Mechanism of asymmetric ovarian development in chick embryos

In most animals, the gonads develop symmetrically, but most birds develop only a left ovary. A possible role for estrogen in this asymmetric ovarian development has been proposed in the chick, but the mechanism underlying this process is largely unknown. Here, we identify the molecular mechanism responsible for this ovarian asymmetry. Asymmetric PITX2 expression in the left presumptive gonad leads to the asymmetric expression of the retinoic-acid (RA)-synthesizing enzyme, RALDH2, in the right presumptive gonad. Subsequently, RA suppresses expression of the nuclear receptors Ad4BP/SF-1 and estrogen receptor alpha in the right ovarian primordium. Ad4BP/SF-1 expressed in the left ovarian primordium asymmetrically upregulates cyclin D1 to stimulate cell proliferation. These data suggest that early asymmetric expression of PITX2 leads to asymmetric ovarian development through up- or downregulation of RALDH2, Ad4BP/SF-1, estrogen receptor alpha and cyclin D1.     

Dax1 suppresses P450arom expression in medaka ovarian follicles

Dax1 is a member of an unusual orphan nuclear receptor family, and is known to regulate P450arom in mammals and is involved in sex differentiation in some vertebrates. To investigate whether Dax1 is involved in the regulation of the steroidogenic pathway for estrogen biosynthesis in medaka ovarian follicles, we isolated Dax1 cDNA from adult medaka ovaries and analyzed its expression pattern in medaka gonads. In adult ovaries, Dax1 mRNA was detected only in postvitellogenic follicles and was not detected in previtellogenic and vitellogenic follicles. In adult testis, Dax1 mRNA was not detected. We compared the expression pattern of Dax1 with that of Foxl2, Ad4BP/Sf-1, P450c17, and P450arom by in situ hybridization using adjacent sections. In contrast to Dax1 expression, these genes were co-expressed in vitellogenic follicles but were not detected in postvitellogenic follicles. Thus, in medaka ovarian follicles, Dax1 did not show any overlapping expression patterns against Foxl2, Ad4BP/Sf-1, P450c17, and P450arom. Moreover, co-transfection experiments demonstrated that Dax1 inhibits Ad4BP/Sf-1- and Foxl2-mediated P450arom expression. On the other hand, during early sex differentiation, Dax1 mRNA was not detected in both males and females. Our results suggest that Dax1 down-regulates Ad4BP/Sf-1- and Foxl2-mediated P450arom expression in medaka ovarian follicles.     

Differential gene dosage effects of Ad4BP/SF-1 on target tissue development

Ad4BP/SF-1 (NR5A1) was identified as a key regulator of the hypothalamus-pituitary-gonadal and -adrenal axes. Loss-of-function studies revealed that Ad4BP/SF-1 is essential for the development of these tissues and spleen. Here, we generated transgenic mouse with BAC recombinants carrying a dual promoter and Tet-off system. These recombinants have a potential to express lacZ and Ad4BP/SF-1 in the tissues where endogenous Ad4BP/SF-1 is expressed. However, protein level of Ad4BP/SF-1 varied among the tissues of the transgenic mice and probably thereby the target tissues are affected differentially. The BAC-transgenic mice were applied to rescue Ad4BP/SF-1 KO mouse. Interestingly, the mice successfully rescued the gonad and spleen but failed to rescue the adrenal gland. This variation might be dependent on in part the protein expression levels among the tissues and in part on differential sensitivities to the gene dosage.     

Heterogeneity of Ovarian Theca and Interstitial Gland Cells in Mice

It has been established that two developmentally and functionally distinct cell types emerge within the mammalian testis and adrenal gland throughout life. Fetal and adult types of steroidogenic cells (i.e., testicular Leydig cells and adrenocortical cells) develop in the prenatal and postnatal period, respectively. Although the ovary synthesizes steroids postnatally, the presence of fetal-type steroidogenic cells has not been described. We had previously established transgenic mouse lines in which fetal Leydig cells were labeled with an EGFP reporter gene by the FLE (fetal Leydig enhancer) of the Ad4BP/SF-1 (Nr5a1) gene. In the present study, we examined the reporter gene expression in females and found that the reporter gene is turned on in postnatal ovaries. A comparison of the expressions of the EGFP and marker genes revealed that EGFP is expressed in not all but rather a proportion of steroidogenic theca and in interstitial gland cells in the ovary. This finding was further supported by experiments using BAC transgenic mice in which reporter gene expression recapitulated endogenous Ad4BP/SF-1 gene expression. In conclusion, our observations from this study strongly suggest that ovarian theca and interstitial gland cells in mice consist of at least two cell types.     

Gonadal structure of the serial-sex changing gobiid fish Trimma okinawae

In order to obtain basic information about the role played by endogenous sex hormones in bringing about sex changes in the serial-sex changing gobiid fish Trimma okinawae, the gonadal structure of male and female phases were observed histologically. Steroid-producing cells (SPC; Leydig cells in a testis) were observed ultrastructurally in the ovaries and testes of both female-phase and male-phase fish. In addition, gonadal expression of P450 cholesterol side-chain-cleavage (scc) was examined immunohistochemically. Gonads of fish in female and male phases were observed to have both ovaries and testes simultaneously. Female-phase fish had matured with many developed vitellogenic oocytes, while male-phase individuals had immature ovaries with many numbers of previtellogenic oocytes at the perinucleolus stage. Testes of fish in different sexual phases had active spermatogenic germ cells. Organellae of SPC in the ovaries of female-phase fish had active structures of steroid production. In contrast, SPC in the ovaries of male-phase fish did not show active structures of steroid production. Immunopositive reactions against the scc antibody in the ovaries of female-phase fish were very strong, but immunoreactions in the ovaries of male-phase fish were very weak. In the testis, moderate immunopositive signals were obtained from dual-phase male/females.     

Structural characterization of the chicken SF-1/Ad4BP gene

SF-1/Ad4BP was identified as a master regulator controlling steroidogenic P-450 genes and belongs to the steroid hormone receptor superfamily. It is expressed in the adrenal cortex, gonads, and pituitary gonadotroph. Targeted disruption of the mouse SF-1/Ad4BP gene showed that it plays a critical role in the development of the steroidogenic tissues and pituitary gonadotroph. We have recently cloned the chicken SF-1/Ad4BP cDNA and have now cloned the chicken SF-1/Ad4BP gene and analyzed its promoter activity. This gene consists of seven exons as well as mammalian counterparts and spans about 15 kb. In mice, the gene encodes another protein, ELP, but we could not find the open reading frame of ELP in the chicken SF-1/Ad4BP gene. The promoter of this gene included five putative cis elements (E, CCAAT, GC and TATA boxes and a GA-rich element), although no TATA box has been found in mammalian counterparts. The E and CCAAT boxes moderately affected promoter activity and the GA-rich element and TATA box were essential for the expression of the chicken SF-1/Ad4BP gene.     

Functional characterization of a new human Ad4BP/SF-1 variation, G146A

Ad4BP/SF-1 plays key roles at all levels of the hypothalamic-pituitary-steroidogenic organ axis and its functional disruption causes endocrine disorders of these organs. However, only three human subjects with Ad4BP/SF-1 mutations have been reported to date, suggesting limited clinical significance as a cause of inborn adrenal or sexual abnormalities. We report the first functional characterization of a new variation found in the hinge region of human Ad4BP/SF-1, G146A. Resulting from a single nucleotide shift (GGG-->GCG), G146A bears slightly diminished transactivation activity evidenced by both adrenal specific cyp11A promoter and ovary specific cyp19 promoter II. The variation does not affect protein expression or stability, exhibiting no dominant negative effect. G146A has a normal interaction pattern with standard co-regulators and subnuclear distribution pattern, and can be considered as a nonsynonymous single nucleotide polymorphism, since it occurs in normals and patients with adrenal diseases. In normal Japanese the allele C frequency is 8%, while in a preliminary population of patients with adrenal diseases it is elevated to 30%; suggesting the G146A variation might be of clinical importance.