Bcl-2 inhibits apoptosis of spermatogonia and growth of spermatogonial stem cells in a cell-intrinsic manner

The growth, differentiation, and death/survival of spermatogonia are precisely regulated for the proper production of spermatozoa. We have previously shown that Bcl-2 ectopically expressed in spermatogonia caused the inhibition of normal spermatogonial apoptosis and the subsequent failure of differentiation in transgenic mice. In addition, the growth of spermatogonial stem cells seemed to be temporally arrested in the transgenic mice. In the present study, we attempted to examine whether the abnormality of spermatogonia described above was caused by Bcl-2 misexpression in the spermatogonia or by an abnormal spermatogenic environment of the transgenic mice. We transplanted testicular cells of transgenic mice to seminiferous tubules of W/Wv mice in which transplanted normal testicular cells can undergo spermatogenesis. We found that the transplanted spermatogonia of the transgenic mice reproduced a series of abnormal changes including temporal growth arrest of spermatogonial stem cells and abnormal accumulation of spermatogonia in tubules, which were also observed in the testes of the transgenic mice. The results indicated that Bcl-2 inhibited apoptosis of spermatogonia and growth of spermatogonial stem cells in a cell-intrinsic manner. We also cultured testicular cells of transgenic mice and found that the spermatogonia of the transgenic mice were better able to survive than were those of wild-type mice but that their differentiation was not affected. The result suggested that failure of differentiation of the accumulated spermatogonia in the transgenic testes is not due to the abnormality of the bcl-2 misexpressing spermatogonia, but may be caused by extrinsic problems including improper interaction of spermatogonia with supporting cells.     

Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF

To study self-renewal and differentiation of spermatogonial stem cells, we have transplanted undifferentiated testicular germ cells of the GFP transgenic mice into seminiferous tubules of mutant mice with male sterility, such as those dysfunctioned at Steel (Sl) locus encoding the c-kit ligand or Dominant white spotting (W) locus encoding the receptor c-kit. In the seminiferous tubules of Sl/Sl(d) or Sl(17H)/Sl(17H) mice, transplanted donor germ cells proliferated and formed colonies of undifferentiated c-kit (-) spermatogonia, but were unable to differentiate further. However, these undifferentiated but proliferating spermatogonia, retransplanted into Sl (+) seminiferous tubules of W mutant, resumed differentiation, indicating that the transplanted donor germ cells contained spermatogonial stem cells and that stimulation of c-kit receptor by its ligand was necessary for maintenance of differentiated type A spermatogonia but not for proliferation of undifferentiated type A spermatogonia. Furthermore, we have demonstrated that their transplantation efficiency in the seminiferous tubules of Sl(17H)/Sl(17H) mice depended upon the stem cell niche on the basement membrane of the recipient seminiferous tubules and was increased by elimination of the endogenous spermatogonia of mutant mice from the niche by treating them with busulfan.     

Availability of subfertile transgenic rats expressing the c-myc gene as recipients for spermatogonial transplantation

The spermatogonial transplantation system was applied to evaluate stem cell kinetics and niche quality and to produce gene-modified animals using the stem cells after homologous recombination-based selection. This study was designed to determine whether the transplanted spermatogonia were able to proliferate and differentiate in male rats expressing the c-myc transgene under control of the human metallothionein IIA promoter (MT-myc Tg rats). Donor testicular cells were prepared from heterozygous chicken beta actin (CAG)/enhanced green fluorescent protein (EGFP)-transgenic rats (EGFP Tg rats) during the second week after birth and injected into the seminiferous tubules of the MT-myc Tg rats (line-A and -B; both subfertile) or rats pretreated with busulfan to remove endogenous spermatogonia. Three to four months after transplantation, cell colonies with EGFP fluorescence were detected in 36% (4/11), 40% (8/20), and 71% (5/7) of the transplanted testes in line-A MT-myc Tg rats, line-B MT-myc Tg rats, and busulfan-treated rats, respectively. No EGFP-positive colonies were detected when wild-type male rats were used as recipients (0/7; testis-basis). The histopathological and immunofluorescent examination of the serial sections from the transplanted testes showed normal spermatogenesis of the donor spermatogonia, but atrophy of the recipient seminiferous tubules. Microinsemination with round spermatids and mature spermatozoa derived from EGFP-positive testes in line-A rats resulted 26% (10/39 transferred) and 23% (11/48 transferred) full-term offspring, respectively. Thus, the MT-myc Tg male rats were suitable as potent recipients for spermatogonial transplantation without any chemical pretreatment to remove the endogenous spermatogonia.     

Proliferation and differentiation of spermatogonial stem cells in the w/wv mutant mouse testis

Mutations in the dominant-white spotting (W; c-kit) and stem cell factor (Sl; SCF) genes, which encode the transmembrane tyrosine kinase receptor and its ligand, respectively, affect both the proliferation and differentiation of many types of stem cells. Almost all homozygous W or Sl mutant mice are sterile because of the lack of differentiated germ cells or spermatogonial stem cells. To characterize spermatogenesis in c-kit/SCF mutants and to understand the role of c-kit signal transduction in spermatogonial stem cells, the existence, proliferation, and differentiation of spermatogonia were examined in the W/Wv mutant mouse testis. In the present study, some of the W/Wv mutant testes completely lacked spermatogonia, and many of the remaining testes contained only a few spermatogonia. Examination of the proliferative activity of the W/Wv mutant spermatogonia by transplantation of enhanced green fluorescent protein (eGFP)-labeled W/Wv spermatogonia into the seminiferous tubules of normal SCF (W/Wv) or SCF mutant (Sl/Sld) mice demonstrated that the W/Wv spermatogonia had the ability to settle and proliferate, but not to differentiate, in the recipient seminiferous tubules. Although the germ cells in the adult W/Wv testis were c-kit-receptor protein-negative undifferentiated type A spermatogonia, the juvenile germ cells were able to differentiate into spermatogonia that expressed the c-kit-receptor protein. Furthermore, differentiated germ cells with the c-kit-receptor protein on the cell surface could be induced by GnRH antagonist treatment, even in the adult W/Wv testis. These results indicate that all the spermatogonial stem cell characteristics of settlement, proliferation, and differentiation can be demonstrated without stimulating the c-kit-receptor signal. The c-kit/SCF signal transduction system appears to be necessary for the maintenance and proliferation of differentiated c-kit receptor-positive spermatogonia but not for the initial step of spermatogonial cell differentiation.     

Commitment of fetal male germ cells to spermatogonial stem cells during mouse embryonic development

The continuous production of mammalian sperm is maintained by the proliferation and differentiation of spermatogonial stem cells that originate from primordial germ cells (PGCs) in the early embryo. Although spermatogonial stem cells arise from PGCs, it is not clear whether fetal male germ cells function as spermatogonial stem cells able to produce functional sperm. In the present study, we examined the timing and mechanisms of the commitment of fetal germ cells to differentiate into spermatogonial stem cells by transplantation techniques. Transplantation of fetal germ cells into the seminiferous tubules of adult testis showed that donor germ cells, at 14.5 days postcoitum (dpc), were able to initiate spermatogenesis in the adult recipient seminiferous tubules, whereas no germ cell differentiation was observed in the transplantation of 12.5-dpc germ cells. These results indicate that the commitment of fetal germ cells to differentiate into spermatogonial stem cells initiates between embryonic days 12.5 and 14.5. Furthermore, the results suggest the importance of the interaction between germ cells and somatic cells in the determination of fetal germ cell differentiation into spermatogonial stem cells, as normal spermatogenesis was observed when a 12.5-dpc whole gonad was transplanted into adult recipient testis. In addition, sperm obtained from the 12.5- dpc male gonadal explant had the ability to develop normally if injected into the cytoplasm of oocytes, indicating that normal development of fetal germ cells in fetal gonadal explant occurred in the adult testicular environment.     

Transplantation of germ cells from rabbits and dogs into mouse testes.

Spermatogonial stem cells of a fertile mouse transplanted into the seminiferous tubules of an infertile mouse can develop spermatogenesis and transmit the donor haplotype to progeny of the recipient mouse. When testis cells from rats or hamsters were transplanted to the testes of immunodeficient mice, complete rat or hamster spermatogenesis occurred in the recipient mouse testes, albeit with lower efficiency for the hamster. The objective of the present study was to investigate the effect of increasing phylogenetic distance between donor and recipient animals on the outcome of spermatogonial transplantation. Testis cells were collected from donor rabbits and dogs and transplanted into testes of immunodeficient recipient mice in which endogenous spermatogenesis had been destroyed. In separate experiments, rabbit or dog testis cells were frozen and stored in liquid nitrogen or cultured for 1 mo before transplantation to mice. Recipient testes were analyzed, using donor-specific polyclonal antibodies, from 1 to >12 mo after transplantation for the presence of donor germ cells. In addition, the presence of canine cells in recipient testes was demonstrated by polymerase chain reaction using primers specific for canine alpha-satellite DNA. Donor germ cells were present in the testes of all but one recipient. Donor germ cells predominantly formed chains and networks of round cells connected by intercellular bridges, but later stages of donor-derived spermatogenesis were not observed. The pattern of colonization after transplantation of cultured cells did not resemble spermatogonial proliferation. These results indicate that fresh and cryopreserved germ cells can colonize the mouse testis but do not differentiate beyond the stage of spermatogonial expansion.     

Xenogeneic transplantation of equine testicular cells into seminiferous tubules of immunocompetent rats

The objectives were to develop a transplantation assay for equine testicular cells using busulfan-treated prepubertal immunocompetent rats as recipients, and to determine if putative equine spermatogonial stem cells (SSCs) could be enriched by flow cytometric cell sorting (based on light scattering properties), thereby improving engraftment efficiency. Four weeks after transplantation of frozen/thawed PKH26-labeled equine testicular cells, 0.029 ± 0.045% (mean ± SD) of viable donor cells transplanted had engrafted. Donor cells were present in seminiferous tubules of all recipient rats forming chains, pairs, mesh structures, or clusters (with two to >30 cells/structure). Cells were localized to the basal compartment by the basement membrane. Although equine cells proliferated within rat seminiferous tubules, no donor-derived spermatogenesis was evident. Furthermore, there was no histologic evidence of acute cellular rejection. No fluorescent cells were present in control testes. When equine testicular cells were sorted based on light scattering properties, the percentage of transplanted donor cells that engrafted was higher after injection of cells from the small, low complexity fraction (II; 0.169 ± 0.099%) than from either the large, high complexity fraction (I; 0.046 ± 0.051%) or unsorted cells (0.009 ± 0.007%; P < 0.05). Seminiferous tubules of busulfan-treated prepubertal immunocompetent rats provided a suitable niche for engraftment and proliferation, but not differentiation, of equine testicular cells. Sorting equine testicular cells based on light scattering properties resulted in a 19-fold improvement in colonization efficiency by cells with high forward scatter and low side scatter, which may represent putative equine SSCs.     

Oligoprogenitor Cells Derived from Spermatogonia Stem Cells Improve Remyelination in Demyelination Model

Embryonic stem (ES) like cells-derived testis represents a possible alternative to replace of neurons and glia. Here, we differentiated spermatogonia cells to oligoprogenitor (OP) like cells and transplanted them to demyelination model and assess their recovery potential in a demyelinated corpus callosum model in rats. ES like cells were differentiated to OP like cells using appropriate inducers and were transplanted in situ to demyelinated corpus callosum. Cell integration as well as demyelination extension and myelination intensity changes were evaluated using histologic studies and immunocytochemistry after 2 and 4 weeks post transplantation. Investigation of Nestin, NF68, Olig2, and NG2 by immunocytochemical technique indicated the differentiation of ES like cells to neuroprogenitor and oligodendrocyte like cells in each induction stage. Histologic findings showed a significant decrease in demyelination extension and a significant increase in remyelination intensity in cell transplanted groups. Also on the base of PLP expression, differentiation of transplanted cells was confirmed to myelinogenic cells using immunocytochemistry technique. We conclude that ES like cells derived from spermatogonia cells can be differentiated to OP like cells that can form myelin after transplantation into the demyelination model in rat, this represents recovery potential of spermatogonia cells which opens new window for cell therapeutic approaches using spermatogonial stem cells.     

Computer assisted image analysis to assess colonization of recipient seminiferous tubules by spermatogonial stem cells from transgenic donor mice

Transplantation of spermatogonial stem cells from fertile, transgenic donor mice to the testes of infertile recipients provides a unique system to study the biology of spermatogonial stem cells. To facilitate the investigation of treatment effects on colonization efficiency an analysis system was needed to quantify colonization of recipient mouse seminiferous tubules by donor stem cell‐derived spermatogenesis. In this study, a computer‐assisted morphometry system was developed and validated to analyze large numbers of samples. Donor spermatogenesis in recipient testes is identified by blue staining of donor‐derived spermatogenic cells expressing the E. coli lacZ structural gene. Images of seminiferous tubules from recipient testes collected three months after spermatogonial transplantation are captured, and stained seminiferous tubules containing donor‐derived spermatogenesis are selected for measurement based on their color by color thresholding. Colonization is measured as number, area, and length of stained tubules. Interactive, operator‐controlled color selection and sample preparation accounted for less than 10% variability for all collected parameters. Using this system, the relationship between number of transplanted cells and colonization efficiency was investigated. Transplantation of 10⁴ cells per testis only rarely resulted in colonization, whereas after transplantation of 10⁵ and 10⁶ cells per testis the extent of donor‐derived spermatogenesis was directly related to the number of transplanted donor cells. It appears that about 10% of transplanted spermatogonial stem cells result in colony formation in the recipient testis. The present study establishes a rapid, repeatable, semi‐interactive morphometry system to investigate treatment effects on colonization efficiency after spermatogonial transplantation in the mouse. Mol. Reprod. Dev. 53:142–148, 1999. © 1999 Wiley‐Liss, Inc.     

E-cadherin can be expressed by a small population of rat undifferentiated spermatogonia in vivo and in vitro

Spermatogonial stem cells (SSCs) maintain gamete production in the testes throughout adult life by balancing self-renewal and differentiation. In vitro culture of SSCs is a crucial technique for gene manipulation of SSCs to generate transgenic animals, for transplantation of SSCs to restore male fertility for infertile man, and for generation of pluripotent stem cells from SSCs to differentiate into various cell lineages. Isolation of highly purified SSCs is an all-important component for development of these techniques. However, definitive markers for SSCs, which purify SSCs (100% enrichment), are unknown. SSCs of many species can colonize the mouse testis; thus, we reasoned that same molecules of SSCs are conserved between species. In mouse, undifferentiated spermatogonia express the surface marker E-cadherin. The hypothesis tested in this work was that E-cadherin (also known as CDH1) can be expressed by undifferentiated spermatogonia of rat testes. In this paper, cross-section immunohistochemistry and whole-mount immunohistochemistry of rat seminiferous tubules were conducted to show that E-cadherin-positive cells were small in number and there are single, paired, and aligned spermatogonia attached along the basement membrane. During in vitro culture period, the undifferentiated rat spermatogonial colonies co-expressed E-cadherin and glial-derived neurotrophic factor family receptor alpha-1 or E-cadherin and promyelocytic leukemia zinc finger. Data collected during the study demonstrate that E-cadherin is expressed by a small population of rat undifferentiated spermatogonia both in vivo and during in vitro culture period.     

EFFECT OF A SPERMATOGONIAL CHALONE ON THE GROWING RAT TESTIS

Immature rats were used in an experiment to test the possible influence of a spermatogonial chalone on the expanding spermatogonial population in their developing testes. An extract from adult rat testes was injected intraperitoneally into 33-day-old rats and control animals were injected with an equal amount of saline. Two groups of normal adult rats similarly injected with the testicular extract and saline solutions served as additional controls. Following these injections, all animals were administered a dose of ³H-thymidine 10 hr before sacrifice. An analysis of the labeling indices of the various types of spermatogonia revealed that in young rats injected with testicular extract the percentage of labeled type A spermatogonia was significantly lower than in control animals. In contrast, the labeling indices of Intermediate and type B spermatogonia were similar in the two groups of young rats. In the two groups of adult animals, there was no difference in the labeling indices of type A or of other types of spermatogonia. These data indicated that the saline extract of adult testes contained a substance, a spermatogonial chalone, inhibiting specifically the proliferation of some type A spermatogonia. The results also support the concept that a spermatogonial chalone may intervene, through its action on the spermatogonial stem cell population, to arrest the growth of the seminiferous tubules as the animal reaches maturity.     

Recipient preparation is critical for spermatogonial transplantation in the rat

Testis cell transplantation from mice or rats into recipient mouse seminiferous tubules results in donor cell-derived spermatogenesis in nearly all host testes. Normal spermatozoa are produced and, in the most successful mouse transplantations, the donor haplotype is transmitted to progeny of the recipient. However, few studies have been performed in other species. In this report, we demonstrate that rat and mouse testis cells will generate donor cell-derived spermatogenesis in recipient rat seminiferous tubules. Depletion of endogenous spermatogenesis before donor cell transplantation was more difficult in rat than reported for mouse recipients. A protocol employing treatment of neonatal rats with busulfan was most effective in preparing recipients and allowed more than 90% of testes to be colonized by donor cells. Transplantation of mouse testis cells into rat seminiferous tubules was most successful in recipients made cryptorchid and treated with busulfan. In the best experiments, about 55% of rat testes were colonized by mouse cells. Both rat and mouse donor cell-derived spermatogenesis were improved by treatment of rat recipients with leuprolide, a gonadotropin-releasing hormone agonist. The studies indicated that recipient preparation for spermatogonial stem cell transplantation was critical in the rat and differs from the mouse. However, modification of currently used techniques should allow male germ line stem cell transplantation in many species.     

Continuously proliferative stem germ cells partially repopulate the aged, atrophic rat testis after gonadotropin-releasing hormone agonist therapy

Aging in the male human is accompanied by testicular atrophy, although relatively little is known about the mechanisms underlying germ cell loss. Testicular atrophy in the aged Brown Norway rat, an animal model for studies of aging in the human, has been attributed to a loss of spermatogonial stem cells. However, examination of testicular cross-sections from 27-mo-old Brown Norway rats indicated that approximately 14% of type A spermatogonia were stem cells. Furthermore, using bromodeoxyuridine labeling, we found that approximately 47% of these stem cells were actively dividing, with a cell cycle time of approximately 12.6 days. Both serum and testicular interstitial fluid testosterone levels were depressed in the aged rat. Therapy with the GnRH agonist, leuprolide, which has been empirically shown to reverse testicular atrophy in other models of germ cell loss, also partially restored spermatogenesis in the aged Brown Norway rat. The extent of testicular atrophy varied considerably, not only within the control and leuprolide-treatment groups but also between the left and right testes of the same animals. No significant difference was found between the mean percentage of populated tubules in 31-mo-old control animals (16.2 +/- 28%, mean +/- SD) and 31-mo-old leuprolide-treated animals (20.9 +/- 19.8%), but categorical comparisons showed that significantly fewer leuprolide-treated animals and testes contained < or = 1% populated tubules, indicating that GnRH agonist therapy stimulates differentiation of type A spermatogonia. An increase in the ratio of soluble to membrane stem cell factor mRNA levels was present in aged rats and partially reversed following leuprolide therapy.     

Expansion of murine spermatogonial stem cells through serial transplantation

Mammalian male germ cells might be generally thought to have infinite proliferative potential based on their life-long production of huge numbers of sperm. However, there has been little substantial evidence that supports this assumption. In the present study, we performed serial transplantation of spermatogonial stem cells to investigate if they expand by self-renewing division following transplantation. The transgenic mouse carrying the Green fluorescent protein gene was used as the donor cell source that facilitated identification and recollection of colonized donor germ cells in the recipient testes. The established colonies of germ cells in the recipient testes were collected and transplanted to new recipients. This serial transplantation of spermatogonial stem cells repopulated the recipient testes, which were successfully performed sequentially up to four times from one recipient to the next. The incubation periods between two sequential transplantations ranged from 55 to 373 days. During these passages, the spermatogonial stem cells showed constant activity to form spermatogenic colonies in the recipient testis. They continued to increase in number for more than a year following transplantation. Colonization efficiency of spermatogonial stem cells was determined to be 4.25% by using Sl/Sl(d) mice as recipients that propagated only undifferentiated type A spermatogonia in their testes. Based on the colonization efficiency, one colony-forming activity was assessed to equate to about 20 spermatogonial stem cells. The spermatogonial stem cells were estimated to expand over 50-fold in 100 days in this experiment.     

Isolation and transplantation of spermatogonia in sheep

Studies in rodents show that spermatogonial transplantation is an excellent new tool for studying spermatogenesis and for preservation and dissemination of genetics. The aim of this study was to adapt the technique to rams. Two issues were addressed: purification of stem cell spermatogonia, and efficient injection of donor spermatogonia into the seminiferous tubules of rams. We compared differential plating and Percoll gradient methods for purifying donor spermatogonia from ram lamb testes. Spermatogonia were identified with an antibody against PGP 9.5, a ubiquitin C-terminal hydrolase. Both purity and total number of spermatogonia recovered were higher after purification by Percoll gradient than by differential plating. Four approaches for injecting cells into the seminiferous tubules of ram testes were compared ex vivo: insertion of a needle into the extra-testicular rete testis after reflection of the head of the epididymis ('surgical' approach), and ultrasound-guided insertion of a needle into the extra-testicular rete, and the proximal and distal parts of the intra-testicular rete testis. 'Surgical' and ultrasound-guided approaches into the extra-testicular rete resulted in highest success rates and best filling of the seminiferous tubules. Finally, the ultrasound guided approach into the extra-testicular rete testis was validated in vivo by transplanting purified spermatogonia previously labeled with a fluorescent molecule (CFDA-SE). In seven of eight testes injected, donor cells were identified within the seminiferous epithelium for up to 2wk after transplantation, indicating the integration of donor cells.     

Juvenile spermatogonial depletion (jsd) mutant seminiferous tubules are capable of supporting transplanted spermatogenesis

In mice, the juvenile spermatogonial depletion (jsd) mutation results in a single wave of spermatogenesis followed by failure of type A spermatogonial stem cells to repopulate the testis, rendering male animals sterile. It is not clear whether the defect in jsd resides in a failure of the somatic component to support spermatogenesis or in a failure that is intrinsic to the mutant's germ cells. To determine if the jsd intratesticular environment is capable of supporting spermatogenesis, germ cell transplantation experiments were performed in which C57BL/6 ROSA germ cells were transplanted into jsd recipients. To determine if jsd spermatogonia are able to develop in a permissive seminiferous environment, jsd germ cells were transplanted into W/W(v) and busulfan-treated C57BL/6 animals. The data demonstrate that up to 7 mo after transplantation of normal germ cells, jsd seminiferous tubules are capable of supporting spermatogenesis. In contrast, when jsd germ cells were transplanted into busulfan-treated C57BL/6 testis, or into testis of W/W(v) mice, no jsd-derived spermatogenesis was observed. The data support the hypothesis that the jsd phenotype is due to a defect in the germ cells themselves, and not in the intratubular environment.     

Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes.

Recently a system was developed in which transplanted donor spermatogonial stem cells establish complete spermatogenesis in the testes of an infertile recipient. To obtain insight into stem cell activity and the behavior of donor germ cells, the pattern and kinetics of mouse spermatogonial colonization in recipient seminiferous tubules were analyzed during the 4 mo following transplantation. The colonization process can be divided into three continuous phases. First, during the initial week, transplanted cells were randomly distributed throughout the tubules, and a small number reached the basement membrane. Second, from 1 wk to 1 mo, donor cells on the basement membrane divided and formed a monolayer network. Third, beginning at about 1 mo and continuing throughout the observation period, cells in the center of the network differentiated extensively and established a colony of spermatogenesis, which expanded laterally by repeating phase two and then three. An average of 19 donor cell-derived colonies developed from 10(6) cells transplanted to the seminiferous tubules of a recipient testis; the number of colonized sites did not change between 1 and 4 mo. However, the length of the colonies increased from 0.73 to 5.78 mm between 1 and 4 mo. These experiments establish the feasibility of studying in a systematic and quantitative manner the pattern and kinetics of the colonization process. Using spermatogonial transplantation as a functional assay, it should be possible to assess the effects of various treatments on stem cells and on recipient seminiferous tubules to provide unique insight into the process of spermatogenesis.