Steroidogenic factor 1 (SF-1), an orphan nuclear receptor, regulates the enzymes that produce sex steroids, and disruption of the Ftz-F1 gene encoding SF-1 precludes adrenal and gonadal development. We now study the role of SF-1 at other levels of the hypothalamic/pituitary/gonadal axis. In Ftz-Fl-disrupted mice, immunohistochemical analyses with antibodies against pituitary trophic hormones showed a selective loss of gonadotrope-specific markers, supporting the role of SF-1 in gonadotrope function. In situ hybridization analyses confirmed these results; pituitaries from Ftz-Fl-disrupted mice lacked transcripts for three gonadotrope-specific markers (LH~, FSH~, and the receptor for gonadotropin-releasing hormone), whereas they exhibited decreased but detectable expression of the ~-subunit of glycoprotein hormones. SF-1 transcripts in the developing mouse pituitary, which first became detectable at embryonic day 13.5-14.5, preceded the appearance of FSH~ and LH~ transcripts. In adult rat pituitary cells, SF-1 transcripts colocalized with immunoreactivity for the gonadotrope-specific LH. Finally, SF-1 interacted with a previously defined promoter element in the glycoprotein hormone ~-subunit gene, providing a possible mechanism for the impaired gonadotropin expression in Ftz-Fl-disrupted mice. These studies establish novel roles of this orphan nuclear receptor in reproductive function.

Hypersecretion of luteinizing hormone (LH) is implicated in infertility and miscarriages in women. A lack of animal models has limited progress in determining the mechanisms of LH toxicity. We (7)], it is difficult to devise protocols for chronic administration of exogenous LH that mimic endogenous pulse patterns of LH. To circumvent this limitation, we report a transgenic mouse model in which elevated hormone levels are maintained chronically, without requiring multiple injections,The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. supraphysiologic dosing, or dampening of the hypothalamicpituitary-gonadal axis.We utilized a two-pronged approach to achieve elevated levels of serum LH. (i) Increased secretion of hormone from the pituitary was achieved by expression of an LH(3 subunit transgene, targeted to the pituitary by a previously characterized bovine a-subunit promoter (8,9). (ii) The LH,B transgene was modified to encode a peptide extension that we proposed would slow the elimination of LH from the serum. This peptide is normally found at the C terminus of the 3 subunit of human chorionic gonadotropin (hCG) (hCG,B) and is referred to as the C-terminal peptide (CTP) (10). The CTP is thought to be a major determinant of the serum t,'2 of hCG and has been shown to increase the til2 of FSH 2-to 3-fold when fused to its (3 subunit (11). Accordingly, we constructed a transgene with the coding region of bovine (b) LH,B fused in-frame to the coding region of CTP (bLH3-CTP). MATERIALS AND METHODSConstruction of the bLH3-CTP Transgene. The bLH,B-CTP minigene was engineered by a two-step PCR. (i) A short fragment containing the C terminus of bLH,B (30 bp) fused in-frame to the CTP (last 87 bp of the hCGj3 gene) was generated. (ii) This fragment was lengthened to contain the entire bLHf3 cDNA fused in-frame to the CTP. This fusion gene was utilized for transfection experiments but was modified to contain the first intron of bLH3 for the transgene construct. The resulting insert was ligated into a BSK-plasmid already containing the bovine a-subunit promoter (positions -315 to +45) and the simian virus 40 late polyadenylylation signal. Transgenic mice were generated by microinjecting the bLH3-CTP insert into fertilized oocytes as described (12). Mice were genotyped by PCR analysis of tail DNA using primers specific to the a-subunit promoter and (3-subunit gene.Analysis of t,,2. Recombinant bLH and bLH-CTP heterodimers were generated by stably cotransfecting CHO cells with viral expression vectors containing the bovine a-subunit gene and either the bLH( or bLH,3-CTP genes as described (13). Serum-free medium was collected and ammonium sulfate-precipitated or concentrated with an Amicon ultrafiltration cell. Female rats were pretreated with 50 ,tg of antide (Sigma) in 20% (vol/vol) propylene glycol, injected subcutaneously 2 h prior to hormone injections t...

The nuclear receptor steroidogenic factor 1 (SF-1) regulates the biosynthesis of the two essential mediators of male sexual differentiation, androgens and Müllerian-inhibiting substance, and is required for adrenal and gonadal development and gonadotropin expression. SF-1 is also expressed in the embryonic ventral diencephalon, subsequently localizing to the ventromedial hypothalamic nucleus, a region important for reproductive behavior. Mice lacking SF-1 secondary to targeted disruption of the Ftz-F1 gene had normal numbers and location of GnRH neurons but exhibited grossly impaired ventromedial hypothalamic nucleus structure. Despite their apparently normal GnRH neurons, treatment of Ftz-F1-disrupted mice with GnRH restored pituitary gonadotropin expression. These studies define SF-1's essential role within a discrete hypothalamic nucleus previously linked to reproduction.