Cytological characterization of the germinal line during testicular differentiation in the lizard Liolaemus gravenhorsti (gray)

The gross morphology, histology, and ultrastructure of Liolaemus gravenhorsti gonads prior to and after differntiation are described. Special emphasis has been given to characterization and changes of the germ cell line throughout intrauterine development and 3 days postpartum. During the pregonadal stage, the primordial germ cell migrates toward gonadal rudiments by way of the mesenchyme. These cells can easily be identified by their great size, voluminous and lobulated nucleus, great quantities of yolk platelets, microtubules, and numerous lipid inclusions. In the undifferentiated gonad, the germ cells (type 1 gonocytes) have an ovoid or spherical shape and autodigestion of yolk platelets, great development of Golgi complex, and mitochondrial aggregation, though fewer liposomes, pseudopodes, and microtubules were noted. Concomitantly with the beginning of mitosis, a third type of germ cell appears, the type 2 gonocytes, which are smaller, with poorly defined membranous systems in various degrees of involution. The seminiferous cords are organized when somatic cells of the medullar portion of the gonad surround type 1 gonocytes. Germinal cells are centrally localized in the cords. Near birth many gonocytes migrate toward the basal lamina of cords and differentiate into spherical prespermatogonia, with few organoids. Sertoli cells eparate them from the basal lamina. In advanced pregnancy, Leyding cells become numerous with morphology typical of androgen-producing cells.     

Identification, isolation, and in vitro culture of porcine gonocytes

Gonocytes are primitive germ cells that reside in the seminiferous tubules of neonatal testes and give rise to spermatogonia, thereby initiating spermatogenesis. Due to a lack of specific markers, the isolation and culture of these cells has proven to be difficult in the pig. In the present study, we show that a lectin, Dolichos biflorus agglutinin (DBA), which has specific affinity for primordial germ cells (PCGs) in the genital ridge, binds specifically to gonocytes in neonatal pig testes. The specific affinity of DBA for germ cells was progressively lost with age. This suggests that DBA binds strongly to primitive germ cells, such as gonocytes, weakly to primitive spermatogonia, and not at all to spermatogonia. The presence of alkaline phosphatase (AP) activity in the germ cells of neonatal pig testis confirmed the existence of primitive germ cells. Gonocytes from neonatal pig testis were purified, and a cell population that consisted of approximately 70% gonocytes was obtained, as indicated by the DBA binding assay. Purified gonocytes were cultured in DMEM/F12 supplemented with 10% FBS in the absence of any specific growth factors for 7 days. The cells remained viable and proliferated actively in culture. Initially, the gonocytes grew as focal colonies that transformed to three-dimensional colonies by 7 days of culture. Cultured germ cells expressed SSEA-1, a marker for embryonic stem (ES) cells, and were negative for the expression of somatic cell markers. These results should help to establish a male germ cell line that could be used for studying spermatogenesis in vitro and for genetic modification of pigs.     

Male differentiation of germ cells induced by embryonic age-specific Sertoli cells in mice

Retinoic acid (RA) is a meiosis-inducing factor. Primordial germ cells (PGCs) in the developing ovary are exposed to RA, resulting in entry into meiosis. In contrast, PGCs in the developing testis enter mitotic arrest to differentiate into prospermatogonia. Sertoli cells express CYP26B1, an RA-metabolizing enzyme, providing a simple explanation for why XY PGCs do not initiate meios/is. However, regulation of entry into mitotic arrest is likely more complex. To investigate the mechanisms that regulate male germ cell differentiation, we cultured XX and XY germ cells at 11.5 and 12.5 days postcoitus (dpc) with an RA receptor inhibitor. Expression of Stra8, a meiosis initiation gene, was suppressed in all groups. However, expression of Dnmt3l, a male-specific gene, during embryogenesis was elevated but only in 12.5-dpc XY germ cells. This suggests that inhibiting RA signaling is not sufficient for male germ cell differentiation but that the male gonadal environment also contributes to this pathway. To define the influence of Sertoli cells on male germ cell differentiation, Sertoli cells at 12.5, 15.5, and 18.5 dpc were aggregated with 11.5 dpc PGCs, respectively. After culture, PGCs aggregated with 12.5 dpc Sertoli cells increased Nanos2 and Dnmt3l expression. Furthermore, these PGCs established male-specific methylation imprints of the H19 differentially methylated domains. In contrast, PGCs aggregated with Sertoli cells at late embryonic ages did not commit to the male pathway. These findings suggest that male germ cell differentiation is induced both by inhibition of RA signaling and by molecule(s) production by embryonic age-specific Sertoli cells.     

Migratory and postmigratory mouse primordial germ cells behave differently in culture

In all vertebrate groups, the progenitors of the germ line, the primordial germ cells (PGCs) arise extragonadally and move to the developing gonad early in embryonic development. We have examined the behavior of isolated pregonadal and gonadal PGCs in vitro on feeder layers of an embryo-derived cell line. Histochemically and serologically identified pregonadal germ cells are found to be actively motile in vitro and, furthermore, show behavior characteristic of invasive cells. PGCs isolated from the developing gonad, however, show little locomotory activity and are not invasive on the same cellular substrate. These observations suggest that PGCs undergo a major change in phenotype at the time of their entry into the gonad anlagen.     

Regulation of germ cell and Sertoli cell development by activin, follistatin, and FSH

We have demonstrated a role for activin A, follistatin, and FSH in male germ cell differentiation at the time when spermatogonial stem cells and committed spermatogonia first appear in the developing testis. Testis fragments from 3-day-old rats were cultured for 1 or 3 days with various combinations of these factors, incubated with bromodeoxyuridine (BrdU) to label proliferating cells, and then processed for stereological analysis and detection of BrdU incorporation. Gonocyte numbers were significantly elevated in cultures treated with activin, while the combination of FSH and the activin antagonist, follistatin, increased the proportion of spermatogonia in the germ cell population after 3 days. All fragment groups treated with FSH contained a significantly higher proportion of proliferating Sertoli cells, while activin and follistatin each reduced Sertoli cell division. In situ hybridization and immunohistochemistry on normal rat testes demonstrated that gonocytes, but not spermatogonia, contain the activin beta(A) subunit mRNA and protein. In contrast, gonocytes first expressed follistatin mRNA and protein at 3 days after birth, concordant with the transition of gonocytes to spermatogonia. Collectively, these data demonstrate that germ cells have the potential to regulate their own maturation through production of endogenous activin A and follistatin. Sertoli cells were observed to produce the activin/inhibin beta(A) subunit, the inhibin alpha subunit, and follistatin, demonstrating that these cells have the potential to regulate germ cell maturation as well as their own development. These findings indicate that local regulation of activin bioactivity may underpin the coordinated development of germ cells and somatic cells at the onset of spermatogenesis.     

RSPO1/β-catenin signaling pathway regulates oogonia differentiation and entry into meiosis in the mouse fetal ovary

Differentiation of germ cells into male gonocytes or female oocytes is a central event in sexual reproduction. Proliferation and differentiation of fetal germ cells depend on the sex of the embryo. In male mouse embryos, germ cell proliferation is regulated by the RNA helicase Mouse Vasa homolog gene and factors synthesized by the somatic Sertoli cells promote gonocyte differentiation. In the female, ovarian differentiation requires activation of the WNT/β-catenin signaling pathway in the somatic cells by the secreted protein RSPO1. Using mouse models, we now show that Rspo1 also activates the WNT/β-catenin signaling pathway in germ cells. In XX Rspo1(-/-) gonads, germ cell proliferation, expression of the early meiotic marker Stra8, and entry into meiosis are all impaired. In these gonads, impaired entry into meiosis and germ cell sex reversal occur prior to detectable Sertoli cell differentiation, suggesting that β-catenin signaling acts within the germ cells to promote oogonial differentiation and entry into meiosis. Our results demonstrate that RSPO1/β-catenin signaling is involved in meiosis in fetal germ cells and contributes to the cellular decision of germ cells to differentiate into oocyte or sperm.     

Neonatal mouse gonocyte proliferation assayed by an in vitro clonogenic method

Survival and proliferation of mouse gonocytes was studied using a single cell clonogenic assay in vitro. The effect of growth factors and extracellular matrix on clonogenic development was quantitated. Fundamental requirements for growth of neonatal gonocytes included addition of fetal calf serum and coating culture wells with collagen IV alone or with added fibronectin. After 4-5 days, colonies ranged in size from four to > 128 cells, and some contained very elongated cells indicating migratory behaviour. Soluble stem cell factor did not have any effect on clonogenicity, although STO (subline of SIM mouse fibroblasts) cells, which produce membrane-bound stem cell factor, reduced colony formation from 79 +/- 5.9% to 20 +/- 3.3% without added growth factor. The majority of gonocytes and type A spermatogonia express the c-kit receptor according to in situ hybridization studies. However, the results indicate that the receptor may not be functional in neonatal gonocytes and their immediate progeny. The current assay for gonocytes can be extended to test new growth factors or proliferation-inducing agents directly, as well as to study cell-cell interactions. This assay and long-term propagation of neonatal germ cells will provide the much needed resources to enable greater understanding of the early development of germ cells.     

Immunolocalization of albumin and transferrin in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis

The localization of albumin and transferrin was examined immunohistochemically in germ cells and Sertoli cells during rat gonadal morphogenesis and postnatal development of the testis. These proteins appeared as early as the 13th day of gestation in migrating primordial germ cells before Sertoli cell differentiation. In the fetal testis, strong immunoreactivity was only detected in the gonocytes. In the prepubertal testis, spermatogonia, primary spermatocytes, and some Sertoli cells accumulate albumin and transferrin. At puberty, different patterns of immunostaining of the germ cells were observed at the various stages of the cycle of the seminiferous epithelium. Diplotene spermatocytes at stage XIII, spermatocytes in division at stage XIV, and round spermatids at stages IV–VIII showed maximal staining. Labeling was evident in the cytoplasm of adult Sertoli cells. Albumin and transferrin staining patterns paralleled each other during ontogenesis.     

Clonogenic culture of normal spermatogonia: in vitro regulation of postnatal germ cell proliferation

The stem cell properties of neonatal germ cells have recently been demonstrated by in vivo transplantation. Regulation of proliferation of these cells, however, is not yet understood, and an in vitro system is needed for directly testing the action of differentiation and proliferation-related factors for germ cells. We developed an in vitro model involving micromanipulation and a single-cell clonogenic assay in which results from independent experiments on spermatogonia and gonocytes have been analyzed and compared. Neonatal germ cells can be distinguished by their large size both in vivo and in vitro in a single-cell suspension. These cells are picked up singly using a micropipette and deposited into a 96-well plate precoated with an extracellular matrix component, e.g., collagen IV. The effect of growth factors or cocultured somatic cells was assayed by counting the percentage of wells containing a colony and comparing this percentage with that of control cultures. Addition of platelet-derived growth factor significantly shifted the modal colony size for gonocytes from >16-64 to >64-128 cells/colony (P < 0.001, chi2) but had no effect on spermatogonia-derived colony size and number. For testis somatic cell underlays, there was a profound inhibition of all colony types, and immunohistochemical staining of testis cell underlays showed inhibin/activinbetaA subunit expression. This finding suggests that negative regulation of germ cell proliferation is mediated by inhibin. Addition of activin A to these cultures resulted in significant recovery (P = 0.046) of gonocyte-derived colony numbers but not spermatogonia-derived colonies, which may reflect the functional regulation by these factors observed in vivo. This proliferation assay also highlights many similarities in the regulation of gonocyte and spermatogonia proliferation in vitro, suggesting that proliferation potential is not noticeably affected by the transition of gonocytes to spermatogonia. For example, the average colony cloning efficiency was 80% for gonocytes and 76% for spermatogonia. This technology forms a basis for optimizing growth of neonatal germ cells for applications such as introduction of genetic material into the germ line to produce transgenic mice and to explore gene therapy.     

Caenorhabditis elegans germline patterning requires coordinated development of the somatic gonadal sheath and the germ line

Interactions between the somatic gonad and the germ line influence the amplification, maintenance, and differentiation of germ cells. In Caenorhabditis elegans, the distal tip cell/germline interaction promotes a mitotic fate and/or inhibits meiosis through GLP-1/Notch signaling. However, GLP-1-mediated signaling alone is not sufficient for a wild-type level of germline proliferation. Here, we provide evidence that specific cells of the somatic gonadal sheath lineage influence amplification, differentiation, and the potential for tumorigenesis of the germ line. First, an interaction between the distal-most pair of sheath cells and the proliferation zone of the germ line is required for larval germline amplification. Second, we show that insufficient larval germline amplification retards gonad elongation and thus delays meiotic entry. Third, a more severe delay in meiotic entry, as is exhibited in certain mutant backgrounds, inappropriately juxtaposes undifferentiated germ cells with cells of the proximal sheath lineage, leading to the formation of a proximal germline tumor derived from undifferentiated germ cells. Tumors derived from dedifferentiated germ cells, however, respond to the proximal interaction differently depending on the mutant background. Our study underscores the importance of strict developmental coordination between neighboring tissues. We discuss these results in the context of mechanisms that may underlie tumorigenesis.     

Germ cell differentiation and proliferation in the developing testis of the South American plains viscacha, Lagostomus maximus (Mammalia, Rodentia)

Cell proliferation and cell death are essential processes in the physiology of the developing testis that strongly influence the normal adult spermatogenesis. We analysed in this study the morphometry, the expression of the proliferation cell nuclear antigen (PCNA), cell pluripotency marker OCT-4, germ cell marker VASA and apoptosis in the developing testes of Lagostomus maximus, a rodent in which female germ line develops through abolished apoptosis and unrestricted proliferation. Morphometry revealed an increment in the size of the seminiferous cords with increasing developmental age, arising from a significant increase of PCNA-positive germ cells and a stable proportion of PCNA-positive Sertoli cells. VASA showed a widespread cytoplasmic distribution in a great proportion of proliferating gonocytes that increased significantly at late development. In the somatic compartment, Leydig cells increased at mid-development, whereas peritubular cells showed a stable rate of proliferation. In contrast to other mammals, OCT-4 positive gonocytes increased throughout development reaching 90% of germ cells in late-developing testis, associated with a conspicuous increase in circulating FSH from mid- to late-gestation. TUNEL analysis was remarkable negative, and only a few positive cells were detected in the somatic compartment. These results show that the South American plains viscacha displays a distinctive pattern of testis development characterized by a sustained proliferation of germ cells throughout development, with no signs of apoptosis cell demise, in a peculiar endocrine in utero ambiance that seems to promote the increase of spermatogonial number as a primary direct effect of FSH.     

Regulation of the proliferation of cocultured gonocytes and Sertoli cells by retinoids,triiodothyronine, and intracellular signaling factors: differences between fetal and neonatal cells

The regulation of early fetal germ cell growth has not been studied in cell culture, probably due to the poor survival of these cells. However, cell culture is the only system in which the control of cell growth can be studied independently of the influence of secreted testicular factors, which are diluted in the medium. We successfully cultured dispersed testicular cells from 16.5-day-old rat fetuses in defined medium and compared the growth of these cells with that of cells from 3-day-old neonates. In this system, fetal gonocytes displayed low levels of mitotic activity and their numbers remained stable. In contrast, neonatal gonocytes displayed high levels of mitotic activity and increased in number, these characteristics resembling those observed in vivo. We found that retinoic acid had deleterious effects on the number of gonocytes but did not affect Sertoli cell proliferation in fetal and neonatal cell cultures. Moreover, in fetal cell cultures, the decrease in the number of gonocytes resulted from a decrease in mitotic activity, probably due to a direct effect of retinoids on fetal gonocytes. Among the selective agonists for the retinoic acid receptor (RARalpha agonist, RARbeta agonist, and RARgamma agonist) and the retinoic X receptor (pan-RXR agonist) tested, only the RARalpha agonist reproduced the effects of retinoic acid at concentrations lower than its Kd value in both fetal and neonatal cell cultures. As both RARalpha and RXRalpha are present in fetal and neonatal gonocytes, we suggest that retinoic acid exerts its effects on gonocytes via a RARalpha-RXRalpha heterodimer, with RARalpha functioning as an active partner and RXRalpha as a passive partner. In this culture system, we show for the first time that triiodothyronine (T3) inhibits testicular fetal Sertoli cell and germ cell growth. We also tested intracellular signaling factors and found that a cAMP analog increased Sertoli cell proliferation and germ cell survival in both fetal and neonatal cells whereas phorbol esters (PMA) strongly inhibited the proliferation of fetal but not of neonatal gonocytes. None of the tested factors (T3, dbcAMP, and PMA) seemed to interact with the all-trans retinoic acid pathway. Thus, fetal gonocytes and neonatal gonocytes differ in intrinsic properties, and their growth is not regulated in the same manner. Despite their low level of mitotic activity, fetal gonocytes were more sensitive to various factors than neonatal gonocytes.     

Expression of DNA methyltransferase (Dnmt1) in testicular germ cells during development of mouse embryo

The DNA methylation pattern is reprogrammed in embryonic germ cells. In female germ cells, the short-form DNA methyltransferase Dnmt1, which is an alternative isoform specifically expressed in growing oocytes, plays a crucial role in maintaining imprinted genes. To evaluate the contribution of Dnmt1 to the DNA methylation in male germ cells, the expression profiles of Dnmt1 in embryonic gonocytes were investigated. We detected a significant expression of Dnmt1 in primordial germ cells in 12.5-14.5 day postcoitum (dpc) embryos. The expression of Dnmt1 was downregulated after 14.5 dpc after which almost no Dnmt1 was detected in gonocytes prepared from 18.5 dpc embryos. The short-form Dnmt1 also was not detected in the 16.5-18.5 dpc gonocytes. On the other hand, Dnmt1 was constantly detected in Sertoli cells at 12.5-18.5 dpc. The expression profiles of Dnmt1 were similar to that of proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, suggesting that Dnmt1 was specifically expressed in the proliferating male germ cells. Inversely, genome-wide DNA methylation occurred after germ cell proliferation was arrested, when the Dnmt1 expression was downregulated. The present results indicate that not Dnmt1 but some other type of DNA methyltransferase contributes to the creation of DNA methylation patterns in male germ cells.     

维甲酸直接诱导鸡原始生殖细胞启动并完成减数分裂

维甲酸（RA）在胚胎期生殖细胞启动减数分裂过程中发挥重要的调控作用,但RA与性腺细胞的作用机制及其能否诱导生殖细胞完成整个减数分裂生成配子的问题尚不清楚.本文以鸡原始生殖细胞体外无饲养层培养体系为模型,避开性腺体细胞的影响,研究RA诱导PGC进入减数分裂的作用机理.研究发现,在无体细胞的情况下,RA显著上调鸡胚PGC中STRA8,SYCP3和DMC1的mRNA和蛋白表达水平,从而促进其进入减数分裂;同时,流式细胞分析和吉姆萨染色结果表明,RA能使鸡胚PGC经历各个减数分裂时期,最终生成36.5%～58.4%单倍体生殖细胞;此外,本实验还对雌性和雄性PGC对RA的应答能力进行了研究,发现两者对RA的敏感程度相似.综上所述,RA能直接诱导PGC启动并完成整个减数分裂过程,生成单倍体生殖细胞,无需体细胞或其他因子的介导.这为临床上治疗不孕不育及配子形成的机理研究提供了基础.     

The role of p63 in germ cell apoptosis in the developing testis

The fetal and neonatal development of male germ cells (gonocytes) is a poorly understood but crucial process for establishing fertility. In rodents, gonocytes go through two phases of proliferation accompanied by apoptosis and separated by a quiescent period during the end of fetal development. P63 is a member of the P53 gene family that yields six isoforms. We detected only the p63 protein and no p53 and p73 in the nucleus of the gonocytes of mouse testes. We report for the first time the ontogeny of each p63 mRNA isoform during testis development. We observed a strong expression of p63gamma mRNA and protein when gonocytes are in the quiescent period. In vitro treatment with retinoic acid prevented gonocytes from entering the quiescent period and was correlated with a reduced production of p63gamma isoform mRNA. We investigated the function of p63 by studying the testicular phenotype of P63-null mice. P63 invalidation slightly, but significantly increased the number of gonocytes counted during the quiescent period. As P63-null animals die at birth we used an original organ culture that mimicked neonatal in vivo development to study further the testicular development. P63 invalidation resulted in a sharply increased number of gonocytes during the culture period due to a decrease in spontaneous apoptosis with no change in proliferation. P63 invalidation also caused abnormal morphologies in the germ cells that were also found in P63(+/-) adult male mice. Thus, p63 appears as an important regulator of germ cell development.     

Differentiation of murine premigratory primordial germ cells in culture.

In the mouse embryo, primordial germ cells first appear in the extraembryonic mesoderm and divide rapidly while migrating to the fetal gonad. Shortly after their arrival in the gonad, germ cells sexually differentiate as proliferation ceases. Previous studies have established that primordial germ cells proliferate and migrate in feeder layer culture. To explore cellular regulation of fetal germ cell development, we have used germ cell nuclear antigen 1 (GCNA1), a marker normally expressed only in postmigratory germ cells, to investigate the developmental potency of both pre- and postmigratory cells in this culture system. We found that explanted premigratory germ cells will initiate expression of this marker and are, therefore, capable of undertaking some aspects of gonocyte differentiation without intimate exposure to the fetal gonad. We have also tested whether postmigratory gonocytes are stable in culture. As detected by either alkaline phosphatase or GCNA1, we did not detect long-term survival of either prospermatogonia or oogonia under conditions that support the survival, proliferation, and differentiation of earlier premigratory cells. These observations are consistent with an autonomous cellular mechanism governing the initial stages of gonocyte differentiation, and suggest that differentiation towards gonocytes is accompanied by a change in requirements for cell survival.     

Spermatogenesis and germ cell transgene expression in xenografted bovine testicular tissue

The present study was conducted to evaluate the development of spermatogenesis and utility of using electroporation to stably transfect germ cells with the beta-galactosidase gene in neonatal bovine testicular tissue ectopically xenografted onto the backs of recipient nude mice. Bull testicular tissue from 4-wk donor calves, which contains a germ cell population consisting solely of gonocytes or undifferentiated spermatogonia, was grafted onto the backs of castrated adult recipient nude mice. Testicular grafts significantly increased in weight throughout the grafting period and the timing of germ cell differentiation in grafted tissue was consistent with postnatal testis development in vivo relative to the bull. Seminiferous tubule diameter also significantly increased with advancing time after grafting. At 1 wk after grafting, gonocytes in the seminiferous cords completed migration to the basement membrane and differentiated germ cell types could be observed 24 wk after grafting. The presence of elongating spermatids at 24 wk confirmed that germ cell differentiation occurred in the bovine tissue. Leydig cells in the grafted bovine tissue were also capable of producing testosterone in the castrated recipient mice from 4 wk to 24 wk after grafting at concentrations that were similar to levels in intact, nongrafted control mice. The testicular tissue that had been electroporated with a beta-galactosidase expression vector showed tubule-specific transgene expression 24 wk after grafting. Histological analysis showed that transgene expression was present in both Sertoli and differentiated germ cells but not in interstitial cells. The system reported here has the potential to be used for generation of transgenic bovine spermatozoa.     

Epigenetic reprogramming in mouse primordial germ cells

Genome-wide epigenetic reprogramming in mammalian germ cells, zygote and early embryos, plays a crucial role in regulating genome functions at critical stages of development. We show here that mouse primordial germ cells (PGCs) exhibit dynamic changes in epigenetic modifications between days 10.5 and 12.5 post coitum (dpc). First, contrary to previous suggestions, we show that PGCs do indeed acquire genome-wide de novo methylation during early development and migration into the genital ridge. However, following their entry into the genital ridge, there is rapid erasure of DNA methylation of regions within imprinted and non-imprinted loci. For most genes, the erasure commences simultaneously in PGCs in both male and female embryos, which is completed within 1 day of development. Based on the kinetics of this process, we suggest that this is an active demethylation process initiated upon the entry of PGCs into the gonadal anlagen. The timing of reprogramming in PGCs is crucial since it ensures that germ cells of both sexes acquire an equivalent epigenetic state prior to the differentiation of the definitive male and female germ cells in which new parental imprints are established subsequently. Some repetitive elements, however, show incomplete erasure, which may be essential for chromosome stability and for preventing activation of transposons to reduce the risk of germline mutations. Aberrant epigenetic reprogramming in the germ line would cause the inheritance of epimutations that may have consequences for human diseases as suggested by studies on mouse models.