Inhibition of Histone Deacetylase Activity Amplifies Retinoic Acid-mediated Induction of Stra8 Expression and Oogenesis in Ovaries of Adult Female Mice. (2008)

Our lab and others have recently reported that, contrary to longstanding beliefs, oogenesis and folliculogenesis occur in the ovaries of adult female mice (Nature 2004 428:145-150; Cell 2005 122:303-315; Reproduction 2006 132:95-109; Cell Cycle 2007 6:2678-2684). Although this work remains controversial (PloS Biol 2007 5:e338 doi:10.1371/journal.pbio.0050338), we have initiated an in-depth analysis of the molecular mechanisms that might be central to the regulation of postnatal oogenesis. Thus far, we have shown that in-vivo inhibition of histone deacetylase (HDAC) activity with trichostatin-A (TSA) rapidly increases primordial follicle numbers in prepubertal as well as in adult female mice (Cell 2005 122:303-315). Others have shown that retinoic acid (RA) is a key mediator of germ cell entry into meiosis in embryonic ovaries and adult testis through transcriptional activation of stimulated by retinoic acid gene 8 (Stra8), which encodes a germline-specific protein required for premeiotic DNA synthesis and early meiotic progression (Nat Genet 2006 38:1430-1434.). We therefore hypothesized that RA signaling may regulate postnatal oogenesis by similar mechanisms, and that this may involve HDAC-dependent epigenetic modification of chromatin structure. To test this, we first analyzed Stra8 expression in ovaries from 60 C57BL/6 female mice at 60 days of age in relation to their immature follicle counts. We found that Stra8 expression was detectable in about 25% of the ovaries examined. Interestingly, ovarian Stra8 expression showed a strong correlation with a lower than average immature oocyte reserve, presumably on the verge of replenishment. In order to gain further insights into RA-mediated Stra8 activation, we cloned 1.4-kb of 5-flanking region of the mouse Stra8 coding sequence. This promoter was ligated upstream of DsRed in an expression vector, which was then permanently transfected into F9 teratoma cells. Treatment of F9pStra8-DsRed cells with either RA or TSA alone marginally induced DsRed expression; however, the two agents together caused a dramatic increase in DsRed expression that was lost when the RA response elements in the Stra8 promoter were inactivated by targeted mutagenesis. These findings were bolstered by results from ChIP assays from which we observed that HDAC6, which is expressed at high levels in the female germline, is involved in suppressing RA-driven Stra8 transcriptional activation by directly associating with the RA response elements in the Stra8 promoter. Finally, adult (6-month-old) female mice given a single injection of TSA without or with RA exhibited a comparable induction of ovarian Stra8 expression along with a significant (approximately 2-fold) increase in the number of primordial follicles within 24 hours. However, TSA treatment failed to activate Stra8 expression or increase primordial follicle numbers in adult female mice maintained on a diet deficient in vitamin A, which is the precursor for endogenous RA production. These findings indicate that, consistent with the outcomes obtained from our analysis of F9pStra8-DsRed cells, TSA requires the presence of RA to activate endogenous Stra8 expression and to enhance primordial follicle formation in ovaries of adult female mice. In conclusion, RA-mediated Stra8 expression represents the first molecular mechanism that may play a central role in the regulation of postnatal oogenesis in mammals (supported by NIH R37-AG012279).