In the Ovary, the Rhox Library Is the Lender and Some Genes Are the Borrowers

The genes of the Rhox homeobox gene cluster of the Hox family of nuclear transcription factors are expressed exclusively in reproductive tissues and have been recognized to play important roles in reproductive processes [1]. In the male, the complexity of regulation by this family and its implications for maturation of spermatozoa in the epididymis have been demonstrated [1] and commented on [2]. The search for master regulators of folliculogenesis has taken an important and complementary turn, as reported in this issue of the Biology of Reproduction, by the McLean laboratory [3]. The work is derived from the hypothesis that, as in the male, the Rhox homeobox gene cluster may serve as master regulators of folliculogenesis. In particular, the Rhox family is one ‘‘library’’ where granulosa cell genes can ‘‘borrow’’ transcription factors for induction and progesterone can serve as the library card. Fertility in the female is partly dependent on follicle stimulating hormone (FSH)-induced maturation of antral follicles to preovulatory follicles as well as the successful maturation of oocytes. FSH initiates this process and induces granulosa cell proliferation, differentiation, and steroidogenesis [4–6]. In response to FSH (or equine chorionic gonadotropin [eCG], which binds FSH receptor), ovarian granulosa cells synthesize steroidogenic acute regulatory protein (StAR) and side-chain cleavage enzyme (Cyp11a1), both of which are involved in rate-limiting steps in the process of progesterone steroidogenesis [7, 8]. Whereas FSH does not induce expression of the progesterone receptor, it does induce luteinizing hormone (LH) receptor mRNA, and early studies have demonstrated that LH/human chorionic gonadotropin (hCG) induces progesterone receptor mRNA specifically in granulosa cells, suggesting an intraovarian role for progesterone [9]. Gene expression profiling of rat and human granulosa cells stimulated by FSH has been carried out in several laboratories. Such studies have demonstrated that FSH can induce a transcriptome that is markedly dependent on protein kinase A (PKA) activation and which also importantly contains elements and scales of expression which cannot be accounted for only by cAMP production [10]. Therefore, although the PKA signaling pathway is sufficient to account for the induction of most genes (as identified by microarray analysis), including those of the progesterone biosynthetic pathway, optimal induction of aromatase, the LH receptor, and other genes requires FSH, suggesting that FSH induces PKA-independent pathways [10]. Likewise, gene expression analysis of LH effects on periovulatory granulosa cells has revealed that many of the genes expressed are transcription factors and that these are, in turn, predicted to modulate granulosa cell gene expression, ovulation, and luteinization [11]. However, it has not always been possible to identify genetic elements that demonstrate direct progesterone receptor-induced transcription of LH-induced genes. This suggests that these genes ‘‘borrow’’ transcription factors from a ‘‘library,’’ and it would seem reasonable that progesterone is the ‘‘library card.’’ What is additionally remarkable about this study by MacLean et al. [3] is that it reveals how this gene family exhibits distinct windows of peak expression, demonstrating temporal relationships, once again, illustrating the complexity of regulation of this family. Of great interest, the studies offer compelling evidence that expression of the Rhox homeobox genes correlates with a biphasic expression pattern linked to stages of folliculogenesis. In essence, Rhox8 induction by FSH is increased by Rhox5, whose expression is concomitantly induced by FSH receptor signaling. However, as Rhox5 expression wanes, FSH-induced LH receptor can signal to induce progesterone receptor A expression, which then induces peak Rhox8 levels that continue through ovulation. Thus, autologous regulation of folliculogenesis by the very steroids which are induced during the process of differentiation and gonadotropin stimulation is revealed in this study to be linked to expression of one member of the family, Rhox8. The results of the present study indicate that the Rhox alpha cluster (Rhox2 and 3) genes are induced in preovulatory follicles by eCG (which binds to the FSH receptor). Likewise the beta subcluster genes (Rhox5, Rhox6, Rhox8) are induced by FSH receptor stimulation but peak later after LH receptor stimulation. The observation that Rhox8 was expressed most highly in the periovulatory phase 8 h after LH receptor activation, led to the insight that progesterone may be a critical factor. Through elegant and compelling detailed studies, the authors were able to discern that whereas in Rhox5 knockout animals there is lowered expression of Rhox8 following eCG treatment, there was a lack of effect of Rhox5 knockout on the second phase of folliculogenesis. This illustrates more particularly the link to Rhox8 control by progesterone and the facilitative role of Rhox5, unlike in the male where Rhox8 expression is dependent on Rhox5. Here again the authors provided compelling data, using animals that were null for progesterone receptor (PR knockout animals), showing that Rhox8 expression was low on this genetic background. Supporting in vitro data using expression plasmids and transient transfections also supported the notion of Rhox5 as an initiating and permissive factor during early folliculoCorrespondence: James A. Dias, University at Albany, State University of New York, University Hall 307, 1400 Washington Avenue, Albany, NY 12222. E-mail: jdias@albany.edu