PROJECT SUMMARY Follicle-stimulating hormone (FSH) is essential for follicular development and fertility in women. Since the FSHβ gene was recently reported to be associated with Polycystic Ovary Syndrome (PCOS) and age of menopause in genome-wide association studies (GWAS), we hypothesize that dysregulation of FSHβ transcription contributes to female infertility including PCOS and Premature Ovarian Failure (POF). The goal of this proposal is to understand the contributions of FSH to the pathophysiology of the female hypothala- mic-pituitary-ovarian axis utilizing mechanistic analyses in immortalized gonadotrope cells, a sophisticated pri- mary pituitary perifusion system, novel genetic mouse models of PCOS and POF, and a new mouse model of PCOS. Aim 1 will focus on the impact of androgen excess on FSHβ transcription and secretion in females. First, we will use primary pituitary cells in an innovative perifusion system to directly test the actions of andro- gens on basal, pulsatile GnRH- and activin-induced regulation of FSH secretion and FSHβ gene expression. Then, female mice lacking AR selectively in gonadotropes will be studied for altered reproductive function including FSHβ transcription and FSH secretion in response to excess androgens using a letrozole-induced PCOS mouse model. Finally, we will investigate whether decreased FSH production in our letrozole PCOS mouse model is dependent on GnRH or due to changes in the activin autocrine feedback loop. Aim 2 will char- acterize the functions of single nucleotide polymorphisms (SNPs) found within the regulatory sequences of the FSHβ gene that are associated with PCOS, age at menopause, and altered LH/FSH ratios in GWAS studies. We have shown that one of these SNPs interferes with LHX3 binding to the proximal FSHβ promoter and reduces FSHβ gene expression. These SNPs will be analyzed for molecular mechanisms altering FSHβ transcription. We will then use CRISPR/Cas9 mutagenesis to create mouse models to determine the effects of these SNP alterations on fertility and FSH secretion in vivo and in primary pituitary cells. Aim 3 will investigate the role of FOXL2 in FSHβ regulation and POF. We have shown that FOXL2 interacts with SMAD proteins for activin induction of FSHβ gene expression, with cJun for synergy between GnRH and activin, and with proges- terone receptor for synergy between activin and progesterone. First, we will investigate the molecular basis for these interactions and their roles in hormonal regulation of FSHβ. Next, FOXL2 mutations found in POF will be studied for their effects on FSHβ transcription in coordination with activin, GnRH, and steroid hormones. Lastly, these mutations will be modeled in mice using CRISPR/Cas9 mutagenesis to study effects on fertility and reproductive senescence in vivo and on FSHβ gene regulation in primary pituitary cells. Together, these aims will delineate the mechanisms by which androgens and SNP mutations within the F...