LAUSR

Abstract Prenatal testosterone (T) excess leads to oligo/anovulation and multifollicular ovary in sheep, characteristics of women with PCOS. Persistence of antral follicles - the result of reduced atresia, follicular growth arrest and premature luteinization, contribute at least in part to the multifollicular ovarian morphology. Decrease in aromatase (CYP19) in granulosa cells and 17alpha-hydroxylase (CYP17) in theca cells and increase in anti-Mullerian hormone (AMH) in granulosa cells may contribute to the antral follicular persistence. Observations that histone H3K9 hypermethylation decreases cumulus cell CYP19 (Journal of Assisted Reproduction and Genetics 33: 1105-1113, 2016), AMH gene hypermethylation reduces granulosa cell AMH (Oncotarget 6: 3627-3643, 2015), and inhibition of histone deacetylation reduces theca cell CYP17 expression (PloS One 7: e49553, 2012) suggest that epigenetic changes are involved. We hypothesized that prenatal T excess induced changes in expression of CYP17, CYP19 and AMH are facilitated by changes in expression of key epigenetic enzymes. Granulosa / theca cells were isolated via laser capture microdissection from antral follicles of 21 months-of-age control (n=5) and prenatal T-treated (100mg im. twice weekly from gestational day 30 to 90; term: 147 days; n=6) sheep. Expression of methylation / demethylation and / or acetylation / deacetylation enzymes were determined by real time PCR and data analyzed by Student’s t-test and Cohen’s effect size analysis. Prenatal T excess decreased (p < 0.05) histone demethylase KDM1A in granulosa and theca cells, histone acetylase (HDAC) 3 in theca cells, and increased histone methyltransferase SMYD3 in the theca cells. Prenatal T excess also induced large magnitude (1) increases in DNA methyltransferase (DNMT) 1, histone methyltransferases EZH2 and SUV39H1, and histone deacetylase HDAC1 in granulosa cells; (2) decrease in DNA methyltransferase DNMT3B, KDM1A, and HDAC3 in granulosa cells; (3) increase in SMYD3 and HDAC1 in theca cells, and (4) decrease in KDM1A and HDAC3 in theca cells. While decrease in KDM1A and increase in SUV39H1 leading to H3K9 hypermethylation may account for reduced granulosa cell expression of CYP19, downregulation of HDAC3 may underlie the decrease in thecal cell expression of CYP17. Decreased DNMT3B expression may contribute to hypomethylation of AMH gene and consequent increase in AMH expression in granulosa cells. These findings suggest that changes in expression of key genes involved in the development of multifollicular phenotype are likely mediated via granulosa and theca cell specific changes in epigenetic enzyme expression. Because the ovarian attributes of prenatal T-treated sheep parallel that seen in PCOS women, these observations may be of translational relevance. Supported by NIH PO1HD44232.