, Genetically Modified , Antibodies , Biology , Cryptorchidism , Epididymis , Germ Cells , Immunohistochemistry , Models, Animal , Orchiopexy , Recovery of Function , Seminiferous Tubules , Sertoli Cells , Spermatocytes , Spermatogenesis , Spermatogonia , Stem Cell Transplantation , Stem Cells , TestisABSTRACT
Objective To explore the dynamic responses of Sertoli cells to depletion of spermatogonial stem cells by busulfan.Methods After intraperitoneal injection of 15, 30 or 44 mg/kg busulfan to mice, the spermatogenesis and the expression of GDNF, PLZF, Nanog and GFRɑ1 mRNA were assessed by real-time quantitative PCR at 5 and 28 days after the busulfan treatment.Results Glial cell line-derived neutrophic factor ( GDNF ) was significantly increased and showed a dose-dependent trend at 5 days after busulfan treatment, but no significant difference was seen in the expression of promyelocytic leukemia zinc finger(PLZF) and GDNF family receptorα-1(GFRα1).The testicular histology also appeared no significant difference at 5 days after busulfan treatment.At 28 days after busulfan treatment, the relative expression lev-els of GDNF, PLZF, Nanog and GFRɑ1 mRNA were drastically increased.Morphological observation showed that spermat-ogenesis damages became even more severe as the busulfan dose increased.Conclusions Sertoli cell response to the de-pletion of spermatogonia occurs as early as the fifth day after busulfan treatment.Production of GDNF in Sertoli cells shows a compensatory increase, which may stimulate spermatogonial stem cells to accelerate their self-renewal, reflected by the enhancing expression of Nanog and PLZF, and ultimately promote the restoration of spermatogenesis.
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Objective To explore the conditions and methods for cryopreservation and proliferation of mouse spermatogonial stem cells (SSCs). Methods SSCs were isolated from six-day-old Kunming mouse using two-step enzymatic digestion and Percoll discontinuous density gradient centrifugation. Cells were frozen with different freezing medium and cooling rate. After thaw, they were cultured in mimimum essential medium alpha (MEMα) supplemented with 10% fetal calf serum (FCS) and 100μg/L glial cell line-derived neurotrophic factor (GDNF). The survived and proliferating SSCs were examined by WST-8 colorimetric assay. Alkaline phosphatase andreverse transcription-polymerase chain reaction (RT-PCR) were performed to confirm if the cultured 96 hours germ cells were still stem cells. Results The best method to cryopreserve SSCs is using cryoprotector containing 10% dimethyl sulfoxide(DMSO), 10% FCS, 0.07mol/L sucrose and 1℃/min cooling rate, and the viability of cells in this method is more than 84%;Although the cell viability in non-programmed freezing method is less than that in the programmed freezing method, it is a simple and effective cropreservation method for mouse SSCs. What is more, the anchoring time of SSCs in this method is 8-12 hours after thaw, SSCs begin to proliferate 24 hours later, and rapid proliferation appears on the 48 hours, colonies are composed by 20-25 cells in 96 hours, when SSCs proliferated nearly 5 times.Conclusion The culture condition we used is suitable for proliferation of frozen-thawed SSCs.
ABSTRACT
Objective Spermatogonial stem cells(SSCs)dissociated from 2～5 days postpartum mice were cultured on Sertoli cells feeders,to study the effect of Sertoli cells feeders on culture of mouse spermatogonial stem cells.Methods Specific markers CD9 of mouse SSCs cultured serum-free StemPro-34 SFM culture medium were identified by immunohistochemical assay.Result During the first week of culture,on Sertoli cells feeders or STO feeders,the biologica1 behaviors of spermatogonial stem cells showed no obvious difference.After a week of culture,compared with control,there were more number of spermatogonial stem cells remained when they were cultured for 60 days.These cells were expressed CD9 positive.In conclusion Sertoli cells can be used as feeders not only to promote survival but also renewal of spermatogonial stem cells.
ABSTRACT
Generally speaking,embryonic stem cells,embryonic germ cells and embryonal carcinoma cells are pluripotent stem cells and they have the potential to differentiate into germ cells.Spermatogonial stem cells is unique adult germline stem cells and can differentiate into sperm.Recent research with stem cells from both embryonic and adult origin will be discussed with particular attention to results that challenge conventional wisdom about the presence of germline stem cells in adults and the plasticity of adult stem cell types.This review focuses on the progress in the research of germline stem cells and introduces the origin and plasticity of adult germline stem cells and its future directions in medical science.