Blanche Capel

In many reptile species, gonadal sex is affected by environmental temperature during a critical period of embryonic development-a process known as temperature-dependent sex determination (TSD).1 The oviparous red-eared slider turtle, Trachemys scripta, has a warm-female/cool-male TSD system and is among the best-studied members of this group.2 When incubated at low temperatures, the somatic cells of the bipotential gonad differentiate into Sertoli cells, the support cells of the testis, whereas at high temperatures, they differentiate into granulosa cells, the support cells of the ovary.3 Here, we report the unexpected finding that temperature independently affects the number of primordial germ cells (GCs) in the embryonic gonad at a time before somatic cell differentiation has initiated. Specifically, embryos incubated at higher, female-inducing temperatures have more GCs than those incubated at the male-inducing temperature. Furthermore, elimination of GCs in embryos incubating at intermediate temperatures results in a strong shift toward male-biased sex ratios. This is the first evidence that temperature affects GC number and the first evidence that GC number influences sex determination in amniotes. This observation has two important implications. First, it supports a new model in which temperature can impact sex determination in incremental ways through multiple cell types. Second, the findings have important implications for a major unresolved question in the fields of ecology and evolutionary biology-the adaptive significance of TSD. We suggest that linking high GC number with female development improves female reproductive potential and provides an adaptive advantage for TSD.

Testicular teratomas and teratocarcinomas are the most common testicular germ cell tumors in early childhood and young men, and they are frequently found unilaterally in the left testis. In 129/SvJ mice carrying a heterozygous copy of the potent modifier of tumor incidence Ter, a point mutation in the dead-end homolog one gene (Dnd1 Ter/+), ∼70% of the unilateral teratomas arise in the left testis. We previously showed that in mice, left/right differences in vascular architecture are associated with reduced hemoglobin saturation and increased levels of the hypoxia inducible factor-1 alpha (HIF-1α) in the left compared to the right testis. To test the hypothesis that systemic reduction of oxygen availability in Dnd1 Ter/+ mice would lead to an increased incidence of bilateral tumors, we placed pregnant females from 129/SvJ Dnd1 Ter/+ intercross matings in a hypobaric chamber for 12-h intervals. Our results show that in 129/SvJ Dnd1 Ter/+ male gonads, the incidence of bilateral teratoma increased from 3.3% to 64% when fetuses were exposed to acute low oxygen conditions for 12-h between E13.8 and E14.3. The increase in tumor incidence correlated with the maintenance of high expression of pluripotency genes Oct4, Sox2 and Nanog, elevated activity of the Nodal signaling pathway, and suppression of germ cell mitotic arrest. We propose that the combination of heterozygosity for the Ter mutation and hypoxia causes a delay in male germ cell differentiation that promotes teratoma initiation.

During mammalian development, gonadal sex determination results from the commitment of bipotential supporting cells to Sertoli or granulosa cell fates. Typically, this decision is coordinated across the gonad to ensure commitment to a single organ fate. When unified commitment fails in an XY mouse, an ovotestis forms in which supporting cells in the center of the gonad typically develop as Sertoli cells, while supporting cells in the poles develop as granulosa cells. This central bias for Sertoli cell fate was thought to result from the initial expression of the drivers of Sertoli cell fate, SRY and/or SOX9, in the central domain, followed by paracrine expansion to the poles. However, we show here that the earliest cells expressing SRY and SOX9 are widely distributed across the gonad. In addition, Sertoli cell fate does not spread among supporting cells through paracrine relay. Instead, we uncover a center-biased pattern of supporting cell precursor ingression that occurs in both sexes and results in increased supporting cell density in the central domain. Our findings prompt a new model of gonad patterning in which a density-dependent organizing principle dominates Sertoli cell fate stabilization.

DND1 is essential to maintain germ cell identity. Loss of Dnd1 function results in germ cell differentiation to teratomas in some inbred strains of mice or to somatic fates in zebrafish. Using our knock-in mouse line in which a functional fusion protein between DND1 and GFP is expressed from the endogenous locus (Dnd1GFP), we distinguished two male germ cell (MGC) populations during late gestation cell cycle arrest (G0), consistent with recent reports of heterogeneity among MGCs. Most MGCs express lower levels of DND1-GFP (DND1-GFP-lo), but some MGCs express elevated levels of DND1-GFP (DND1-GFP-hi). A RNA-seq time course confirmed high Dnd1 transcript levels in DND1-GFP-hi cells along with 5-10-fold higher levels for multiple epigenetic regulators. Using antibodies against DND1-GFP for RNA immunoprecipitation (RIP)-sequencing, we identified multiple epigenetic and translational regulators that are binding targets of DND1 during G0 including DNA methyltransferases (Dnmts), histone deacetylases (Hdacs), Tudor domain proteins (Tdrds), actin dependent regulators (Smarcs), and a group of ribosomal and Golgi proteins. These data suggest that in DND1-GFP-hi cells, DND1 hosts coordinating mRNA regulons that consist of functionally related and localized groups of epigenetic enzymes and translational components.