Mammalian gonadal sex-determining (GSD) genes are expressed in a unique population

Mammalian gonadal sex-determining (GSD) genes are expressed in a unique population of somatic cells that differentiate into granulosa cells in XX gonads or Sertoli cells in XY gonads. resource for the spatial-temporal expression pattern of genes that are part of the genetic network underlying the early stages of mammalian fetal gonadal development, including the development of granulosa and Sertoli cells. [3, 4] followed by the upregulation of the transcription factor [5, 6]. Ovarian development appears to rely on a complex interaction of the signaling molecules and upregulation [7C9]. The molecular events that are critical for differentiation of XX and XY bipotential gonads occur in a specific population of somatic cells, the somatic support cells (SSCs). The SSCs originate from coelomic epithelium cells covering the genital ridge [10]. The SSCs differentiate as granulosa cells in the XX gonad and Sertoli cells in the XY gonad [11]. In the developing ovary, precursor SSCs differentiate into fetal granulosa cells that 1) associate with germ cell clusters to form ovigerous cords, 2) likely play a role in the breakdown of these cords, and 3) form primordial follicles at the time of birth [12]. In the developing testis, precursor SSCs express (((originally were identified as GSD genes because when mutated, they caused abnormal gonadal development, including sex reversal (reviewed in Brennan and Capel [17], Park and Jameson [18], and Wilhelm et al. [19]). The fact that many cases of human gonadal sex reversal remain unexplained indicates that additional GSD genes await discovery. We reasoned that the rate of GSD gene discovery would improve if the gene expression profile of SSCs was determined during the early stages of gonad development. We chose to conduct these experiments in mice because the necessary genetic tools were available. Not only would the planned approach provide a powerful resource for identifying additional GSD genes, it would provide a list of potential genes to examine in cases Droxinostat IC50 of unexplained human abnormal GSD. Two previous studies have revealed the expression profile of isolated is expressed in XX and XY SSCs. Because is expressed in additional cell types, including precursor steroidogenic cells, using the transgene introduced the possibility that expression of genes unique to the SSCs was masked in the mixed cell population. For example, neither study Droxinostat IC50 detected the Rabbit Polyclonal to KAL1 expression of the critical GSD gene transgene expressed only in SSCs and an X-linked trangene that distinguishes XX and XY individuals by eye pigmentation. A microarray analysis of the transcriptional profile of E11.5 and E12.5 XX versus XY SSCs identified the spatial-temporal sexual dimorphic expression pattern of many new genes. Real-time RT-PCR analysis verified that 53 of 57 genes tested were expressed in a sexual dimorphic pattern, and whole-mount in situ hybridization (WISH) analysis confirmed the expression pattern of six genes in XX and XY fetal E11.5, E12.5, and E13.5 gonads. Combined, these data provide an important resource for determining the genetic network controlling fetal gonad development and differentiation. MATERIALS AND METHODS The Jackson Laboratory is American Association for Laboratory Animal Science accredited, and all animal procedures were approved by The Jackson Laboratory Animal Care and Use Committee. Mouse Strains FVB.Cg-Tg(Tyr)3412ARpw Tg(Sry-EGFP)92Ei. To Droxinostat IC50 allow XX and XY SSCs to be efficiently isolated from fetal gonads, we constructed a special FVB inbred strain carrying two transgenes. The origin of both transgenes and the method used to construct this strain are presented below. For brevity, this strain is designated FVB-Tg3412A,Tg92. FVB/NJ-Tg3412A. Visual inspection of the gonads cannot be used to determine the sex of individual fetuses prior to E12.5. To circumvent this problem, we obtained FVB-Tg3412A transgenic Droxinostat IC50 mice from Dr. Richard Woychik, then at the Oak Ridge National Laboratory (Oak Ridge, TN). FVB-Tg3412A transgenic mice were created by injecting a tyrosinase minigene (1.95-kb cDNA sequence; Tg3412A) into FVB/N fertilized eggs [22, 23]. Normally, FVB/N mice are homozygous for the albino allele of the tyrosinase gene, and thus are unpigmented. Because Tg3412A inserted into the X chromosome, FVB females carrying a single copy of Tg3412A had a mosaic pigmented pattern, whereas FVB females homozygous for Tg3412A and males hemizygous for Tg3412A were pigmented. By mating normal (albino) FVB/NJ females to FVB/NJ males carrying Tg3412A, the sex of each offspring can be determined as Droxinostat IC50 early as E11: XY offspring lacked eye pigmentation (Tg3412A absent),.