Cassy M. Spiller

FGF9 suppresses meiosis and promotes male germ cell fate in mice.

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.

Endogenous Nodal signaling regulates germ cell potency during mammalian testis development

Germ 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 TGFβ 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.

Loss of Wnt5a Disrupts Primordial Germ Cell Migration and Male Sexual Development in Mice

ABSTRACT 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.

Identification of Suitable Normalizing Genes for Quantitative Real-Time RT-PCR Analysis of Gene Expression in Fetal Mouse Gonads

In biological research, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assays are commonly employed to study mRNA abundance in cells and tissues. This type of assay usually relies on assessing transcript abundance relative to constitutively expressed endogenous reference genes. Therefore, it is important that the reference genes themselves are stably expressed in the cells or tissues analyzed, independent of factors such as age, sex, disease or experimental manipulations. Since no gene is expressed at the same level in all cells at all times, suitable reference genes must be identified for the specific cellular system or tissue being investigated. Here, we sought to identify stably expressed endogenous reference genes during embryonic gonad development in the mouse. We measured the transcript abundance of 10 frequently employed normalizing genes, of which 4 were stably expressed in fetal gonads from 11.5 to 14.5 dpc irrespective of sex. Based on our analysis, we suggest that Rn18s, Rps29, Tbp and Sdha are suitable reference genes for qRT-PCR expression studies during early gonad differentiation in the mouse.

Retinoblastoma 1 Protein Modulates XY Germ Cell Entry into G1/G0 Arrest During Fetal Development in Mice

Abstract During mouse germ cell development, the first sign of sex differentiation occurs when XY germ cells enter G1/G0 arrest from 12.5 days postcoitum (dpc). Retinoblastoma 1 (RB1), a potent cell cycle regulator, was investigated in XY germ cell arrest by studying germ cell proliferation in Rb1−/− mutant mouse embryos. Because mice homozygous for the Rb1 deletion die in utero around 14.5 dpc, we used ex vivo culture techniques to allow analysis of developing gonads to 16.5 dpc. In Rb1−/− gonads, we observed normal somatic cell development, assessed by immunofluorescence for markers HSD3B1 and anti-Müllerian hormone. However, at 14.5 dpc, when wild-type XY germ cells had arrested, we could detect actively proliferating germ cells using the proliferation markers MKI67, pHH3, and bromodeoxyuridine incorporation. The increased proliferation was reflected with a trend of increased germ cell number and expression of germ cell markers Ddx4 and Pou5f1 in the Rb1−/− testes. By 16.5 dpc, this phenotype was resolved such that the entire germ cell population had entered G1/G0 arrest, although the total germ cell number remained elevated. At each stage analyzed, we saw no increase in expression of RB1 family members Rbl1 and Rbl2 in the Rb1−/− testes, but we saw a significant increase of cyclin-dependent kinase (CDK) inhibitor Cdkn1b and Cdkn2b expression. We conclude that Rb1 is required for correct germ cell entry into G1/G0 arrest in the wild-type gonad at 14.5 dpc, but in its absence, upregulation of other cell cycle suppressors, including Cdkn1b and Cdkn2b, can induce delayed germ cell arrest.

Gonadal defects in Cited2-mutant mice indicate a role for SF1 in both testis and ovary differentiation.

Sex determination is regulated by a molecular antagonism between testis- and ovary-determining pathways in the supporting cell lineage of the gonadal primordia. Genes important for maintaining this lineage play critical roles in early gonadal development, but their influence on testis and ovary differentiation is unclear due to the severity of loss-of-function phenotypes. The transcription factor SF1 (Nr5a1/Ad4BP) is one such factor, required for establishing the supporting cell lineage, and for propagating the male pathway. In the gonad, Sf1 expression is enhanced by the transcriptional co-factor Cited2. We have used the reduced levels of Sf1 expression in Cited2(-/-) mice as a hypomorphic model to gain insight into the sex-specific roles of SF1 function in gonadal development. In XY mutant mice, we found that testis development was delayed in Cited2(-/-) gonads, and that testis structure was permanently disrupted. In XX Cited2(-/-) gonads, ectopic cell migration was observed which correlated with a transient upregulation of Fgf9, and a delay in Wnt4 then Foxl2 expression. These data suggest a novel role for SF1 in promoting ovarian development in addition to its roles in testis 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.

Cell cycle analysis of fetal germ cells during sex differentiation in mice

Background information. Primordial germ cells in developing male and female gonads are responsive to somatic cell cues that direct their sex‐specific differentiation into functional gametes. The first divergence of the male and female pathways is a change in cell cycle state observed from 12.5 dpc (days post coitum) in mice. At this time XY and XX germ cells cease mitotic division and enter G1/G0 arrest and meiosis prophase I respectively. Aberrant cell cycle regulation at this time can lead to disrupted ovarian development, germ cell apoptosis, reduced fertility and/or the formation of germ cell tumours.

ALDH1A1 provides a source of meiosis-inducing retinoic acid in mouse fetal ovaries.

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.

Sex determination in mammalian germ cells

Germ cells are the precursors of the sperm and oocytes and hence are critical for survival of the species. In mammals, they are specified during fetal life, migrate to the developing gonads and then undergo a critical period during which they are instructed, by the soma, to adopt the appropriate sexual fate. In a fetal ovary, germ cells enter meiosis and commit to oogenesis, whereas in a fetal testis, they avoid entry into meiosis and instead undergo mitotic arrest and mature toward spermatogenesis. Here, we discuss what we know so far about the regulation of sex-specific differentiation of germ cells, considering extrinsic molecular cues produced by somatic cells, as well as critical intrinsic changes within the germ cells. This review focuses almost exclusively on our understanding of these events in the mouse model.