Flow-cytometric isolation of testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions

There is a need to isolate different populations of spermatogenic cells to investigate the molecular events that occur during spermatogenesis. Here we developed a new method to identify and purify testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions (pvasa-GFP) at various stages of spermatogenesis. Rainbow trout piwi-like (rtili), rainbow trout scp3 (rt-scp3), and rainbow trout shippo1 (rt-shippo1) were identified as molecular markers for spermatogonia, spermatocytes, and spermatids, respectively. The testicular cells were separated into five fractions (A-E) by flow cytometry (FCM) according to their GFP intensities. Based on the molecular markers, fractions A and B were found to contain spermatogonia, while fractions C and D contained spermatocytes, and fraction E contained spermatids. We also classified the spermatogonia into type A, which contained spermatogonial stem cells (SSCs), and type B, which did not. As none of the molecular markers tested could distinguish between the two types of spermatogonia, we subjected them to a transplantation assay. The results indicated that cells with strong GFP fluorescence (fraction A) colonized the recipient gonads, while cells with weaker GFP fluorescence (fraction B) did not. As only SSCs could colonize the recipient gonads, this indicated that fraction A and fraction B contained mainly type A and type B spermatogonia, respectively. These findings confirmed that our system could identify and isolate various populations of testicular cells from rainbow trout using a combination of GFP-dependent FCM and a transplantation assay.     

Spermatogonial transplantation in fish: A novel method for the preservation of genetic resources

Recent progress in genome-based breeding has created various fish strains carrying desirable genetic traits; however, methods for the long-term preservation of their genetic resources have not yet been developed, mainly due to the lack of cryopreservation techniques for fish eggs and embryos. Recently, we established an alternative cryopreservation technique for fish spermatogonia using a slow-freezing method. Furthermore, we developed a transplantation system to produce functional eggs and sperm derived from spermatogonia. Spermatogonia isolated from the testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout (Oncorhynchus mykiss) were transplanted into the peritoneal cavity of triploid masu salmon (Oncorhynchus masou) hatchlings of both genders. The transplanted trout spermatogonia migrated towards the gonadal anlagen of the recipient salmon, into which they were subsequently incorporated. We confirmed that the donor-derived spermatogonia resumed gametogenesis, and produced sperm and eggs in male and female recipient salmon, respectively. Fertilization of the resultant eggs and sperm produced only rainbow trout in the first filial (F₁) generation, suggesting that the sterile triploid recipient salmon produced functional eggs and sperm derived from the trout donors. A combination of spermatogonial transplantation and cryopreservation could be a powerful tool for preserving valuable fish strains with desirable genetic traits and endangered species.     

Enrichment of transplantable germ cells in salmonids using a novel monoclonal antibody by magnetic‐activated cell sorting

In the fish germ cell transplantation system, only type A spermatogonia (ASGs) and oogonia are known to be incorporated into the recipient genital ridges, where they undergo gametogenesis. Therefore, high colonization efficiency can be achieved by enriching undifferentiated germ cells out of whole testicular cells. In this study, we used magnetic‐activated cell sorting (MACS) for enriching undifferentiated germ cells of rainbow trout using a monoclonal antibody that recognizes a specific antigen located on the germ cell membrane. We screened the antibodies to be used for MACS by performing immunohistochemistry on rainbow trout gonads. Two antibodies, nos. 172 and 189, showed strong signals for ASGs and oogonia. Next, we performed MACS with antibody no. 172 using gonadal cells isolated from vasa‐gfp rainbow trout showing GFP in undifferentiated germ cells. We found that GFP‐positive cells are highly enriched in antibody no. 172‐positive fractions. Finally, to examine the transplantability of MACS‐enriched cells, we intraperitoneally transplanted sorted or unsorted cells into recipient larvae. We observed that transplantability of sorted cells, particularly ovarian cells, were significantly higher than that of unsorted cells. Therefore, MACS with antibody no. 172 could enrich ASGs and oogonia and become a powerful tool to improve transplantation efficiency in salmonids.     

Intraperitoneal Germ Cell Transplantation in the Nile Tilapia Oreochromis niloticus

Germ cell transplantation offers promising applications in finfish aquaculture and the preservation of endangered species. Here, we describe an intraperitoneal spermatogonia transplantation procedure in the Nile tilapia Oreochromis niloticus. Through histological analysis of early gonad development, we first determined the best suitable stage at which exogenous germ cells should be transplanted into the recipients. For the transplantation procedure, donor testes from a transgenic Nile tilapia strain carrying the medaka β-actin/enhanced green fluorescent protein (EGFP) gene were subjected to enzymatic dissociation. These testicular cells were then stained with PKH26 and microinjected into the peritoneal cavity of the recipient fish. To confirm colonization of the donor-derived germ cells, the recipient gonads were examined by fluorescent and confocal microscopy. PKH26-labeled cells exhibiting typical spermatogonial morphology were incorporated into the recipient gonads and were not rejected within 22 days posttransplantation. Long-term survival of transgenic donor-derived germ cells was then verified in the gonads of 5-month-old recipients and in the milt and vitelogenic oocytes of 1-year-old recipients, by means of PCR using EGFP-specific primers. EGFP-positive milt from adult male recipients was used to fertilize non-transgenic oocytes and produced transgenic offspring expressing the donor-derived phenotype. These results imply that long-term survival, proliferation, and differentiation of the donor-derived spermatogonia into vitelogenic oocytes and functional spermatozoa are all possible. Upon further improvements in the transplantation efficiency, this intraperitoneal transplantation system could become a valuable tool in the conservation of genetic resources for cichlid species.     

The radiation-induced block in spermatogonial differentiation is due to damage to the somatic environment, not the germ cells

Radiation and chemotherapeutic drugs cause permanent sterility in male rats, not by killing most of the spermatogonial stem cells, but by blocking their differentiation in a testosterone-dependent manner. However, it is not known whether radiation induces this block by altering the germ or the somatic cells. To address this question, we transplanted populations of rat testicular cells containing stem spermatogonia and expressing the green fluorescent protein (GFP) transgene into various hosts. Transplantation of the stem spermatogonia from irradiated adult rats into the testes of irradiated nude mice, which do not show the differentiation block of their own spermatogonia, permitted differentiation of the rat spermatogonia into spermatozoa. Conversely transplantation of spermatogonial stem cells from untreated prepubertal rats into irradiated rat testes showed that the donor spermatogonia were able to colonize along the basement membrane of the seminiferous tubules but could not differentiate. Finally, suppression of testosterone in the recipient irradiated rats allowed the differentiation of the transplanted spermatogonia. These results conclusively show that the defect caused by radiation in the rat testes that results in the block of spermatogonial differentiation is due to injury to the somatic compartment. We also observed colonization of tubules by transplanted Sertoli cells from immature rats. The present results suggest that transplantation of spermatogonia, harvested from prepubertal testes to adult testes that have been exposed to cytotoxic therapy might be limited by the somatic damage and may require hormonal treatments or transplantation of somatic elements to restore the ability of the tissue to support spermatogenesis.     

Basic features of bovine spermatogonial culture and effects of glial cell line-derived neurotrophic factor

Spermatogonial stem cells (SSC) are a small self-renewing subpopulation of type A spermatogonia, which for the rest are composed of differentiating cells with a very similar morphology. We studied the development of primary co-cultures of prepubertal bovine Sertoli cells and A spermatogonia and the effect of glial cell line-derived neurotropic factor (GDNF) on the numbers and types of spermatogonia, the formation of spermatogonial colonies and the capacity of the cultured SSC to colonize a recipient mouse testis. During the first week of culture many, probably differentiating, A spermatogonia entered apoptosis while others formed pairs and chains of A spermatogonia. After 1 week colonies started to appear that increased in size with time. Numbers of single (A(s)) and paired (A(pr)) spermatogonia were significantly higher in GDNF treated cultures at Days 15 and 25 (P < 0.01 and 0.05, respectively), and the ratio of A(s) to A(pr) and spermatogonial chains (A(al)) was also higher indicating enhanced self-renewal of the SSC. Furthermore, spermatogonial outgrowths in the periphery of the colonies showed a significantly higher number of A spermatogonia with a more primitive morphology under the influence of GDNF (P < 0.05). Spermatogonial stem cell transplantation experiments revealed a 2-fold increase in stem cell activity in GDNF treated spermatogonial cultures (P < 0.01). We conclude that GDNF rather than inducing proliferation, enhances self-renewal and increases survival rates of SSC in the bovine spermatogonial culture system.     

Histology of salmonid testes during maturation

The commonly applied classification systems of fish gonad maturity divide the maturation process into certain stages. However, the scales do not entirely reflect the continuity of the maturation process. Based on light microscope observations, the paper describes a comprehensive pattern of testicular transformations during maturation. The study was carried out on precocious underyearling and 1-year-old males of sea trout (Salmo trutta m. trutta L.), 1-year-old males of salmon (Salmo salar L.), and males of brown trout (Salmo trutta m. fario L.) aged from 7 months to 4 years. A total of 821 gonads collected during all seasons of the year were examined. The fish were fixed in Bouin's fluid. Histological slides of the mid-part of the gonad were made using the standard paraffin technique. The 3-6 microm sections were stained with Heidenhain haematoxylin. Histological changes of testes during maturation were similar in the three species studied. Immature and resting gonads contained type A spermatogonia in lobules only. The appearance of cystic structures containing type B spermatogonia in the lobules signalled the beginning of the sexual cycle in male gonads. Type B spermatogonia underwent synchronous mitotic divisions resulting in an increase in the total number of spermatogonia. As the spermatogenesis continued, the gonads showed a gradual increase in the number of cysts containing cells at all the spermatogenetic stages: type B spermatogonia, primary and secondary spermatocytes, spermatids, and spermatozoa. The well-formed spermatozoa were released to the lobule lumen once the Sertoli cells and spermatozoa connections broke up and the cyst disappeared. This was a continuous process observed throughout the spawning season. The spermatozoa were moved to the efferent duct. While some of the germ cells were completing spermatogenesis, the lobules contained less and less cysts with type B spermatogonia, primary and secondary spermatocytes, and spermatids; eventually all the cells completed spermatogenesis. At the end of maturation, vacuoles, up to 18.9 microm in final diameter (brown trout), appeared in the Sertoli cells. The vacuoles were visible in the lobule wall epithelium for a prolonged period of time. In most salmonid individuals examined, the reproductive cycles were observed to overlap. In some fish, the preparation for another cycle began very early, i.e., at the and of preceding spermatogenesis, which had not been observed before. Gonad maturation in some males was incomplete.     

Dorsomorphin Promotes Survival and Germline Competence of Zebrafish Spermatogonial Stem Cells in Culture

Zebrafish spermatogonial cell cultures were established from Tg(piwil1:neo);Tg(piwil1:DsRed) transgenic fish using a zebrafish ovarian feeder cell line (OFC3) that was engineered to express zebrafish Lif, Fgf2 and Gdnf. Primary cultures, initiated from testes, were treated with G418 to eliminate the somatic cells and select for the piwil1:neo expressing spermatogonia. Addition of dorsomorphin, a Bmp type I receptor inhibitor, prolonged spermatogonial stem cell (SSC) survival in culture and enhanced germline transmission of the SSCs following transplantation into recipient larvae. In contrast, dorsomorphin inhibited the growth and survival of zebrafish female germline stem cells (FGSCs) in culture. In the presence of dorsomorphin, the spermatogonia continued to express the germ-cell markers dazl, dnd, nanos3, vasa and piwil1 and the spermatogonial markers plzf and sox17 for at least six weeks in culture. Transplantation experiments revealed that 6 week-old spermatogonial cell cultures maintained in the presence of dorsomorphin were able to successfully colonize the gonad in 18% of recipient larvae and produce functional gametes in the resulting adult chimeric fish. Germline transmission was not successful when the spermatogonia were cultured 6 weeks in the absence of dorsomorphin before transplantation. The results indicate that Bmp signaling is detrimental to SSCs but required for the survival of zebrafish FGSCs in culture. Manipulation of Bmp signaling could provide a strategy to optimize culture conditions of germline stem cells from other species.     

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.     

Flow-cytometric isolation and enrichment of teleost type A spermatogonia based on light-scattering properties

The transplantation of germ cells is a powerful tool both for studying their development and for reproductive biotechnology. An intraperitoneal germ cell transplantation system was recently developed for use in several teleost species. Donor germ cells transplanted into the peritoneal cavity of hatchlings migrated toward and were incorporated into the recipient's genital ridges, where they underwent gametogenesis. Among male germ cells, only type A spermatogonia were capable of colonizing the recipient gonads, unlike those at more advanced stages. The enrichment of type A spermatogonia is therefore important to achieve efficient donor-cell incorporation and subsequent donor-derived gametogenesis. Here we established a simple and rapid system of isolation and enrichment for fish type A spermatogonia, using flow cytometry. Type A spermatogonia were found to have distinctive forward and side light scatter properties compared to that with other types of testicular cell. Based on these characteristics, we were able to isolate and enrich type A spermatogonia by using flow cytometry. After intraperitoneal transplantation, the enriched type A spermatogonia could be successfully incorporated into the recipient genital ridges. This flow cytometry approach using forward and side light scatter was also found to be applicable to other salmonid and sciaenid species, suggesting that it could be a powerful tool for isolating and enriching transplantable type A spermatogonia in a wide range of teleosts. We expect this method to contribute significantly to germ cell biology and biotechnology.     

Real-time observation of transplanted 'green germ cells': proliferation and differentiation of stem cells

To elucidate the mechanism of proliferation and differentiation of testicular germ cells, donor testicular germ cells labeled with enhanced green fluorescent protein (eGFP) were transplanted to recipient seminiferous tubules. The kinetics of colonization as well as of differentiation of the donor cells was followed in the same transplanted tubules (alive) under ultraviolet light. One week after transplantation, clusters of fluorescent cells were randomly spread as dots in the recipient seminiferous tubule, whereas non-homed cells flowed out from the testis to the epididymis. By 4 weeks after transplantation, green germ cells were observed with weak and moderate fluorescence along the recipient seminiferous tubule. By 8 weeks, proliferation and differentiation of the germ cells occurred, resulting in strong fluorescence in the middle part of the seminiferous tubule but in weak and moderate fluorescence at both terminals. The length of the fluorescent positive seminiferous tubule became longer. Detailed histological analyses of the recipient tubules indicated that the portions of the seminiferous tubule in weak, moderate, and strong fluorescence contained the spermatogonia, spermatogonia with spermatocytes, and all types of germ cells including spermatids, respectively. Thus, testicular stem cells colonized first as dots within 1 week, and then proliferated along the basement membrane of the seminiferous tubules followed by differentiation.     

Germ line chimera produced by transfer of cultured chick primordial germ cells.

Intrinsic primordial germ cells (PGCs) from stage 27 (5-day-old) chick embryonic germinal ridges were cultured in vitro for a further 5 days, and shown to proliferate on stroma cells derived from the germinal ridge. To determine whether these cultured PGCs could colonize and contribute to the germ-line, PGCs were isolated by gentle pipetting, labeled with PKH26 fluorescent dye and injected into the blood stream of stage 17 (2.5-day-old) chick embryos. The recipient embryos were incubated until they reached stage 28. Thin sections of these embryos were analysed by fluorescent confocal laser microscopy. These analyses showed that the labeled donor PGCs had migrated into the germinal ridges of the recipient embryos, and transplanted PGCs had undergone at least 3-7 divisions. These results suggest that PGCs that had passed far beyond the migration stage in vivo were still able to migrate, colonize and proliferate in recipient chick embryonic gonads.     

Estimation of Surfagon Influence on the Gonadal State of Rainbow Trout <Emphasis Type="Italic">Parasalmo mykiss</Emphasis> (=<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) Juveniles at the Background of Temperature Stress

Regulatory effect of surfagon—a synthetic analog of gonadotropin-releasing hormone—on the gonad state of rainbow trout (Kamchatka steelhead Parasalmo mykiss (=Oncorhynchus mykiss)) juveniles is investigated. The juveniles were 2.5 months old and were exposed to brief (4 days) heat stress (19–20°C). The increase of water temperature was followed by anomalies of gonads after 1 month (destruction of sex cells and hypertrophy of connective tissue). In females, the increased water temperature activates sex redetermination: in ovaries, cysts containing destroyed spermatogonia are found. In 1.5 months, the exogenous application of surfagon was followed by acceleration of spermatogenesis in gonads of experimental fish (2.5 months old) and by a slight decrease of fraction of anomalies in structure of their testes compared with the gonads of fish not exposed to surfagon. Exposure of rainbow trout to surfagon prior to sex differentiation is more efficient than after it is completed.     

Testis transplantation in male rainbow trout (Oncorhynchus mykiss)

The objective of the present study was to establish a procedure for the transplantation of an intact testis from one male rainbow trout (Oncorhynchus mykiss) to another individual and evaluate the reproductive function of the transplanted testis at sexual maturity. Isogenic (cloned) male rainbow trout were produced by crossing a completely homozygous male (YY) with a homozygous female (XX) to eliminate any problem of tissue rejection. Transplantation was performed on four pairs of sexually immature animals (n = 8); each served both as a donor and recipient. The left testis was removed by making a ventral midline incision to expose the body cavity and gonads. The left testis was disconnected at the anterior and posterior points of attachment and transferred to the recipient fish where it was placed in position adjacent to the pyloric cecae. The right testis was left intact. After 4 wk, the fish were injected (i.p.) twice weekly for 8 or 9 wk with salmon pituitary extract (1.5 mg/kg) to induce precocious sexual maturation. A similar number of untreated fish were maintained as controls. Following this treatment, all the fish were killed, and the right (intact) and left (transplanted) testes were removed, weighed, and sampled for sperm. Although the mean weights of the left, transplanted testes were significantly (P: < 0.05) smaller than the intact testes (transplants = 1.2 g; intact = 3.9 g), transplanted testes were present in all animals, had increased in mass, and were sexually mature containing sperm. The mean fertility, as measured by the proportion of eggs completing first cleavage, of sperm derived from transplanted testes (92%) was no different from the sperm obtained from intact testes (84%). Similarly, there was no difference in the number of embryos attaining the eyed stage of development, after 18 days of incubation, that were derived from transplanted (84%) or intact testes (85%).     

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 1994^1,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal². The use of donor males carrying the bacterial β-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals¹. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located³. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation^4,5, to study Sertoli cell-germ cell interaction^6,7, SSC homing and colonization^3,8, as well as SSC self-renewal and differentiation^9,10.Germ cell transplantation is also feasible in large species¹¹. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species¹². An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm¹³. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice¹⁴.     

Production of donor-derived offspring by allogeneic transplantation of Spermatogonia in the yellowtail (Seriola quinqueradiata)

Although the yellowtail (Seriola quinqueradiata) is the fish most commonly farmed in Japan, breeding of this species has not yet started. This is primarily due to the lack of sufficiently sophisticated methods for manipulating gametogenesis, which makes it difficult to collect gametes from specific dams and sires. If it were possible to produce large numbers of surrogate fish by transplanting germ cells isolated from donor individuals harboring desirable genetic traits, then the probability of acquiring gametes carrying the donor-derived haplotype would increase, and breeding programs involving this species might increase as a result. As a first step, we established a method for the allogeneic transplantation of yellowtail spermatogonia and the production of donor-derived offspring. Donor cells were collected from immature (10-month-old) yellowtail males with testes containing abundant type A spermatogonia, labeled with PKH26 fluorescent dye, and transferred into the peritoneal cavities of 8-day-old larvae. Fluorescence observation at 28 days post-transplantation revealed that PKH26-labeled cells were incorporated into recipients' gonads. To assess whether donor-derived spermatogonia could differentiate into functional gametes in the allogeneic recipient gonads, gametes collected from nine male and four female adult recipients were fertilized with wild-type eggs and milt. Analysis of microsatellite DNA markers confirmed that some of the first filial (F(1)) offspring were derived from donor fish, with the average contribution of donor-derived F(1) offspring being 66% and the maximum reaching 99%. These findings confirmed that our method was effective for transplanting yellowtail spermatogonia into allogeneic larvae to produce donor-derived offspring.     

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.     

Functional demonstration of the ability of a primary spermatogonium as a stem cell by tracing a single cell destiny in Xenopus laevis

In Xenopus, although primary spermatogonium (PG), the largest cell in the testis, is believed to be spermatogonial stem cell by histological observations, functional evidence has never been obtained. In the present study, we first indicated that culture of juvenile testis in a medium supplemented with follicle stimulating hormone resulted in no proliferation of PG. In this culture system, early secondary spermatogonia could undergo mitotic divisions with a concomitant decrease in their size, so that they became distinguishable in size from PG. Because the subcutaneous environment of juveniles permitted aggregates of the dissociated testicular cells to reconstruct the normal testis structure, we next inserted a genetically marked PG isolated from cultured testes into the aggregate and transplanted it subcutaneously. In this system, 73.9% of the aggregates contained a marked PG. When we observed the aggregates 12 weeks after transplantation, most aggregates (70.0%) contained marked PG that had self-renewed. Among these, fully growing aggregates contained many spermatogenic cells at the later developmental stage. These results suggested that isolated PG from the cultured testes had the ability as stem cells, and that purification of the spermatogenic stem cells became reliable in Xenopus.     

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.     

Testicular development in the sea trout (Salmo trutta morpha trutta L.) after sex differentiation, with a reference to precocious maturation

Testicular development was followed in juvenile sea trout (Salmo trutta morpha trutta L.) stocked in a river near Szczecin, Poland in 1992. Fish age was between 3 and 6.5 months post‐hatch. Fish were sampled monthly. Sex‐dependent differences in gonad structure and timing of their differentiation were observed after dissection under light microscope. In 3‐month‐old fry, when female gonads were differentiating (morphologically and cytologically), gonads of potential males remained undifferentiated. Development of a gonad into a male was primarily indicated by the formation of seminiferous tubules (lobules). In the sea trout under study, the lobules formed between the fourth and fifth month post‐hatch (July–August) (fork length >5.6 cm). There were no significant differences in body fork length between fish with and without lobules, although mean length of the former was higher. Early spermatogenesis began once the type B spermatogonia appeared. The timing of their appearance differed widely among individuals. Type B spermatogonia were found for the first time in a 5‐month‐old male (late August). Spermatocytes and cells of subsequent stages appeared in an incompletely matured 6.5‐month‐old male as ‘attempted spermatogenesis’ (fork length = 8.8 cm). Most examined males remained immature, their germ cells not having passed the type A spermatogonium level. In 6.5‐month‐old alevins, no significant differences in fish size between individuals beginning spermatogenesis (stage II) and those at stage I were detected, although those at stage II were longer. As the male gonad structures were forming, the quantitative gonad parameters were gradually increasing, even when referring to the unit area; only the gonocyte size gradually decreased. Generally, each observed monthly or bimonthly difference was statistically significant (P < 0.05).