PROJECT SUMMARY Follicle-stimulating hormone (FSH) is essential for follicular development and fertility in women. Since the FSH? 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 FSH? 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 FSH? 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 FSH? gene expression. Then, female mice lacking AR selectively in gonadotropes will be studied for altered reproductive function including FSH? 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 FSH? 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 FSH? promoter and reduces FSH? gene expression. These SNPs will be analyzed for molecular mechanisms altering FSH? 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 FSH? regulation and POF. We have shown that FOXL2 interacts with SMAD proteins for activin induction of FSH? 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 FSH?. Next, FOXL2 mutations found in POF will be studied for their effects on FSH? 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 FSH? gene regulation in primary pituitary cells. Together, these aims will delineate the mechanisms by which androgens and SNP mutations within the FSH? regulatory region alter FSH? gene expression and contribute to PCOS, and how dysregulation of FSH? by mutant FOXL2 proteins contributes to POF. These studies may provide insight for diagnosis and treatment of female infertility.