Steroidogenic Capacity of Residual Ovarian Tissue in 4-Vinylcyclohexene Diepoxide-Treated Mice Short title: Steroidogenesis in Ovarian Stroma Summary sentence: Residual ovarian tissue of follicle-depleted mice expresses genes involved in steroidogenesis and produces androgens.

Menopause is an important public health issue because of its association with a number of disorders. Androgens produced by residual ovarian tissue after menopause could impact the development of these disorders. However, it has been unclear whether the post-menopausal ovary retains steroidogenic capacity. Thus, an ovary-intact mouse model for menopause which uses the occupational chemical 4-vinylcyclohexene diepoxide (VCD) was used to characterize the expression of steroidogenic genes in residual ovarian tissue of follicle-depleted mice. Female B6C3F1 mice (28d) were dosed daily for 20d with vehicle or VCD (160 mg/kg/day) to induce ovarian failure. Ovaries were collected on d181 and analyzed for mRNA and protein. Acyclic aged mice were used as controls for natural ovarian senescence. Relative to cycling controls, expression of mRNA encoding steroidogenic acute regulatory protein (Star), cholesterol side chain cleavage (Cyp11a1), 3beta-hydroxysteroid dehydrogenase (Hsd3b), 17alpha-hydroxylase (Cyp17a1), scavenger receptor class B, type 1 (Scarb1), low density lipoprotein receptor (Ldlr) and luteinizing hormone receptor (Lhcgr) was enriched in VCD-treated ovaries. In acyclic aged ovaries mRNA for only Cyp17a1 and Lhcgr was greater than controls. Compared to cycling controls, ovaries from VCD-treated and aged mice had similar levels of HSD3B, CYP17A1 and LHCGR protein. The pattern of protein immunofluorescence staining for HSD3B in follicle-depleted (VCD-treated) ovaries was homogeneous while that for CYP17A1 was only seen in residual interstitial cells. Circulating levels of FSH and LH were increased, and androstenedione levels were detectable following follicle depletion in VCD-treated mice. These findings support that residual ovarian tissue in VCD-treated mice retains androgenic capacity.