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.     

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.     

Germ cell transplantation in goats

Transplantation of spermatogonial stem cells provides a unique approach for the study of spermatogenesis and manipulation of the male germ line. This technique may also offer an alternative to the currently inefficient methods of producing transgenic domestic animals. We have recently established the technique of spermatogonial transplantation, originally developed in laboratory rodents, in pigs, and this study was aimed to extend the technique to the goat. Isolated donor testis cells were infused into the seminiferous tubules of anesthetized recipient goats through an ultrasonographically-guided catheter inserted into the rete testis. Donor cells were obtained by enzymatic digestion of freshly collected testes from immature goats (either from the recipients' contralateral testis or from unrelated donors). Prior to transplantation, testis cells were labeled with a fluorescent marker to allow identification after transplantation. Recipient testes were examined for the presence and localization of labeled donor cells at 3-week intervals up to 12 weeks after transplantation. Labeled donor cells were found in the seminiferous tubules of all testes, comprising 10-35% of the examined tubules. Histological examination of the recipient testes did not reveal evident tissue damage, except for limited fibrotic changes at the site of needle insertion. Likewise there were no detectable local or systemic signs of immunologic reactions to the transplantations. These results indicate that germ cell transplantation is technically feasible in immature male goats and that donor-derived cells are retained in the recipient testis for at least three months and through puberty. This study represents the first report of germ cell transplantation in goats.     

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.     

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.     

Germ cell transplantation in pigs.

Spermatogonial stem cells form the foundation of spermatogenesis, and their transplantation provides a unique opportunity to study spermatogenesis and may offer an alternative approach for animal transgenesis. This study was designed to extend the technique of spermatogonial transplantation to an economically important, large-animal model. Isolated immature pig testes were used to develop the intratesticular injection technique. Best results of intratubular germ cell transfer were obtained when a catheter was inserted into the rete testis under ultrasound guidance. The presence of infused dye or labeled cells was confirmed in the seminiferous tubules from 70 of 89 injected isolated testes. Infusion of 3-6 ml of dye solution or cell suspension could fill the rete and up to 50% of seminiferous tubules. The technique was subsequently applied in vivo. Donor cells included testis cells from 1- or 10-wk-old boars (from the recipients' contralateral testis or unrelated donors) and those from mice carrying a marker gene. Porcine testis cells were labeled with a fluorescent marker before transplantation. Testes were examined for the presence and localization of labeled donor cells immediately after transplantation or every week for 4 wk. Labeled porcine donor cells were found in numerous seminiferous tubules from 10 of 11 testes receiving pig cells. These results indicate that germ cell transplantation is feasible in immature pigs, and that porcine transplanted cells are retained in the recipient testis for at least 1 mo. This study represents a first step toward successful spermatogonial transplantation in a farm animal species.     

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.     

Development of the stages of the cycle in mouse seminiferous epithelium after transplantation of green fluorescent protein-labeled spermatogonial stem cells

To study the mechanism of male germ cell differentiation, testicular germ cells carrying green fluorescent protein (GFP) as a transgene marker were transplanted into infertile mouse testis. Fluorescence-positive seminiferous tubule segments colonized with GFP-labeled donor germ cells were isolated and measured, and differentiated germ cells were analyzed in living squashed preparations. Cell associations in normal stages of the seminiferous epithelial cycle were also studied and used as a reference. Two months after transplantation, the average length of the colonies was 1.3 mm. The cell associations of transplanted colonies were consistent with those of normal stages of the cycle. However, stages of the cycle were not necessarily identical in different colonies. Three months after transplantation, the average length of transplanted colonies was 3.4 mm, and the cell association in every portion of a colony was similar to that of the corresponding stage of the cycle. Even in long fused colonies made by transplantation of a higher concentration of male germ cells, the cell association patterns in various regions of a single colony were similar and consistent with those of some of the normal stages of the cycle. Development of different stages inside the colony was observed by 6 mo after transplantation. These results indicate that the commencement of spermatogonial stem cell differentiation occurs randomly to develop different stages of the cycle in different colonies. Then, each colony shows one single stage of the cycle for a long time, even if it becomes a very large colony or fuses with other colonies. These observations indicate the existence of some kind of synchronization mechanism. By 6 mo, however, normal development of the stages of the cycle appeared in seminiferous tubules.     

Primate spermatogonial stem cells colonize mouse testes

In mice, transplantation of spermatogonial stem cells from a fertile male to the seminiferous tubules of an infertile recipient male results in progeny with donor-derived haplotype. Attempts to extend this approach by transplanting human testis cells to mice have led to conflicting claims that no donor germ cells persisted or that human spermatozoa were produced in the recipient. To examine this issue we used the baboon, a primate in which testis cell populations of several ages could be obtained for transplantation, and demonstrate that donor spermatogonial stem cells readily establish germ cell colonies in recipient mice, which exist for periods of at least 6 mo. However, differentiation of germ cells toward the lumen of the tubule and production of spermatozoa did not occur. The presence of baboon spermatogonial stem cells and undifferentiated spermatogonia in mouse seminiferous tubules for long periods after transplantation indicates that antigens, growth factors, and signaling molecules that are necessary for interaction of these cells and the testis environment have been preserved for 100 million years of evolutionary separation. Because germ cell differentiation and spermatogenesis did not occur, the molecules necessary for this process appear to have undergone greater divergence between baboon and mouse.     

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.     

Spermatogonial stem cell transplantation, cryopreservation and culture.

Testis cells of a fertile male mouse can be transplanted to the seminiferous tubules of an infertile male, where the donor spermatogonial stem cells will establish spermatogenesis and produce spermatozoa that transmit the donor haplotype to progeny. In addition, stem cells can be cryopreserved for long periods, thereby making male germ lines immortal. Recently, mouse testis cells have been cultured for longer than 3 months and, following transplantation, produced spermatogenesis. These techniques are likely to be applicable to many species, since rat testis cells can be cryopreserved and generate spermatogenesis in the seminiferous tubules of immunodeficient mice.     

Spontaneous degeneration of germ cells in normal rat testis: assessment of cell types and frequency during the spermatogenic cycle.

The occurrence of degenerating germ cells in the cycle of the seminiferous epithelium was measured in testicular tissues from eight normal adult rats. Testes were perfusion fixed, embedded in epoxy resin and, after sectioning a total of 180 randomly selected blocks at 1 microns, stained sections were examined by light microscopy; all cross-sectioned seminiferous tubules were categorized into one of 14 stages of the spermatogenic cycle. The number of degenerating cells per tubule was recorded in 2103 tubules. Degenerating germ cells were not detected at stages II-VI, and only rarely at stage VII (n = 366 tubules) in which one primary spermatocyte and one step 19 spermatid degenerated. All other stages exhibited a greater incidence of degenerative germ cells, particularly at stage XIV where, on average, the frequency of degenerating cells per round seminiferous tubule was about 40 times greater than at stage VII. The results indicated that, in the normal adult rat testis, the germ cells are least at risk of degeneration as they pass through stage VII.     

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.     

Characterization of germ cells from pre-pubertal bull calves in preparation for germ cell transplantation

Although methods to assess testis cell populations are established in mice, the detailed validation of similar methods for bovine testis cells is necessary for the development of emerging technologies such as male germ cell transplantation. As young calves provide donor cells for germ cell transplantation, we characterized cell populations from three key pre-pubertal stages. Nine Angus bull calves were selected to represent three stages of testis development at ages (and testis weights) of 2–3 months (Stage 1, 10 g), 4–5 months (Stage 2, 35 g), and 6–7 months (Stage 3, 70 g). The proportion and absolute numbers of germ and somatic cells in fixed sections and from enzymatically dissociated seminiferous tubules were assessed. Germ cells were identified by DBA and PGP9.5 staining, and Sertoli cells by vimentin and GATA-4 staining. Staining of serial sections confirmed that DBA and PGP9.5 identified similar cells, which were complementary to those stained for vimentin and GATA-4. In fixed tubules, the proportion of cells within tubules that were positive for DBA and PGP9.5 increased nearly three-fold from Stage 1 to Stage 2 with no further increase at Stage 3. Absolute numbers of spermatogonia also increased between Stages 1 and 2. After enzymatic dissociation of tubules, three times more DBA- and PGP9.5-positive cells were isolated from Stage 3 testes than from either Stage 1 or 2 testes. A higher proportion of spermatogonia was observed after enzymatic isolation than were present in seminiferous tubules. These data should help to predict the yield and expected proportions of spermatogonia from three distinct stages of testis development in pre-pubertal bull calves.     

Germ cell transplantation from large domestic animals into mouse testes

Donor-derived spermatogenesis after spermatogonial transplantation to recipient animals could serve as a novel approach to manipulate the male germ line in species where current methods of genetic modification are still inefficient. The objective of the present study was to investigate germ cell transplantation from boars, bulls, and stallions, which are economically important domestic animals, to mouse recipients. Donor testis cells (fresh, cryopreserved, or cultured for 1 month) were transplanted into testes of immunodeficient recipient mice in which endogenous spermatogenesis had been destroyed. Recipient testes were analyzed from 1 to > 12 months after transplantation for the presence of donor germ cells by donor-specific immunohistochemistry. Donor cells were present in most recipient testes with species-dependent differences in pattern and extent of colonization. Porcine donor germ cells formed chains and networks of round cells connected by intercellular bridges but later stages of donor-derived spermatogenesis were not observed. Transplanted bovine testis cells initially appeared similar but then developed predominantly into fibrous tissue within recipient seminiferous tubules. Few equine germ cells proliferated in mouse testes with no obvious difference between cells recovered from a scrotal or a cryptorchid donor testis. The pattern of colonization after transplantation of cultured cells did not resemble spermatogonial proliferation. These results indicate that fresh or cryopreserved germ cells from large animals can colonize the mouse testis but do not differentiate beyond the stage of spermatogonial expansion. Species-specific differences in the compatibility of large animal donors and mouse recipients were detected which cannot be predicted solely on the basis of phylogenetic distance between donor and recipient 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.     

The distribution of sex-specific (H-Y) antigens within the seminiferous tubules of the testis: an immunohistochemical study

Summary The cellular distribution of H-Y antigen within the seminiferous tubules of testes from both 20-day-old and adult rats has been examined immunohistochemically. Large amounts of diffuse-staining material surrounding the germ cells were observed within the tubules of 20-day-old rats while the germ cells appeared to have little H-Y positive material on them. In the sexually mature rat, the seminiferous tubules contained cells at various stages of development. Peroxidase staining was evident on many, but not all of these cells. On spermatids and spermatozoa with cytoplasmic droplets attached, peroxidase staining appeared to be present in only a proportion of these cells.     

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.     

Seminiferous tubule involution in elderly men

The observation of different types of seminiferous tubules (from tubules with normal spermatogenesis to sclerosed tubules) in aging human testes points to the progressive stages of tubular involution in elderly men. The tubules with hypospermatogonesis (reduced number of elongated spermatids) show numerous morphological anomalies in the germ cells, including multinucleated cells. Abnormal germ cells degenerate, causing Steroli cell vacuolation. These vacuoles correspond to dilations of the extracellular spaces resulting from the premature exfoliation of germ cells. Degenerating cells that are phagocytized by Sertoli cells lead to an accumulation of lipid droplets in the Sertoli cell cytoplasm. The loss of germ cells begins with spermatids, but progressively affects the preceding germ cell types, and tubules with maturation arrested at the level of spermatocytes or spermatogonia are observed. Simultaneously, an enlargement of the tunica propria occurs. This leads to the formation of sclerosed tubules, some of which display a low seminiferous epithelium consisting of a few cells--including lipid-loaded Sertoli cells and both Ap and Ad spermatogonia--and others, showing complete sclerosis, are devoid of seminiferous epithelium. The development of tubular involution is similar to that reported after experimental ischemia, which also seems to cause nonspecific effects on the testis such as multinucleate cells, vacuoles, and increased lipids in Sertoli cells.     

Restoration of spermatogenesis in infertile mice by Sertoli cell transplantation

The niche is considered to play an important role in stem cell biology. Sertoli cells are the only somatic cells in the seminiferous tubule that closely interact with germ cells to create a favorable environment for spermatogenesis. However, little is known about how Sertoli cells develop to form the male germ line niche. We report here that Sertoli cells recovered and dissociated from testes of donor male mice can be microinjected into recipient testes, form mature seminiferous tubule structures, and support spermatogenesis. Sertoli cells from perinatal donors had a dramatically greater capacity for generating seminiferous tubules than those from adult donors. Furthermore, transplantation of wild-type Sertoli cells into infertile Steel/Steel(dickie) testes created a permissive testicular microenvironment for generating spermatogenesis and spermatozoa. Thus, our results demonstrate that the male germ line stem cell niche can be transferred between animals. In addition, the technique provides a novel tool with which to analyze spermatogenesis and might provide a mechanism for correcting fertility in males suffering from supporting cell defects.