Germ cell development in the XXY mouse: evidence that X chromosome reactivation is independent of sexual differentiation

Prior to entry into meiosis, XX germ cells in the fetal ovary undergo X chromosome reactivation. The signal for reactivation is thought to emanate from the genital ridge, but it is unclear whether it is specific to the developing ovary. To determine whether the signals are present in the developing testis as well as the ovary, we examined the expression of X-linked genes in germ cells from XXY male mice. To facilitate this analysis, we generated XXY and XX fetuses carrying X chromosomes that were differentially marked and subject to nonrandom inactivation. This pattern of nonrandom inactivation was maintained in somatic cells but, in XX as well as XXY fetuses, both parental alleles were expressed in germ cell-enriched cell populations. Because testis differentiation is temporally and morphologically normal in the XXY testis and because all germ cells embark upon a male pathway of development, these results provide compelling evidence that X chromosome reactivation in fetal germ cells is independent of the somatic events of sexual differentiation. Proper X chromosome dosage is essential for the normal fertility of male mammals, and abnormalities in germ cell development are apparent in the XXY testis within several days of X reactivation. Studies of exceptional germ cells that survive in the postnatal XXY testis demonstrated that surviving germ cells are exclusively XY and result from rare nondisjunctional events that give rise to clones of XY cells.     

Efficiently accumulating germ-like stem cells from mouse postnatal ovary by in situ tissue culture

Although recent studies have revealed that germline stem cells (GSCs) exist in the mouse postnatal ovary, how to efficiently obtain GSCs for regenerating neo-oogenesis is still a technical challenge. Here, we report that using in situ tissue culture we can efficiently accumulate large amounts of proliferating germ-like cells from mouse postnatal ovaries. Usually, more than 10,000 germ-like cells can be derived from one ovary by this method, and over 20% of these cells can grow into germ-like cells with self-renewal, which thus can serve as a good cell pool to isolate GSCs by other cell assorting methods such as FACS. This method is simple and time-saving, which should be useful for in future studies on mouse GSCs.     

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.     

Retinoic acid derived from the fetal ovary initiates meiosis in mouse germ cells

Meiotic initiation of germ cells at 13.5 dpc (days post‐coitus) indicates female sex determination in mice. Recent studies reveal that mesonephroi‐derived retinoic acid (RA) is the key signal for induction of meiosis. However, whether the mesonephroi is dispensable for meiosis is unclear and the role of the ovary in this meiotic process remains to be clarified. This study provides data that RA derived from fetal ovaries is sufficient to induce germ cell meiosis in a fetal ovary culture system. When fetal ovaries were collected from 11.5 to 13.5 dpc fetuses, isolated and cultured in vitro, germ cells enter meiosis in the absence of mesonephroi. To exclude RA sourcing from mesonephroi, 11.5 dpc urogenital ridges (UGRs; mesonephroi and ovary complexes) were treated with diethylaminobenzaldehyde (DEAB) to block retinaldehyde dehydrogenase (RALDH) activity in the mesonephros and the ovary. Meiosis occurred when DEAB was withdrawn and the mesonephros was removed 2 days later. Furthermore, RALDH1, rather than RALDH2, serves as the major RA synthetase in UGRs from 12.5 to 15.5 dpc. DEAB treatment to the ovary alone was able to block germ cell meiotic entry. We also found that exogenously supplied RA dose‐dependently reduced germ cell numbers in ovaries by accelerating the entry into meiosis. These results suggest that ovary‐derived RA is responsible for meiosis initiation. J. Cell. Physiol. 228: 627–639, 2013. © 2012 Wiley Periodicals, Inc.     

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.     

Smurf-mediated differential proteolysis generates dynamic BMP signaling in germline stem cells during Drosophila testis development

Germline stem cells (GSCs) produce gametes throughout the reproductive life of many animals, and intensive studies have revealed critical roles of BMP signaling to maintain GSC self-renewal in Drospophila adult gonads. Here, we show that BMP signaling is downregulated as testes develop and this regulation controls testis growth, stem cell number, and the number of spermatogonia divisions. Phosphorylated Mad (pMad), the activated Drosophila Smad in germ cells, was restricted from anterior germ cells to GSCs and hub-proximal cells during early larval development. pMad levels in GSCs were then dramatically downregulated from early third larval instar (L3) to late L3, and maintained at low levels in pupal and adult GSCs. The spatial restriction and temporal down-regulation of pMad, reflecting the germ cell response to BMP signaling activity, required action in germ cells of E3 ligase activity of HECT domain protein Smurf. Analyses of Smurf mutant testes and dosage-dependent genetic interaction between Smurf and mad indicated that pMad downregulation was required for both the normal decrease in stem cell number during testis maturation in the pupal stage, and for normal limit of four rounds of spermatogonia cell division for control of germ cell numbers and testis size. Smurf protein was expressed at a constant low level in GSCs and spermatogonia during development. Rescue experiments showed that expression of exogenous Smurf protein in early germ cells promoted pMad downregulation in GSCs in a stage-dependent but concentration-independent manner, suggesting that the competence of Smurf to attenuate response to BMP signaling may be regulated during development. Taken together, our work reveals a critical role for differential attenuation of the response to BMP signaling in GSCs and early germ cells for control of germ cell number and gonad growth during development.     

多倍体银鲫nanos2等位多态性、共线性和表达模式分析

为研究生殖干细胞(Germline stem cells, GSCs)的标记基因nanos2的功能, 在银鲫(Carassius gibelio)中克隆了两个nanos2的同源基因(Homologs), 将其命名为Cgnanos2a和Cgnanos2b, 等位、系统进化树和共线性分析表明, 在银鲫进化历程中发生了额外的两轮多倍化事件, 是一个异源六倍体。qPCR分析表明Cgnanos2a和Cgnanos2b在5月龄银鲫精巢中的表达水平最高, 其次是卵巢; 对在孵化后25—190d的卵巢中的表达动态分析表明, 孵化后25d的卵巢中的Cgnanos2a和Cgnanos2b转录水平最高, 随后表达水平急剧下降; 并且Cgnanos2a和Cgnanos2b呈现出偏向表达的特征。切片RNA原位杂交实验结果表明, Cgnanos2a和Cgnanos2b均特异地在银鲫卵巢邻近生殖上皮的胞囊(Cyst)中一类直径小于20 μm的细胞中表达, Cgnanos2a在精巢精小囊边缘一类单个或两个紧紧相邻的细胞中高表达, 推测是银鲫的GSCs。此外, 还可在精原细胞和初级精母细胞中检测到Cgnanos2a和Cgnanos2b的转录本。研究通过对nanos2阳性细胞的追踪描绘了银鲫卵巢早期胞囊的发育过程, 为后续分离银鲫GSCs奠定了基础; 同时对银鲫nanos2两个歧化的部分同源基因的序列特征、进化及表达特征分析, 为研究鱼类多次多倍化事件后重复基因的进化提供了一个典型例子。     

Eggs forever?

A group of scientists from Harvard Medical School (Johnson et al., 2004) claims to have "established the existence of proliferative germ cells that sustain oocyte and follicle production in the postnatal mammalian ovary," expressing no doubts about their methods, results and conclusion. Johnson et al. based their conclusions of oocyte and follicular renewal from existing germline stem cells (GSC) in the postnatal mouse ovary on three types of observations: (1) A claimed discordance in follicle loss versus follicle atresia in the neonatal period and in the following pubertal and adult period; (2) immunohistochemical detection of proliferating GSC with meiotic capacity using combined markers for meiosis, germline, and mitosis; and (3) neo-folliculogenesis in ovarian chimeric grafting experiments with adult mice. Oogenesis is the process that transforms the proliferative oogonium into an oocyte through meiosis, followed by folliculogenesis and follicular and oocyte maturation. The most crucial part in producing a functional oocyte is firstly, initiation and completion of the first meiotic prophase, and secondly, enclosure of the resulting diplotene oocyte in a follicle. Neither of these two events has been shown to take place in Johnson et al.'s study of the postnatal mouse ovary. We hereby address the observations underpinning their hypothesis and conclude that it is premature to replace the paradigm that adult mammalian neo-oogenesis/folliculogenesis does not take place.     

Uncovering gene regulatory networks during mouse fetal germ cell development

The commitment of germ cells to either oogenesis or spermatogenesis occurs during fetal gonad development: germ cells enter meiosis or mitotic arrest, depending on whether they reside within an ovary or a testis, respectively. Despite the critical importance of this step for sexual reproduction, gene networks underlying germ cell development have remained only partially understood. Taking advantage of the W(v) mouse model, in which gonads lack germ cells, we conducted a microarray study to identify genes expressed in fetal germ cells. In addition to distinguishing genes expressed by germ cells from those expressed by somatic cells within the developing gonads, we were able to highlight specific groups of genes expressed only in female or male germ cells. Our results provide an important resource for deciphering the molecular pathways driving proper germ cell development and sex determination and will improve our understanding of the etiology of human germ cell tumors that arise from dysregulation of germ cell differentiation.     

Identification of novel markers of mouse fetal ovary development

In contrast to the developing testis, molecular pathways driving fetal ovarian development have been difficult to characterise. To date no single master regulator of ovarian development has been identified that would be considered the female equivalent of Sry. Using a genomic approach we identified a number of novel protein-coding as well as non-coding genes that were detectable at higher levels in the ovary compared to testis during early mouse gonad development. We were able to cluster these ovarian genes into different temporal expression categories. Of note, Lrrc34 and AK015184 were detected in XX but not XY germ cells before the onset of sex-specific germ cell differentiation marked by entry into meiosis in an ovary and mitotic arrest in a testis. We also defined distinct spatial expression domains of somatic cell genes in the developing ovary. Our data expands the set of markers of early mouse ovary differentiation and identifies a classification of early ovarian genes, thus providing additional avenues with which to dissect this process.     

Pelota controls self-renewal of germline stem cells by repressing a Bam-independent differentiation pathway

In the Drosophila ovary, germline stem cell (GSC) self-renewal is controlled by both extrinsic and intrinsic factors. The Bmp signal from niche cells controls GSC self-renewal by directly repressing a Bam-dependent differentiation pathway in GSCs. pelota (pelo), which has been previously shown to be required for Drosophila male meiosis, was identified in our genetic screen as a dominant suppressor of the dpp overexpression-induced GSC tumor phenotype. In this study, we reveal the unexpected new role of Pelo in controlling GSC self-renewal by repressing a Bam-independent differentiation pathway. In pelo mutant ovaries, GSCs are lost rapidly owing to differentiation. Results from genetic mosaic analysis and germ cell-specific rescue show that it functions as an intrinsic factor to control GSC self-renewal. In pelo mutant GSCs, Bmp signaling activity detected by Dad-lacZ expression is downregulated, but bam expression is still repressed. Furthermore, bam mutant germ cells are still able to differentiate into cystocytes without pelo function, indicating that Pelo is involved in repressing a Bam-independent differentiation pathway. Consistent with its homology to the eukaryotic translation release factor 1alpha, we show that Pelo is localized to the cytoplasm of the GSC. Therefore, Pelo controls GSC self-renewal by repressing a Bam-independent differentiation pathway possibly through regulating translation. As Pelo is highly conserved from Drosophila to mammals, it may also be involved in the regulation of adult stem cell self-renewal in mammals, including humans.     

Neuropeptide Y-containing nerves in rat gonads: sex difference and development

The objectives of the present study were 1) to evaluate for a sex difference in innervation of adult rat gonads by neuropeptide Y-immunoreactive (NPY-I) nerves and 2) to examine the development of innervation of rat gonads by NPY-I nerves during the fetal and neonatal periods. With fluorescence immunocytochemistry, NPY-I nerves were profuse in adult ovarian tissues. Ovarian blood vessels were particularly well innervated by NPY-I nerves, and nerves were also detected in interstitial gland tissues. No nerves were found within the testis, and NPY-I nerves were only rarely located within the tunica albuginea. During fetal life, ovaries were devoid of NPY-I nerves; however, nerves were visualized within the connective tissue immediately peripheral to the ovary on fetal Day 22. As early as postnatal Day 2, NPY-I nerves were observed in connective tissue septa of the developing ovary. By postnatal Day 12, NPY-I nerves surrounded developing follicles and blood vessels of the ovarian cortex. In the developing testis after postnatal Day 5, NPY-I nerves were limited to the tunica albuginea and surrounding large subcapsular blood vessels. Structures within the testis lacked innervation by NPY-I nerves. These anatomical studies suggest that NPY-I nerves are absent in the gonads during fetal life and grow into the ovary and not the testis during the perinatal period and that NPY-I nerves may play a role in the functioning of the rat ovary, but may not be important in control of testicular function.     

A study of meiosis in chimeric mouse fetal gonads

The influence of somatic environment on the onset and progression of meiosis in fetal germ cells was studied in chimeric gonads produced in vitro by dissociation-reaggregation experiments. Germ cells isolated from testes or ovaries of 11.5-13.5 days post coitum (dpc) CD-1 mouse embryos were loaded with the fluorescent supravital dye 5-6 carboxyfluorescein diacetate succinimyl ester (CFSE) and mixed with a cell suspension obtained by trypsin-EDTA treatment of gonads of various ages and of the same or opposite sex. Whereas 11.5 dpc donor germ cells appeared unable to survive in the chimeric gonads obtained, about 76% of the CFSE-labeled female germ cells obtained from 12.5 dpc donor embryos (premeiotic germ cells) found viable within host ovarian tissues showed a meiotic nucleus. In contrast, a smaller number (about 19%) were in meiosis in chimeric testes. None or very few of donor male germ cells entered meiosis in testes or ovarian host tissues. Aggregation of meiotic 13.5 dpc female germ cells with testis tissues from 13.5 to 14.5 dpc embryos resulted in inhibition of meiotic progression and pyknosis in most donor germ cells. These results support the existence of a meiosis-preventing substance or a factor causing oocyte degeneration in the fetal mouse testis, but not of a meiosis-inducing substance in the fetal ovary.     

Retinoic acid signaling in regulation of meiosis during embryonic development in mice

In the embryonic gonads of mice, the genetic and epigenetic regulatory programs for germ cell sex specification and meiosis induction or suppression are intertwined. The quest for garnering comprehensive understanding of these programs has led to the emergence of retinoic acid (RA) as an important extrinsic factor, which regulates initiation of meiosis in female fetal germ cells that have attained a permissive epigenetic ground state. In contrast, germ cells in fetal testis are protected from the exposure to RA due to the activity of CYP26B1, an RA metabolizing enzyme, which is highly expressed in fetal testis. In this review, we provide an overview of the molecular mechanisms operating in fetal gonads of mice, which enable regulation of meiosis via RA signaling.     

Induction of meiosis in fetal mouse testis in vitro

Fetal mouse testes and ovaries with their urogenital connections were cultured singly or in pairs on Nuclepore filters. When a testis in which the sex was not yet morphologically detectable was cultured together with older ovaries containing germ cells which were progressing through the meiotic prophase, the male germ cells were triggered to enter meiosis. When older fetal testes in which the testicular cords have developed were cultured together with ovaries of the same age with germ cells in meiosis, the oocytes were prevented from reaching diplotene stage. It was concluded that the fetal male and female gonads secrete diffusable substances which influence germ cell differentiation. The male gonad secretes a "meiosis-preventing substance" (MPS) which can arrest the female germ cells within the meiotic prophase. The female gonad secretes a "meiosis-inducing substance" (MIS) which can trigger the nondifferentiated male germ cells to enter meiosis.