Cnot3 is required for male germ cell development and spermatogonial stem cell maintenance.

The foundation of spermatogenesis and lifelong fertility is provided by spermatogonial stem cells (SSCs). SSCs divide asymmetrically to either replenish their numbers (self-renewal) or produce undifferentiated progenitors that proliferate before committing to differentiation. However, regulatory mechanisms governing SSC maintenance are poorly understood. Here, we show that the CCR4-NOT mRNA deadenylase complex subunit CNOT3 plays a critical role in maintaining spermatogonial populations in mice. Cnot3 is highly expressed in undifferentiated spermatogonia, and its deletion in spermatogonia resulted in germ cell loss and infertility. Single cell analyses revealed that Cnot3 deletion led to the de-repression of transcripts encoding factors involved in spermatogonial differentiation, including those in the glutathione redox pathway that are critical for SSC maintenance. Together, our study reveals that CNOT3 - likely via the CCR4-NOT complex - actively degrades transcripts encoding differentiation factors to sustain the spermatogonial pool and ensure the progression of spermatogenesis, highlighting the importance of CCR4-NOT-mediated post-transcriptional gene regulation during male germ cell development.

Apoptosis in the fetal testis eliminates developmentally defective germ cell clones.

Many germ cells are eliminated during development, long before oogenesis or spermatogenesis. In mouse fetal testes, the majority of germ cell apoptosis coincides with the onset of male differentiation, suggesting coordination of these processes. We studied fetal germ-cell fates and discovered that both apoptosis and differentiation initiate in clonally related clusters. Lineage tracing confirmed that germ cells die as clones independent of intercellular bridges, suggesting that shared intrinsic properties are apoptotic determinants. We identified transcriptional heterogeneity among fetal germ cells that included an apoptosis-susceptible population characterized by failure to differentiate, whereas successful differentiation to prospermatogonia occurred through the expression of epigenetically regulated genes, including LINE1. Our results indicate that the fetal germ-cell fate is based on discrete cell-heritable identities. Elevated DNA methylation in the apoptosis-susceptible subpopulation supports our hypothesis that earlier errors in germ-cell epigenetic reprogramming derail differentiation in cellular progeny, leading to fetal apoptotic selection that ultimately improves the gamete quality.

Maximizing the ovarian reserve in mice by evading LINE-1 genotoxicity.

Female reproductive success critically depends on the size and quality of a finite ovarian reserve. Paradoxically, mammals eliminate up to 80% of the initial oocyte pool through the enigmatic process of fetal oocyte attrition (FOA). Here, we interrogate the striking correlation of FOA with retrotransposon LINE-1 (L1) expression in mice to understand how L1 activity influences FOA and its biological relevance. We report that L1 activity triggers FOA through DNA damage-driven apoptosis and the complement system of immunity. We demonstrate this by combined inhibition of L1 reverse transcriptase activity and the Chk2-dependent DNA damage checkpoint to prevent FOA. Remarkably, reverse transcriptase inhibitor AZT-treated Chk2 mutant oocytes that evade FOA initially accumulate, but subsequently resolve, L1-instigated genotoxic threats independent of piRNAs and differentiate, resulting in an increased functional ovarian reserve. We conclude that FOA serves as quality control for oocyte genome integrity, and is not obligatory for oogenesis nor fertility.

Multiple Mechanisms Cooperate to Constitutively Exclude the Transcriptional Co-Activator YAP from the Nucleus During Murine Oogenesis.

Reproduction depends on the generation of healthy oocytes. Improving therapeutic strategies to prolong or rescue fertility depends on identifying the inter- and intracellular mechanisms that direct oocyte development under physiological conditions. Growth and proliferation of multiple cell types is regulated by the Hippo signaling pathway, whose chief effectors are the transcriptional co-activator YAP and its paralogue WWTR1. To resolve conflicting results concerning the potential role of Hippo in mammalian oocyte development, we systematically investigated the expression and localization of YAP in mouse oocytes. We report that that YAP is expressed in the germ cells beginning as early as Embryonic Day 15.5 and subsequently throughout pre- and postnatal oocyte development. However, YAP is restricted to the cytoplasm at all stages. YAP is phosphorylated at serine-112 in growing and fully grown oocytes, identifying a likely mechanistic basis for its nuclear exclusion, and becomes dephosphorylated at this site during meiotic maturation. Phosphorylation at serine-112 is regulated by a mechanism dependent on cyclic AMP and protein kinase A, which is known to be active in oocytes prior to maturation. Growing oocytes also contain a subpopulation of YAP, likely dephosphorylated, that is able enter the oocyte nucleus, but it is not retained there, implying that oocytes lack the cofactors required to retain YAP in the nucleus. Thus, although YAP is expressed throughout oocyte development, phosphorylation-dependent and -independent mechanisms cooperate to ensure that it does not accumulate in the nucleus. We conclude that nuclear YAP does not play a significant physiological role during oocyte development in mammals.

A Whole-Mount Approach for Accurate Quantitative and Spatial Assessment of Fetal Oocyte Dynamics in Mice.

Depletion of oocytes from the embryonic ovary is a key feature of mammalian oogenesis; however, the rational and molecular bases for this phenomenon remain poorly understood. Presently in the field, the most systematic analysis used to understand the effect of a given molecular pathway on fetal oocyte attrition is to count the number of oocytes in ovaries at different stages of development. This analysis is commonly done using a sampling method based on sectioning of the ovary, a technique that includes many laborious steps culminating in an inaccurate estimate of oocyte number contained within that ovary. This inability to generate data that are directly comparable between labs hinders the field and raises questions about the timing and rate of oocyte depletion. Therefore, we set out to implement a robust method that can be easily used by most research laboratories to study the dynamics of oogenesis during fetal mouse ovary development in both normal and experimental conditions. Here we describe an approach to accurately count the total number of oocytes in embryonic ovaries. This method is based on whole-mount immunofluorescence, tissue clearing with sucrose and ScaleA2 reagent, and automatic detection and counting of germ cells in intact ovaries using confocal microscopy and three-dimensional software analyses. We demonstrate the power of the method by assessing variation of fetal oocyte numbers between left and right ovaries and between litters of mice. Finally, we anticipate that the method could be adopted to the analysis of substages of meiotic prophase I and ovarian somatic cells.

A role for retrotransposon LINE-1 in fetal oocyte attrition in mice.

Fetal oocyte attrition (FOA) is a conserved but poorly understood process of elimination of more than two-thirds of meiotic prophase I (MPI) oocytes before birth. We now implicate retrotransposons LINE-1 (L1), activated during epigenetic reprogramming of the embryonic germline, in FOA in mice. We show that wild-type fetal oocytes possess differential nuclear levels of L1ORF1p, an L1-encoded protein essential for L1 ribonucleoprotein particle (L1RNP) formation and L1 retrotransposition. We demonstrate that experimental elevation of L1 expression correlates with increased MPI defects, FOA, oocyte aneuploidy, and embryonic lethality. Conversely, reverse transcriptase (RT) inhibitor AZT has a profound effect on the FOA dynamics and meiotic recombination, and it implicates an RT-dependent trigger in oocyte elimination in early MPI. We propose that FOA serves to select oocytes with limited L1 activity that are therefore best suited for the next generation.

Synaptonemal complex length variation in wild-type male mice.

Meiosis yields haploid gametes following two successive divisions of a germ cell in the absence of intervening DNA replication. Balanced segregation of homologous chromosomes in Meiosis I is aided by a proteinaceous structure, the synaptonemal complex (SC). The objective of this study was to determine total average autosomal SC lengths in spermatocytes in three commonly used mouse strains (129S4/SvJae, C57BL/6J, and BALB/c). Our experiments revealed that the total autosomal SC length in BALB/c spermatocytes is 9% shorter than in the two other strains. Shorter SCs are also observed in spermatocytes of (BALB/c × 129S4/SvJae) and (C57BL/6J × BALB/c) F1 hybrids suggesting a genetic basis of SC length regulation. Along these lines, we studied expression of a selected group of genes implicated in meiotic chromosome architecture. We found that BALB/c testes express up to 6-fold less of Rec8 mRNA and 4-fold less of REC8 protein. These results suggest that the mechanism that defines the SC length operates via a REC8­dependent process. Finally, our results demonstrate that genetic background can have an effect on meiotic studies in mice.

Shuttling of SOX proteins.

The control of access of SOX proteins to their nuclear target genes is a powerful strategy to activate or repress complex genetic programs. The sub-cellular targeting sequences of SOX proteins are concentrated within the DNA binding motif, the HMG (for high mobility group) domain. Each SOX protein displays two different nuclear localization signals located at the N-terminal and C-terminal part of their highly conserved DNA binding domain. The N-terminal nuclear localization signal binds calmodulin and is potentially regulated by intracellular calcium signalling, while the C-terminal nuclear localization signal, which binds importin-beta, responds to other signalling pathways such as cyclic AMP/protein kinase A. Mutations inducing developmental disorders like sex reversal have been reported in both NLSs of SRY, interfering with its nuclear localization and suggesting that both functional nuclear localization signal are required for its nuclear activity. A nuclear export signal is also present in the HMG box of SOX proteins. Group E SOX proteins harbour a perfect consensus nuclear export signal sequence in contrast to all other SOX proteins, which display only imperfect ones. However, observations made during mouse embryonic development suggest that non-group E SOX proteins could also be regulated by a nuclear export mechanism. The presence of nuclear localization and nuclear export signal sequences confers nucleocytoplasmic shuttling properties to SOX proteins, and suggests that cellular events regulated by SOX proteins are highly dynamic.

The PGD2 pathway, independently of FGF9, amplifies SOX9 activity in Sertoli cells during male sexual differentiation.

Activation by the Y-encoded testis determining factor SRY and maintenance of expression of the Sox9 gene encoding the central transcription factor of Sertoli cell differentiation are key events in the mammalian sexual differentiation program. In the mouse XY gonad, SOX9 upregulates Fgf9, which initiates a Sox9/Fgf9 feedforward loop, and Sox9 expression is stimulated by the prostaglandin D2 (PGD2) producing lipocalin prostaglandin D synthase (L-PGDS, or PTDGS) enzyme, which accelerates commitment to the male pathway. In an attempt to decipher the genetic relationships between Sox9 and the L-Pgds/PGD2 pathway during mouse testicular organogenesis, we found that ablation of Sox9 at the onset or during the time window of expression in embryonic Sertoli cells abolished L-Pgds transcription. By contrast, L-Pgds(-/-) XY embryonic gonads displayed a reduced level of Sox9 transcript and aberrant SOX9 protein subcellular localization. In this study, we demonstrated genetically that the L-Pgds/PGD2 pathway acts as a second amplification loop of Sox9 expression. Moreover, examination of Fgf9(-/-) and L-Pgds(-/-) XY embryonic gonads demonstrated that the two Sox9 gene activity amplifying pathways work independently. These data suggest that, once activated and maintained by SOX9, production of testicular L-PGDS leads to the accumulation of PGD2, which in turn activates Sox9 transcription and nuclear translocation of SOX9. This mechanism participates together with FGF9 as an amplification system of Sox9 gene expression and activity during mammalian testicular organogenesis.

[Prostaglandin D2: new roles in the embryonic and pathological gonad]. / La prostaglandine D2: nouveaux rôles dans la gonade embryonnaire et pathologique.

Prostaglandin D2 (PGD2) belongs to the superfamily of ubiquitous signalling molecules, the prostaglandins ; these bind to specific G-coupled transmembrane receptors, inducing various transduction pathways. Prostaglandins PGE2 and PGF2alpha have several identified functions during ovulation, fecondation and embryo implantation. However, the roles of PGD2 within the male or female reproductive organs are still largely unknown, even though the PGD2-producing enzyme, prostaglandin D synthase (PGDS), is detected in these organs. In this study, we summarize recent data highlighting new functions of PGD2 in the onset of testicular embryogenesis and in the growth inhibition of ovarian cancer cells. In both cases, PGD2 acts by activating the function of the Sertoli cell differentiating factor SOX9.

Expression and biological role of the prostaglandin D synthase/SOX9 pathway in human ovarian cancer cells.

New therapeutic strategies for ovarian cancer include the identification of involved signaling pathways that could potentially serve as a source of biomarkers for early stages of the disease. In this study, we show that the embryonic male prostaglandin D synthase (Pgds)/SOX9 pathway is expressed at both the RNA and protein levels in different types of human ovarian tumors, pointing to Pgds and SOX9 as possible diagnostic markers for ovarian carcinomas. Using ovarian cancer cell lines, we found, first, that components of the Pgds/SOX9 pathway are expressed in these cells, and second, that treatment of these cells with prostaglandin D2 (PGD2) can inhibit their growth via its DP1 receptor and induce apoptosis. Finally, using siRNA and overexpression strategies, we demonstrate that SOX9 expression is induced by PDG2 and is responsible for PDG2-mediated growth inhibition. Accordingly, as stimulating the PGD2/DP1 signal transduction pathway upregulates SOX9 expression, either activators of this pathway or DP1 agonists may be useful as new therapeutic agents.

Transient expression of SOX9 protein during follicular development in the adult mouse ovary.

SOX9 is an essential activating transcription factor that plays a critical role in Sertoli cell differentiation and subsequent testis cord formation. Cytoplasmic SOX9 is present in both sexes during early gonadal embryogenesis. While in males the protein is later translocated into the nucleus of pre-Sertoli cells, its expression is rapidly turned off in females. In mammalian male gonads, SOX9 activates the expression of anti-Müllerian hormone (AMH), a male hormone that initiates Müllerian ducts regression and that is also expressed in postnatal ovarian follicles. Here, we confirm that the SOX9 protein is not present in the immature ovary but also show that SOX9 is transiently expressed in the mature ovary depending on the follicular cycle. Indeed, SOX9 protein was found in the nuclear compartment of the inner cells of the theca interna cell layer which surrounds the pre-antral/antral follicles. In contrast, no expression was detected in the AMH expressing granulosa cells. While these findings exclude the possibility that SOX9 regulates AMH expression in the ovary, they show that SOX9 could nevertheless play a role in the developing follicle.

Gene expression during sex determination reveals a robust female genetic program at the onset of ovarian development.

The primary event in mammalian sexual development is the differentiation of the bipotential gonads into either testes or ovaries. Our understanding of the molecular pathways specifying gonadal differentiation is still incomplete. To identify the initial molecular changes accompanying gonadal differentiation in mice, we have performed a large-scale transcriptional analysis of XX and XY Sf1-positive gonadal cells during sex determination. In both male and female genital ridges, a robust genetic program is initiated pre-dating the first morphological changes of the differentiating gonads. Between E10.5 and E13.5, 2306 genes were expressed in a sex-specific manner in the somatic compartment of the gonads; 1223 were overexpressed in XX embryos and 1083 in XY embryos. Although sexually dimorphic genes were scattered throughout the mouse genome, we identified chromosomal regions hosting clusters of genes displaying similar expression profiles. The cyclin-dependent kinase inhibitors Cdkn1a and Cdkn1c are overexpressed in XX gonads at E11.5 and E12.5, suggesting that the increased proliferation of XY gonads relative to XX gonads may result from the overexpression of cell cycle inhibitors in the developing ovaries. These studies define the major characteristics of testicular and ovarian transcriptional programs and reveal the richness of signaling processes in differentiation of the bipotential gonads into testes and ovaries.

NHERF2/SIP-1 interacts with mouse SRY via a different mechanism than human SRY.

In mammals, male sex determination is controlled by the SRY protein, which drives differentiation of the bipotential embryonic gonads into testes by activating the Sertoli cell differentiation program. The morphological effects of SRY are well documented; however, its molecular mechanism of action remains unknown. Moreover, SRY proteins display high sequence variability among mammalian species, which makes protein motifs difficult to delineate. We previously isolated SIP-1/NHERF2 as a human SRY-interacting protein. SIP-1/NHERF2, a PDZ protein, interacts with the C-terminal extremity of the human SRY protein. Here we showed that the interaction of SIP-1/NHERF2 and SRY via the SIP-1/NHERF2 PDZ1 domain is conserved in mice. However, the interaction occurs via a domain that is internal to the mouse SRY protein and involves a different recognition mechanism than human SRY. Furthermore, we show that mouse and human SRY induce nuclear accumulation of the SIP-1/NHERF2 protein in cultured cells. Finally, a transgenic mouse line expressing green fluorescent protein under the control of the mouse Sry promoter allowed us to show that SRY and SIP-1/NHERF2 are co-expressed in the nucleus of pre-Sertoli cells during testis determination. Taken together, our results suggested that the function of SIP-1/NHERF2 as an SRY cofactor during testis determination is conserved between human and mouse.

Cytoplasmic retention of the sex-determining factor SOX9 via the microtubule network.

SOX9 is a sex-determining factor which induces Sertoli cell differentiation and subsequent testis cord formation. It is expressed both in male and female undifferentiated gonads in the cytoplasmic compartment of pre-Sertoli cells. At the time of sexual differentiation, SOX9 moves into the nucleus of male pre-Sertoli cells whereas in female, it remains in the cytoplasm and then its expression decreases. To study the cytoplasmic localization of SOX9, we have analyzed its interaction with the cytoskeleton components. By treatment of NT2/D1 and transfected NIH3T3 cell lines and embryonic gonads with nocodazole, a drug depolymerizing the microtubules, we show that cytoplasmic retention of SOX9 requires the integrity of the microtubule network. Using biochemical experiments, we demonstrated that SOX9 is able to interact with microtubules in vitro and in vivo. On the other hand, we observed a complete male-specific reorganization of the microtubule network in epithelial Sertoli cells of the male embryonic gonad at the time of sexual differentiation and testis cord formation.

Prostaglandin D2 induces nuclear import of the sex-determining factor SOX9 via its cAMP-PKA phosphorylation.

During mammalian gonadal development, nuclear import/export of the transcription factor SOX9 is a critical step of the Sry-initiated testis-determining cascade. In this study, we identify a molecular mechanism contributing to the SOX9 nuclear translocation in NT2/D1 cells, which is mediated by the prostaglandin D2 (PGD2) signalling pathway via stimulation of its adenylcyclase-coupled DP1 receptor. We find that activation of cAMP-dependent protein kinase A (PKA) induces phosphorylation of SOX9 on its two S64 and S181 PKA sites, and its nuclear localization by enhancing SOX9 binding to the nucleocytoplasmic transport protein importin beta. Moreover, in embryonic gonads, we detect a male-specific prostaglandin D synthase expression and an active PGD2 signal at the time and place of SOX9 expression. We thus propose a new step in the sex-determining cascade where PGD2 acts as an autocrine factor inducing SOX9 nuclear translocation and subsequent Sertoli cell differentiation.