Cyp26b1 Expression in Murine Sertoli Cells Is Required to Maintain Male Germ Cells in an Undifferentiated State during Embryogenesis

In mammals, germ cells within the developing gonad follow a sexually dimorphic pathway. Germ cells in the murine ovary enter meiotic prophase during embryogenesis, whereas germ cells in the embryonic testis arrest in G0 of mitotic cell cycle and do not enter meiosis until after birth. In mice, retinoic acid (RA) signaling has been implicated in controlling entry into meiosis in germ cells, as meiosis in male embryonic germ cells is blocked by the activity of a RA-catabolizing enzyme, CYP26B1. However, the mechanisms regulating mitotic arrest in male germ cells are not well understood. Cyp26b1 expression in the testes begins in somatic cells at embryonic day (E) 11.5, prior to mitotic arrest, and persists throughout fetal development. Here, we show that Sertoli cell-specific loss of CYP26B1 activity between E15.5 and E16.5, several days after germ cell sex determination, causes male germ cells to exit from G0, re-enter the mitotic cell cycle and initiate meiotic prophase. These results suggest that male germ cells retain the developmental potential to differentiate in meiosis until at least at E15.5. CYP26B1 in Sertoli cells acts as a masculinizing factor to arrest male germ cells in the G0 phase of the cell cycle and prevents them from entering meiosis, and thus is essential for the maintenance of the undifferentiated state of male germ cells during embryonic development.     

Retinoic Acid signalling and the control of meiotic entry in the human fetal gonad

The development of mammalian fetal germ cells along oogenic or spermatogenic fate trajectories is dictated by signals from the surrounding gonadal environment. Germ cells in the fetal testis enter mitotic arrest, whilst those in the fetal ovary undergo sex-specific entry into meiosis, the initiation of which is thought to be mediated by selective exposure of fetal ovarian germ cells to mesonephros-derived retinoic acid (RA). Aspects of this model are hard to reconcile with the spatiotemporal pattern of germ cell differentiation in the human fetal ovary, however. We have therefore examined the expression of components of the RA synthesis, metabolism and signalling pathways, and their downstream effectors and inhibitors in germ cells around the time of the initiation of meiosis in the human fetal gonad. Expression of the three RA-synthesising enzymes, ALDH1A1, 2 and 3 in the fetal ovary and testis was equal to or greater than that in the mesonephros at 8-9 weeks gestation, indicating an intrinsic capacity within the gonad to synthesise RA. Using immunohistochemistry to detect RA receptors RARα, β and RXRα, we find germ cells to be the predominant target of RA signalling in the fetal human ovary, but also reveal widespread receptor nuclear localization indicative of signalling in the testis, suggesting that human fetal testicular germ cells are not efficiently shielded from RA by the action of the RA-metabolising enzyme CYP26B1. Consistent with this, expression of CYP26B1 was greater in the human fetal ovary than testis, although the sexually-dimorphic expression patterns of the germ cell-intrinsic regulators of meiotic initiation, STRA8 and NANOS2, appear conserved. Finally, we demonstrate that RA induces a two-fold increase in STRA8 expression in cultures of human fetal testis, but is not sufficient to cause widespread meiosis-associated gene expression. Together, these data indicate that while local production of RA within the fetal ovary may be important in regulating the onset of meiosis in the human fetal ovary, mechanisms other than CYP26B1-mediated metabolism of RA may exist to inhibit the entry of germ cells into meiosis in the human fetal testis.     

Differentiation of mouse primordial germ cells into female or male germ cells.

Mouse primordial germ cells (PGCs) migrate from the base of the allantois to the genital ridge. They proliferate both during migration and after their arrival, until initiation of the sex-differentiation of fetal gonads. Then, PGCs enter into the prophase of the first meiotic division in the ovary to become oocytes, while those in the testis become mitotically arrested to become prospermatogonia. Growth regulation of mouse PGCs has been studied by culturing them on feeder cells. They show a limited period of proliferation in vitro and go into growth arrest, which is in good correlation with their developmental changes in vivo. However, in the presence of multiple growth signals, PGCs can restart rapid proliferation and transform into pluripotent embryonic germ (EG) cells. Observation of ectopic germ cells and studies of reaggregate cultures suggested that both male and female PGCs show cell-autonomous entry into meiosis and differentiation into oocytes if they were set apart from the male gonadal environments. Recently, we developed a two-dimensional dispersed culture system in which we can examine transition from the mitotic PGCs into the leptotene stage of the first meiotic division. Such entry into meiosis seems to be programmed in PGCs before reaching the genital ridges and unless it is inhibited by putative signals from the testicular somatic cells.     

The Enzymatic Activity of Cu/Zn Superoxide Dismutase Does Not Fluctuate in Mouse Spermatogenic Cells Despite mRNA Changes

In the mammalian testis, multiple mRNAs encoding the copper zinc superoxide dismutase (SOD-1) are expressed in postmeiotic male germ cells. Here we relate SOD-1 mRNA levels to SOD-1 protein and enzyme activity levels in mouse spermatogenic cells. Although the sizes and relative amounts of the multiple SOD-1 mRNAs vary as male germ cells enter meiosis and proceed into the postmeiotic stages of spermatogenesis, the amount of SOD-1 protein and enzyme activity does not fluctuate significantly, suggesting a precise control of SOD-1 activity in male germ cells.     

New testicular mechanisms involved in the prevention of fetal meiotic initiation in mice

In mammals, early fetal germ cells are unique in their ability to initiate the spermatogenesis or oogenesis programs dependent of their somatic environment. In mice, female germ cells enter into meiosis at 13.5 dpc whereas in the male, germ cells undergo mitotic arrest. Recent findings indicate that Cyp26b1, a RA-degrading enzyme, is a key factor preventing initiation of meiosis in the fetal testis. Here, we report evidence for additional testicular pathways involved in the prevention of fetal meiosis. Using a co-culture model in which an undifferentiated XX gonad is cultured with a fetal or neonatal testis, we demonstrated that the testis prevented the initiation of meiosis and induced male germ cell differentiation in the XX gonad. This testicular effect disappeared when male meiosis starts in the neonatal testis and was not directly due to Cyp26b1 expression. Moreover, neither RA nor ketoconazole, an inhibitor of Cyp26b1, completely prevented testicular inhibition of meiosis in co-cultured ovary. We found that secreted factor(s), with molecular weight greater than 10 kDa contained in conditioned media from cultured fetal testes, inhibited meiosis in the XX gonad. Lastly, although both Sertoli and interstitial cells inhibited meiosis in XX germ cells, only interstitial cells induced mitotic arrest in germ cell. In conclusion, our results demonstrate that male germ cell determination is supported by additional non-retinoid secreted factors inhibiting both meiosis and mitosis and produced by the testicular somatic cells during fetal and neonatal life.     

Gonadal cell proliferation dimorphism

Dimorphism between testis and ovary in germ cells proliferative behavior, shows remarkable differences in foetal and neonatal period [14.5 days post conception (dpc)--7 days post partum (dpp)]. Immunostaining of the foetal testis, with the PCNA and Ki-67 antibodies [estimation of Labeling Index (LI)], reveals increasing germ cells population until birth. Afterwards, a sharp decline in the first 3 days of postnatal life and a transient increase, between 3 and 5 dpp, is observed. Then, the mitotic activity of germ cells ceases. In the foetal ovary, germ cells proliferation reaches a peak value before birth, decreasing thereafter Somatic (Sertoli or follicular) cells behave similarly in both sexes. Increased mitotic activity is observed throughout the examined period. Thus, the gonadal dimorphism in proliferative behavior, concerns only germ cell lineage and is established during the foetal and neonatal period.     

The effect of retinoic acid on the meiosis in the chick embryo (Gallus gallus domesticus)

In this study it was shown that the injection of retinoic acid (RA) into incubated eggs on day 9 or 14 induced entry the males germ cells into preleptotene stage of prophase I on day 17, which are absent in the control embryos. At the same time the meiosis marker SCP3 was detected in the germ cells. Which was also absent at control embryos. On day 19 in male embryos the number of male germ cells at the stage preleptoteny increased, but there were no germ cells in the following stages of the prophase of meiosis. In 20-day-old chicks meiotic germ cells were absent. Thus, white it is shown that the influence of RA on the developing chicken embryos induces the entry of germ cells into preleptotene stage of prophase I meiosis. However, further meiotic transformations don't occur. Thus RA is only one of many factors providing meiotic cell division.     

Gonadal morphogenesis and sex differentiation in the viviparous fish Chapalichthys encaustus (Teleostei, Cyprinodontiformes, Goodeidae)

This study describes the structural and ultrastructural characteristics of gonadal sex differentiation and expression of Vasa, a germline marker, in different developmental stages of embryos and newborn fry of the barred splitfin Chapalichthys encaustus, a viviparous freshwater teleost endemic to Mexico. In stage 2 embryos, the gonadal crest was established; gonadal primordia were located on the coelomic epithelium, formed by scarce germ and somatic cells. At stage 3, the undifferentiated gonad appeared suspended from the mesentery of the developing swimbladder and contained a larger number of germ and somatic cells. At stages 4 and 5, the gonads had groups of meiotic and non-meiotic germ cells surrounded by somatic cells; meiosis was evident from the presence of synaptonemal complexes. These stages constituted a transition towards differentiation. At stage 6 and at birth, the gonad was morphologically differentiated into an ovary or a testis. Ovarian differentiation was revealed by the presence of follicles containing meiotic oocytes, and testicular differentiation by the development of testicular lobules containing spermatogonia in mitotic arrest, surrounded by Sertoli cells. Nuage, electron-dense material associated with mitochondria, was observed in germ cells at all gonadal stages. The Vasa protein was detected in all of the previously described stages within the germ-cell cytoplasm. This is the first report on morphological characteristics and expression of the Vasa gene during sexual differentiation in viviparous species of the Goodeidae family. Chapalichthys encaustus may serve as a model to study processes of sexual differentiation in viviparous fishes and teleosts.     

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.     

检控蛋白Rad17在小鼠睾丸精子发生过程中的表达及其磷酸化变化

Rad17是细胞周期检控点信号转导过程中的一个关键检控蛋白,在DNA损伤检控和DNA复制检控中具有重要功能。但Rad17在细胞减数分裂中的检控作用还不是很清楚。因细胞减数分裂在睾丸组织中非常活跃,应用Western印迹检测Rad17在不同发育时期的小鼠睾丸组织中的表达及其磷酸化水平,并应用免疫组化的方法检测小鼠睾丸组织不同时期生殖细胞内Rad17的表达变化。结果显示Rad17在小鼠睾丸组织内高表达,而在肝、肾等组织中表达水平较低;Rad17在不同周龄的小鼠睾丸组织中均高水平表达,但在4周龄以后的小鼠睾丸组织中其磷酸化水平明显升高;免疫组化结果显示Rad17在精原细胞、精母细胞的细胞核中高表达,但在成熟精子细胞中消失。这些结果提示Rad17在小鼠睾丸生殖细胞减数分裂过程中也起重要检控作用。     

Surface location and stage-specificity of differentiation antigens on germ cells in the common carp (Cyprinus carpio), as revealed with monoclonal antibodies and immunogold staining

During development of juvenile and young adult carp (Cyprinus carpio, L., Teleostei) three differentiation stages were distinguished in the testis: the prespermatogenic, the early spermatogenic and the advanced spermatogenic testis. Carp testis tissue of these stages was dissociated by enzymatic digestion and viable testis cells with well preserved morphological features were obtained. The surface location and stage-specificity of differentiation antigens on these germ cells was investigated using monoclonal antibodies (MAbs) raised against carp spermatozoa. Binding of MAbs to cells was visualized with immunofluorescence as well as in the immunogold staining assay. Both methods revealed that antigenic determinants defined by seven MAbs were located on the outer surface of testis cells. Four MAbs, i.e. WCS 3, 17, 28 and 29, reacted with germ cells from both pre-spermatogenic testes (WCS 28 weakly) and spermatogenic testes. The antigenic determinants defined by three other MAbs, i.e. WCS 7, 11 and 12, appeared only after the onset of spermatogenesis. In the immunogold staining assay a post-fixation and nuclear staining procedure was developed which allowed identification of isolated germ cells, revealing clearly, for all seven MAbs, that the determinants were expressed on germ cells but not on somatic cells and, for WCS 7, 11 and 12 only, that the determinants first appeared on small spermatogonia prior to meiosis. A survey of the immunogold assay on the binding of the seven MAbs with isolated germ cells from ovaries, is included.     

Gene expression of the tyrosine kinase receptor c-kit during ovarian development in the brushtail possum (Trichosurus vulpecula).

Ovarian development and function have been extensively studied in eutherian species, with stem cell factor and its receptor, c-kit, having been shown to play key roles at various stages of these processes. In contrast, relatively little is known regarding ovarian development in marsupials. The aims of this study were, first, to establish the timing of key events during germ cell maturation and follicular development and, second, to determine the timing and cellular localization of gene expression for c-kit in the ovaries of a marsupial, the brushtail possum (Trichosurus vulpecula). For this study, ovaries were collected from possums ranging in age from Day 1 after birth to adult. Using stereology, the number of germ cells was found to increase rapidly during the first 60-100 days of life. This was followed by a sharp decline in number, wherein almost 90% of germ cells had disappeared by Day 180. From histological examinations, the time of initiation of meiosis, follicular formation, and follicular growth were determined to occur on Days 35, 50, and 60, respectively. Using in situ hybridization, c-kit gene expression was localized to germ cells and somatic cells during the first 15 days of life; however, after Day 30 and into adult life, c-kit expression was exclusive to germ cells. Results from this study suggest that the pattern of ovarian development is similar in marsupials to eutherians, and that c-kit may play a key role in germ cell development at various stages throughout life.     

Meiotic germ cells antagonize mesonephric cell migration and testis cord formation in mouse gonads

The developmental fate of primordial germ cells in the mammalian gonad depends on their environment. In the XY gonad, Sry induces a cascade of molecular and cellular events leading to the organization of testis cords. Germ cells are sequestered inside testis cords by 12.5 dpc where they arrest in mitosis. If the testis pathway is not initiated, germ cells spontaneously enter meiosis by 13.5 dpc, and the gonad follows the ovarian fate. We have previously shown that some testis-specific events, such as mesonephric cell migration, can be experimentally induced into XX gonads prior to 12.5 dpc. However, after that time, XX gonads are resistant to the induction of cell migration. In current experiments, we provide evidence that this effect is dependent on XX germ cells rather than on XX somatic cells. We show that, although mesonephric cell migration cannot be induced into normal XX gonads at 14.5 dpc, it can be induced into XX gonads depleted of germ cells. We also show that when 14.5 dpc XX somatic cells are recombined with XY somatic cells, testis cord structures form normally; however, when XX germ cells are recombined with XY somatic cells, cord structures are disrupted. Sandwich culture experiments suggest that the inhibitory effect of XX germ cells is mediated through short-range interactions rather than through a long-range diffusible factor. The developmental stage at which XX germ cells show a disruptive effect on the male pathway is the stage at which meiosis is normally initiated, based on the immunodetection of meiotic markers. We suggest that at the stage when germ cells commit to meiosis, they reinforce ovarian fate by antagonizing the testis pathway.     

Surface location and stage-specificity of differentiation antigens on germ cells in the common carp (Cyprinus carpio), as revealed with monoclonal antibodies and immunogold staining

Summary During development of juvenile and young adult carp (Cyprinus carpio, L., Teleostei) three differentiation stages were distinguished in the testis: the prespermatogenic, the early spermatogenic and the advanced spermatogenic testis. Carp testis tissue of these stages was dissociated by enzymatic digestion and viable testis cells with well preserved morphological features were obtained. The surface location and stage-specificity of differentiation antigens on these germ cells was investigated using monoclonal antibodies (MAbs) raised against carp spermatozoa. Binding of MAbs to cells was visualized with immunofluorescence as well as in the immunogold staining assay. Both methods revealed that antigenic determinants defined by seven MAbs were located on the outer surface of testis cells. Four MAbs, i.e. WCS 3, 17, 28 and 29, reacted with germ cells from both pre-spermatogenic testes (WCS 28 weakly) and spermatogenic testes. The antigenic determinants defined by three other MAbs, i.e. WCS 7, 11 and 12, appeared only after the onset of spermatogenesis. In the immunogold staining assay a post-fixation and nuclear staining procedure was developed which allowed identification of isolated germ cells, revealing clearly, for all seven MAbs, that the determinants were expressed on germ cells but not on somatic cells and, for WCS 7, 11 and 12 only, that the determinants first appeared on small spermatogonia prior to meiosis. A survey of the immunogold assay on the binding of the seven MAbs with isolated germ cells from ovaries, is included.     

Cell proliferation and hormonal changes during postnatal development of the testis in the pig

Histometrical evaluation of the testis was performed in 36 Piau pigs from birth to 16 mo of age to investigate Sertoli cell, Leydig cell, and germ cell proliferation. In addition, blood samples were taken in seven animals from 1 wk of age to adulthood to measure plasma levels of FSH and testosterone. Sertoli cell proliferation in pigs shows two distinct phases. The first occurs between birth and 1 mo of age, when the number of Sertoli cells per testis increases approximately sixfold. The second occurs between 3 and 4 mo of age, or just before puberty, which occurs between 4 to 5 mo of age, when Sertoli cells almost double their numbers per testis. The periods of Sertoli cell proliferation were concomitant with high FSH plasma levels and prominent elongation in the length of seminiferous cord/tubule per testis. Leydig cell volume increased markedly from birth to 1 mo of age and just before puberty. In general, during the first 5 mo after birth, Leydig cell volume growth showed a similar pattern as that observed for testosterone plasma levels. Also, the proliferation of Leydig cells per testis before puberty showed a pattern similar to that observed for Sertoli cells. However, Leydig cell number per testis increased up to 16 mo of age. Substantial changes in Leydig cell size were also observed after the pubertal period. From birth to 4 mo of age, germ cells proliferated continuously, increasing their number approximately two- to fourfold at each monthly interval. A dramatic increase in germ cells per cross-section of seminiferous tubule was observed from 4 to 5 mo of age; their number per tubule cross-section stabilized after 8 mo. To our knowledge, this is the first longitudinal study reporting the pattern of Sertoli cell, germ cell, and Leydig cell proliferative activity in pigs from birth to adulthood and the first study to correlate these events with plasma levels of FSH and testosterone.     

Estrogen-induced gonadal sex reversal in the tammar wallaby

Estrogens have a feminizing effect on gonadal differentiation in fish, amphibians, reptiles, and birds. However, the role of estrogen during gonadal differentiation in mammals is less clear. We investigated the effect of estrogen on gonadal differentiation of male tammar wallabies. Male pouch young were treated orally with estradiol benzoate or oil from the day of birth, before seminiferous cords develop, to Day 25 postpartum and were killed at Day 50 postpartum. In all estrogen-treated neonates, a decrease in gonadal volume, volume of the seminiferous cords, thickness of the tunica albuginea, and number of germ cells was found. The stage of treatment affected the magnitude of the response. Two of three male young born prematurely after 25 days of gestation and treated subsequently with estradiol had ovary-like gonads, with well-developed cortical and medullary regions and primordial follicle formation. Furthermore, at Day 50 postpartum, many (21%) of the germ cells in these sex-reversed ovaries were in the leptotene and zygotene stages of meiosis, similar to female germ cells at the same stage of development. In the other males born on Day 26 of gestation or later, estradiol treatment from the day of birth caused development of dysgenetic testes, with abnormal Sertoli cells, atrophy of the seminiferous tubules and tunica albuginea, and absence of meiotic germ cells. In this marsupial, therefore, estradiol can induce either partial or complete transformation of the male gonads into an ovary with meiotic germ cells. These results confirm that estrogen can inhibit early testicular development, and that testis determination occurs during a narrow window of time.