The pluripotency factor LIN28 in monkey and human testes: a marker for spermatogonial stem cells?

Mammalian spermatogenesis is maintained by spermatogonial stem cells (SSCs). However, since evidentiary assays and unequivocal markers are still missing in non-human primates (NHPs) and man, the identity of primate SSCs is unknown. In contrast, in mice, germ cell transplantation studies have functionally demonstrated the presence of SSCs. LIN28 is an RNA-binding pluripotent stem cell factor, which is also strongly expressed in undifferentiated mouse spermatogonia. By contrast, two recent reports indicated that LIN28 is completely absent from adult human testes. Here, we analyzed LIN28 expression in marmoset monkey (Callithrix jacchus) and human testes during development and adulthood and compared it with that in mice. In the marmoset, LIN28 was strongly expressed in migratory primordial germ cells and gonocytes. Strikingly, we found a rare LIN28-positive subpopulation of spermatogonia also in adult marmoset testis. This was corroborated by western blotting and quantitative RT-PCR. Importantly, in contrast to previous publications, we found LIN28-positive spermatogonia also in normal adult human and additional adult NHP testes. Some seasonal breeders exhibit a degenerated (involuted) germinal epithelium consisting only of Sertoli cells and SSCs during their non-breeding season. The latter re-initiate spermatogenesis prior to the next breeding-season. Fully involuted testes from a seasonal hamster and NHP (Lemur catta) exhibited numerous LIN28-positive spermatogonia, indicating an SSC identity of the labeled cells. We conclude that LIN28 is differentially expressed in mouse and NHP spermatogonia and might be a marker for a rare SSC population in NHPs and man. Further characterization of the LIN28-positive population is required.     

Derivation and morphological characterization of mouse spermatogonial stem cell lines

Spermatogonial stem cells (SSCs), having yet to possess decisive markers, can only be detected retrospectively by transplantation assay. It was reported recently that mouse gonocytes collected from DBA/2 and ICR neonates propagated in vitro. This cultured germ cell, named the germline stem cell (GS cell), produced functional sperm to make progeny when transplanted into recipient mouse testes. Here we show that GS cell lines can be established not only from neonatal testes but also from the testis of adult mice. We also confirmed that GS cells once transplanted into a host testis can be recovered to resume in vitro expansion, indicating that they are convertible mutually with SSCs in adult testes. Confocal laser microscopic examination showed GS cells resemble undifferentiated spermatogonia in the adult testis. This unique cell line could be useful for research in germ cell biology and applicable as a new tool for the genetic engineering of animals.     

Identification of the starting point for spermatogenesis and characterization of the testicular stem cell in adult male rhesus monkeys

BACKGROUND: Spermatogonial expansion in man and non-human primates has been studied for decades. Controversy persists about the cell type representing the testicular stem cell and the exact kinetics of spermatogonial proliferation. We recently determined the starting point of spermatogenesis and proposed a model for clonal expansion of spermatogonia in adult macaques. Here we want to confirm the initiation event, study and compare the details of the kinetics of spermatogonial expansion in vivo and in vitro, and characterize a population of A spermatogonia acting as testicular stem cells. METHODS and RESULTS: We localized BrdU-positive spermatogonia in whole mounts and sections of adult rhesus monkey testes. Culture of testicular tissue was used to determine the expansion and differentiation of premeiotic germ cells. We confirm that A(pale) spermatogonia divide equally at stage VII and produce two types of progeny after mitosis at stage IX of the seminiferous cycle following defined clonal patterns. Small numbers of proliferating single A spermatogonia exist which present a population of label-retaining cells. CONCLUSIONS: In the rhesus monkey the population of A(pale) spermatogonia cycle continuously and initiate spermatogenesis by a self-renewing division at stage VII of the seminiferous epithelial cycle. Rarely dividing single A spermatogonia exist which potentially are the male germline stem cells in the primate testis.     

Stem cell based therapeutical approach of male infertility by teratocarcinoma derived germ cells

Infertility affects 13-18% of couples and growing evidence from clinical and epidemiological studies suggests an increasing incidence of male reproductive problems. There is a male factor involved in up to half of all infertile couples. The pathogenesis of male infertility can be reflected by defective spermatogenesis due to failure in germ cell proliferation and differentiation. We report here in vitro generation of a germ cell line (SSC1) from the pluripotent teratocarcinoma cells by a novel promoter-based sequential selection strategy and show that the SSC1 cell line form mature seminiferous tubule structures, and support spermatogenesis after transplantation into recipient testes. To select differentiated germ cell population, we generated a fusion construct (Stra8-EGFP) harbouring the 1.4 kb promoter region of germ line specific gene Stra8 and coding region of enhanced green fluorescence protein. This region was sufficient to direct gene expression to the germinal stem cells in testis of transgenic mice. The purified cells expressed the known molecular markers of spermatogonia Rbm, cyclin A2, Tex18, Stra8 and Dazl and the beta1- and alpha6-integrins characteristic of the stem cell fraction. This cell line undergoes meiosis and can develop into sperm when transplanted into germ cell depleted testicular tubules. Sperm were viable and functional, as shown by fertilization after intra-cytoplasmic injection into mouse oocytes. This approach provides the basis that is essential for studying the development and differentiation of male germ line stem cell, as well as for developing new approaches to reproductive engineering and infertility treatment.     

GDNF-mediated signaling via RET tyrosine 1062 is essential for maintenance of spermatogonial stem cells

Well-organized spermatogenesis, including the maintenance of spermatogonial stem cells (SSCs), is indispensable for continuous male fertility. Signaling by glial cell line-derived neurotrophic factor (GDNF) via the RET/GDNF family receptor α1 (GFRα1) receptor complex is essential for self-renewal of murine SSCs and may also regulate their differentiation. When phosphorylated, tyrosine 1062 in RET presents a binding site for the phosphotyrosine-binding domains of several adaptor and effector proteins that are important for activation of a variety of intracellular signaling pathways. In this study, we investigated the role of signaling via RET tyrosine 1062 in spermatogenesis using RET Y1062F knockin mice (Y1062F mice), in which tyrosine 1062 was replaced with phenylalanine. Homozygous Y1062F mice showed marked atrophy of testes due to reduced production of germ cells. RET-expressing spermatogonia in seminiferous tubules of homozygous Y1062F mice decreased after postnatal day (P) 7 and germ cells were almost undetectable by P21. These phenomena appeared to be due to a lack of SSC self-renewal and inability to maintain the undifferentiated state. Our findings suggest that RET signaling via tyrosine 1062 is essential for self-renewal of SSCs and regulation of their differentiation.     

Testicular development in cynomolgus monkeys

The testes from 136 male cynomolgus monkeys were examined histopathologically in order to investigate the relationship between the development of spermatogenesis and testis weight, age, and body weight. At Grade 1 (immature), Sertoli cells and spermatogonia were the only cell classes in the testis. At Grade 2 (pre-puberty), no elongated spermatids were observed in the testis, although a few round spermatids and small lumen formation were observed. At Grade 3 (onset of puberty), all classes of germ cells were observed in the testis, although seminiferous tubule diameters and numbers of germ cells were small. Slight debris in the epididymis was observed in almost all animals. At Grade 4 (puberty), almost complete spermatogenesis was observed in the seminiferous tubules and it was possible to ascertain the spermatogenesis stage as described by Clermont, although tubule diameters and numbers of germ cells were small. There was less debris in the epididymis than at Grade 3. At Grade 5 (early adult), complete spermatogenesis was observed in the seminiferous tubules. At Grade 6 (adult), complete spermatogenesis in the seminiferous tubules and a moderate or large number of sperm in the epididymis were observed. Moreover, sperm analysis using ejaculated sperm was possible. Logistic regression analysis showed that testis weight is a good indicator of testicular maturity.     

Germ cell transfer into rat, bovine, monkey and human testes.

Germ cell transplantation is a potentially valuable technique offering oncological patients gonadal protection by reinitiating spermatogenesis from stem cells which were reinfused into the seminiferous tubules. In order to achieve an intratubular germ cell transfer, intratubular microinjection, efferent duct injections and rete testis injections were applied on dissected testes of four different species: rat, bull, monkey and man. Ultrasound-guided intratesticular rete testis injection was the best and least invasive injection technique with maximal infusion efficiency for larger testes. Deep infiltration of seminiferous tubules was only achieved in immature or partially regressed testes. This technique was applied in vivo on two cynomolgus monkeys. In the first monkey a deep infusion of injected cells and dye into the lumen of the seminiferous tubules was achieved. In the second, transplanted germ cells were present in the seminiferous epithelium 4 weeks after the transfer. These cells were morphologically identified as B-spermatogonia and located at the base of the seminiferous epithelium. In summary, this paper describes a promising approach for germ cell infusion into large testes. The application of this technique is the first successful attempt of a germ cell transfer in a primate.     

Germ cell transplantation into X-irradiated monkey testes.

BACKGROUND: An intense debate is ongoing regarding options for fertility protection in oncological patients. Germ cell transplantation has been applied to restore mouse spermatogenesis. Here, an attempt to apply autologous germ cell transplantation to a primate animal model is described. METHODS: Five adult male cynomolgus monkeys were biopsied to retrieve and cryopreserve germ cells before both testes were irradiated (dose 2 Gy). Six weeks later, each monkey received an infusion of its own cell suspension into the right testis, while the left testes were infused with saline. Testis size, sperm counts and serum concentrations of inhibin, FSH and testosterone were analysed weekly for 9 months. Spermatogenic recovery was determined histologically at the end of the study. RESULTS: In four monkeys, the germ cell-infused right testes showed a slight to moderate increase in the rate of regrowth in comparison with the left testes. In two monkeys the right testis proceeded to recover more prominently, resulting in larger right testis volumes and better or full spermatogenic recovery at the study end. Restoration of spermatogenesis occurred as an all-or-nothing event. Inhibin B concentrations increased, while FSH and testosterone concentrations decreased with testicular regrowth. Sperm counts did not recover. CONCLUSIONS: The present study demonstrates the immaturity and complexity of germ cell transplantation as a clinical approach.     

Identification of stem cells during prepubertal spermatogenesis via monitoring of nucleostemin promoter activity

The nucleostemin (NS) gene encodes a nucleolar protein found at high levels in several types of stem cells and tumor cell lines. The function of NS is unclear but it may play a critical role in S-phase entry by stem/progenitor cells. Here we characterize NS expression in murine male germ cells. Although NS protein was highly expressed in the nucleoli of all primordial germ cells, only a limited number of gonocytes showed NS expression in neonatal testes. In adult testes, NS protein was expressed at high levels in the nucleoli of spermatogonia and primary spermatocytes but at only low levels in round spermatids. To evaluate the properties of cells expressing high levels of NS, we generated transgenic reporter mice expressing green fluorescent protein (GFP) under the control of the NS promoter (NS-GFP Tg mice). In adult NS-GFP Tg testes, GFP and endogenous NS protein expression were correlated in spermatogonia and spermatocytes but GFP was also ectopically expressed in elongated spermatids and sperm. In testes of NS-GFP Tg embryos, neonates, and 10-day-old pups, however, GFP expression closely coincided with endogenous NS expression in developing germ cells. In contrast to a previous report, our results support the existence in neonatal testes of spermatogonial stem cells with long-term repopulating capacity. Furthermore, our data show that NS expression does not correlate with cell-cycle status during prepuberty, and that strong NS expression is essential for the maintenance of germline stem cell proliferation capacity. We conclude that NS is a marker of undifferentiated status in the germ cell lineage during prepubertal spermatogenesis.     

The application of biomarkers of spermatogonial stem cells for restoring male fertility

Spermatogonial stem cells (SSCs) are defined by their ability to both self-renew and produce differentiated germ cells that will develop into functional spermatozoa. Because of this ability, SSCs can reestablish spermatogenesis after testicular damage caused by cytotoxic agents or after transplantation into an infertile recipient. Therefore, SSCs are an important target cell for restoring male fertility, particularly for cancer patients who have to undergo sterilizing cancer therapies. In the mouse, the identification of SSC markers allows for the isolation of a highly enriched population of stem cells. This enriched stem cell population can be expanded in culture for an indefinite period of time, cryopreserved, and transplanted into infertile recipients to restore fertility. Thus, the identification of markers and the establishment of a long-term culture system for human SSCs will be crucial for realizing the potential of these cells in a clinical setting. In this article, we focus on the markers that have been identified for mouse SSCs and discuss how human SSC markers may be used in the restoration of fertility.     

Expression of plasminogen activator and inhibitor, urokinase receptor and inhibin subunits in rhesus monkey testes

The expression and localization of mRNA's for tissue plasminogen activator (tPA), urokinase PA (uPA), uPA receptor (uPAR) and inhibin subunits, alpha, beta A and beta B in monkey testes was investigated. Using in-situ hybridization with digoxigenin-labelled cRNA probes (dig- cRNA), we demonstrated that tPA and plasminogen activator inhibitor type 1 (PAI-1) were expressed in testes of both immature and mature rhesus monkeys. tPA mRNA was localized predominantly in Sertoli cells. Expression level was low in immature testis, increased dramatically in the adult and varied with seminiferous cycle. PAI-1 mRNA was localized mainly in germ cells except late spermatids. uPA mRNA was expressed stage-specifically in Sertoli cells of adult testis. uPA receptor mRNA was localized in germ cells of mature testis but not in spermatogonia or late spermatids. Assayed by fibrin overlay technique, PA activity in conditioned media of purified Sertoli cells (Sc) was negligible, PA activity in media obtained from co-cultured Sertoli and Leydig cells (LS), however, was significantly increased, although Leydig cells alone were not capable of producing any PA activity. Addition of follicle stimulating hormone (FSH) to the incubation medium remarkably increased PA secretion in both Sc and LS cultures. Human chronic gonadotrophin (HCG) had no significant effect on PA activity in the Sc culture but dramatically stimulated PA activity in the co-culture system. Dihydrotestosterone (DHT) did not mimic the effect of HCG. PAI-1 activity was secreted mainly by germ cells and did not differ between the two culture systems. FSH and forskolin inhibited PAI-1 secretion. Inhibin alpha, beta A and beta B subunit mRNAs were localized in Sertoli cells of adult monkey testes, with no obvious difference in the expression levels. These data suggest that PA/PAI-1 and other related factors are expressed in rhesus monkey testis under the control of various hormones, seminiferous cycle and cell-cell interactions through paracrine or autocrine regulation. Locally generated fibrinolysis may play an important role in the process of spermatogenesis.      

The genetic control and germ cell kinetics of the female and male germ line in mammals including man.

The female germ line (germ cell lineage, Keimbahn) is provided with only one proliferation wave, the oogenic, whereas male gametogenesis involves two successive waves: prespermatogenic, which corresponds to the female proliferation wave, and spermatogenesis, which is responsible for the immense number of male gametes produced in mature testes. Both male proliferation systems are linked by the transitional or T prospermatogonia. Using the reverse percentage of labelled metaphases method, it has been shown that the first differences between female and male germ cells can be identified by the end of the first wave, when oogonia and multiplying or M prospermatogonia are proliferating. This prenatal first wave of proliferation of male germ cells was also demonstrated in man and ceases around the 22nd week of pregnancy. Spermatogenesis involves a stock of stem cells (stem spermatogonia), a flexibly reacting pool of undifferentiated spermatogonia and several generations of differentiating spermatogonia, which proliferate almost exponentially. Furthermore, it consists of spermatocytes and haploid spermatids transforming into spermatozoa. The oocytes pass through the preleptotene stage, synthesizing DNA, and thereafter traverse the meiotic prophase up to the diplotene stage. In mammals they act as 'pre-embryos' in a similar but to a lesser degree than oocytes of amphibia and insects. The maternal chromosomes are largely responsible for the development of the embryo, the paternal genome for the development of the extra-embryonic tissue. The synthesis of transgenic animals is a powerful weapon in the armoury of geneticists, as has recently been demonstrated: a 14 kb genomic DNA fragment (Sry) is sufficient to induce testis differentiation and subsequent male development when introduced into chromosomally female mouse embryos.     

In vitro culture of testicular germ cells: regulatory factors and limitations

Spermatogenesis is regulated mainly by endocrine factors and also by testicular paracrine/autocrine growth factors. These factors are produced by Sertoli cells, germ cells, peritubular cells and interstitial cells, mainly Leydig cells and macrophages. The interactions and the ratio between Sertoli and germ cells in the seminiferous tubules ensure successful spermatogenesis. In order to culture spermatogonial stem cells (SSCs) in vitro, researchers tried to overcome some of the obstacles -- such as the low number of stem cells in the testis, absence of specific markers to identify SSCs -- in addition to difficulties in keeping the SSCs alive in culture. Recently, some growth factors important for the proliferation and differentiation of SSCs were identified, such as glial cell line derived neurotrophic factor (GDNF), stem cell factor (SCF) and leukemia inhibitory factor (LIF); also, markers for SSCs at different stages were reported. Therefore, some groups succeeded in culturing SSCs (under limitations), or more differentiated cells and even were able to produce in vitro germ cells from embryonic stem cells. Thus, success in culturing SSCs is dependent on understanding the molecular mechanisms behind self-renewal and differentiation. Culture of SSCs should be a good tool for discovering new therapeutic avenue for some infertile men or for patients undergoing chemotherapy/radiotherapy (pre-puberty or post-puberty).     

In vitro culture of testicular germ cells: Regulatory factors and limitations

Spermatogenesis is regulated mainly by endocrine factors and also by testicular paracrine/autocrine growth factors. These factors are produced by Sertoli cells, germ cells, peritubular cells and interstitial cells, mainly Leydig cells and macrophages. The interactions and the ratio between Sertoli and germ cells in the seminiferous tubules ensure successful spermatogenesis. In order to culture spermatogonial stem cells (SSCs) in vitro, researchers tried to overcome some of the obstacles—such as the low number of stem cells in the testis, absence of specific markers to identify SSCs—in addition to difficulties in keeping the SSCs alive in culture. Recently, some growth factors important for the proliferation and differentiation of SSCs were identified, such as glial cell line derived neurotrophic factor (GDNF), stem cell factor (SCF) and leukemia inhibitory factor (LIF); also, markers for SSCs at different stages were reported. Therefore, some groups succeeded in culturing SSCs (under limitations), or more differentiated cells and even were able to produce in vitro germ cells from embryonic stem cells.

Thus, success in culturing SSCs is dependent on understanding the molecular mechanisms behind self-renewal and differentiation. Culture of SSCs should be a good tool for discovering new therapeutic avenue for some infertile men or for patients undergoing chemotherapy/radiotherapy (pre-puberty or post-puberty).     

Cloning of cDNAs and the differential expression of A-type cyclins and Dmc1 during spermatogenesis in the Japanese eel, a teleost fish.

We cloned A-type cyclins (cyclins A1 and A2) and Dmc1 cDNAs from the eel testis. Cyclin A1 mRNA was predominantly expressed in the livers, ovaries, and testes of the eels. In contrast to cyclin A1 mRNA, a very high expression of cyclin A2 mRNA was observed in the brains, livers, kidneys, spleens, ovaries, and testes of the eels. Dmc1 mRNA was predominantly expressed in the testes and ovaries; expression in the brain was also detected. In the eel testis, a few type-A spermatogonia incorporating 5-bromo-2'-deoxyuridine (BrdU) were seen before the initiation of spermatogenesis by hormonal induction. On day 1 after hormonal induction, the number of BrdU-labeled spermatogonia increased remarkably, and after 3 and 6 days, many labeled type-B spermatogonia were also observed. The expression of cyclin A2 increased 1 day after the induction of spermatogenesis and reached a plateau after 6 days, when many type-B spermatogonia with high proliferative activity were found. In contrast, the expression of cyclin A1 mRNA was detected after 9 days, coincident with the first appearance of spermatocytes. Cyclin A1 mRNA was localized in germ cells of all stages, from primary spermatocytes to round spermatids, whereas cyclin A2 mRNA was specifically localized in spermatogonia, secondary spermatocytes, round spermatids, and testicular somatic cells, including Sertoli cells. Dmc1 was localized only in the earlier stages of primary spermatocytes; before this stage, cyclin A1 mRNA was not detectable. Overall, cyclin A2, Dmc1, and cyclin A1 are expressed in spermatogenic cells sequentially before and during meiosis in the eel testis.     

Spermatogonia,Germline Cells,and Testicular Organization in the Characiform Prochilodus lineatus Studied Using Histological,Stereological, and Morphometric Approaches

Prochilodus lineatus is an important representative of the order Characiformes and a species that offers great advantages to fish farming. Therefore, detailed knowledge of its reproductive biology can be applied to various fields of production and biotechnology. In this study, we have identified testicular germ cells during spermatogenesis and have evaluated the volumetric proportion of the testes occupied by structures of the tubular and intertubular compartments. In addition, the individual volume of type A spermatogonia was measured and used to estimate the mean number of these cells per testis. Gonads of adult P. lineatus males were extracted and fixed. Light and transmission electron microscopy were applied to fragments of three testicular regions. Histological, stereological, and morphometric analyses were performed. The stereological data suggest that components of the tubular and intertubular compartments of the P. lineatus testes present a uniform distribution in all three regions and therefore reflect regions with similar distributions of cell types. In addition, P. lineatus testes showed ～0.6% of type A spermatogonia, as well as a predominance of cysts of primary spermatocytes and spermatids during the reproductive phase evaluated. The results from this study provide a better understanding of the morphology and structure of the testis and of the characterization of the type A spermatogonia in P. lineatus. The nuclear diameter of germ cells also decreases significantly during spermatogenesis. The data presented herein are the first of its kind for the order Characiformes and may be useful for future biotechnology studies on fish reproduction. Anat Rec, 300:589–599, 2017. © 2016 Wiley Periodicals, Inc.     

BMP4/Smad Signaling Pathway Induces the Differentiation of Mouse Spermatogonial Stem Cells via Upregulation of Sohlh2

Spermatogonial stem cells (SSCs) capable of self‐renewal and differentiation are the foundation for spermatogenesis. Although several factors that govern these processes have been investigated, the underlying molecular mechanisms have not been fully elucidated. Here, we investigated the role of BMP4 in mouse SSC differentiation, and found that SSCs cultured in the presence of BMP4 underwent differentiation, characterized by downregulation of SSC self‐renewal markers, Plzf, and upregulation of SSC differentiation marker, c‐kit. Smad1/5/8 proteins were phosphorylated during BMP4‐induced differentiation. The effects of BMP4 on SSCs were blocked by BMP4 inhibitor (Dorsomorphin). The activation of BMP4/Smad signaling pathway in SSCs increased the expression of Sohlh2, which is involved in the early differentiation of spermatogonia. Knockdown sohlh2 expression by RNA interference abolished the effect of BMP4 on SSC differentiation and the upregulation of c‐kit expression. Overall, our results suggest that BMP4 plays an important role during the early differentiation of SSCs via upregulation of sohlh2. Anat Rec, 297:749–757, 2014. © 2014 Wiley Periodicals, Inc.