Sex determination of mammalian germ cells occurs during fetal development and depends on signals from gonadal somatic cells. Previous studies have established that retinoic acid (RA) triggers ovarian germ cells to enter meiosis and thereby commit to oogenesis, whereas in the developing testis, the enzyme CYP26B1 degrades RA and germ cells are not induced to enter meiosis. Using in vitro and in vivo models, we demonstrate that fibroblast growth factor 9 (FGF9) produced in the fetal testis acts directly on germ cells to inhibit meiosis; in addition, FGF9 maintains expression of pluripotency-related genes and upregulates markers associated with male germ cell fate. We conclude that two independent and mutually antagonistic pathways involving RA and FGF9 act in concert to determine mammalian germ cell sexual fate commitment and support a model in which the mitosis/meiosis switch is robustly controlled by both positive and negative regulatory factors.
SUMMARYGerm cells, the embryonic precursors of sperm or oocytes, respond to molecular cues that regulate their sex-specific development in the fetal gonads. In males in particular, the balance between continued proliferation and cell fate commitment is crucial: defects in proliferation result in insufficient spermatogonial stem cells for fertility, but escape from commitment and prolonged pluripotency can cause testicular germ cell tumors. However, the factors that regulate this balance remain unidentified. Here, we show that signaling by the TGF morphogen Nodal and its co-receptor Cripto is active during a crucial window of male germ cell development. The Nodal pathway is triggered when somatic signals, including FGF9, induce testicular germ cells to upregulate Cripto. Germ cells of mutant mice with compromised Nodal signaling showed premature differentiation, reduced pluripotency marker expression and a reduced ability to form embryonic germ (EG) cell colonies in vitro. Conversely, human testicular tumors showed upregulation of NODAL and CRIPTO that was proportional to invasiveness and to the number of malignant cells. Thus, Nodal signaling provides a molecular control mechanism that regulates male germ cell potency in normal development and testicular cancer.
Substantial evidence exists that during fetal ovarian development in mammals, retinoic acid (RA) induces germ cells to express the pre-meiotic marker Stra8 and enter meiosis, and that these effects are prevented in the fetal testis by the RA-degrading P450 enzyme CYP26B1. Nonetheless, the role of RA has been disputed principally because germ cells in embryos lacking two major RA-synthesizing enzymes, ALDH1A2 and ALDH1A3, remain able to enter meiosis. Here we show that a third RA-synthesizing enzyme, ALDH1A1, is expressed in fetal ovaries, providing a likely source of RA in the absence of ALDH1A2 and ALDH1A3. In ovaries lacking ALDH1A1, the onset of germ cell meiosis is delayed. Our data resolve the conundrum posed by conflicting published data sets and reconfirm the model that meiosis is triggered by endogenous RA in the developing ovary.
Disruptions in the regulatory pathways controlling sex determination and differentiation can cause disorders of sex development, often compromising reproductive function. Although extensive efforts have been channeled into elucidating the regulatory mechanisms controlling the many aspects of sexual differentiation, the majority of disorders of sex development phenotypes are still unexplained at the molecular level. In this study, we have analyzed the potential involvement of Wnt5a in sexual development and show in mice that Wnt5a is male-specifically upregulated within testicular interstitial cells at the onset of gonad differentiation. Homozygous deletion of Wnt5a affected sexual development in male mice, causing testicular hypoplasia and bilateral cryptorchidism despite the Leydig cells producing factors such as Hsd3b1 and Insl3. Additionally, Wnt5a-null embryos of both sexes showed a significant reduction in gonadal germ cell numbers, which was caused by aberrant primordial germ cell migration along the hindgut endoderm prior to gonadal colonization. Our results indicate multiple roles for Wnt5a during mammalian reproductive development and help to clarify further the etiology of Robinow syndrome (OMIM 268310), a disease previously linked to the WNT5A pathway.
Mammalian sex determination hinges on sexually dimorphic transcriptional programs in developing fetal gonads. A comprehensive view of these programs is crucial for understanding the normal development of fetal testes and ovaries and the etiology of human disorders of sex development (DSDs), many of which remain unexplained. Using strand-specific RNA-sequencing, we characterized the mouse fetal gonadal transcriptome from 10.5 to 13.5 days post coitum, a key time window in sex determination and gonad development. Our dataset benefits from a greater sensitivity, accuracy and dynamic range compared to microarray studies, allows global dynamics and sex-specificity of gene expression to be assessed, and provides a window to non-transcriptional events such as alternative splicing. Spliceomic analysis uncovered female-specific regulation of Lef1 splicing, which may contribute to the enhanced WNT signaling activity in XX gonads. We provide a user-friendly visualization tool for the complete transcriptomic and spliceomic dataset as a resource for the field.
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