Control of oocyte release by progesterone receptor-regulated gene expression

Abstract

The progesterone receptor (PGR) is a nuclear receptor transcription factor that is essential for female fertility, in part due to its control of oocyte release from the ovary, or ovulation. In all mammals studied to date, ovarian expression of PGR is restricted primarily to granulosa cells of follicles destined to ovulate. Granulosa cell expression of PGR is induced by the pituitary Luteinizing Hormone (LH) surge via mechanisms that are not entirely understood, but which involve activation of Protein Kinase A and modification of Sp1/Sp3 transcription factors on the PGR promoter. Null mutations for PGR or treatment with PGR antagonists block ovulation in all species analyzed, including humans. The cellular mechanisms by which PGR regulates ovulation are currently under investigation, with several downstream pathways having been identified as PGR-regulated and potentially involved in follicular rupture. Interestingly, none of these PGR-regulated genes has been demonstrated to be a direct transcriptional target of PGR. Rather, in ovarian granulosa cells, PGR may act as an inducible coregulator for constitutively bound Sp1/Sp3 transcription factors, which are key regulators for a discrete cohort of ovulatory genes.

Figure 1. The LH surge transcriptionally regulates PGR expression in rodent granulosa cells.

LH binding to its receptor activates adenylate cyclise (AC), stimulating the production of cAMP and activation of protein kinase A (PKA). The LH surge also activates protein kinase C (PKC), which acts synergistically with PKA to induce PGR mRNA expression. PKA activates MAP kinase (MAPK) and its downstream kinases (ERK1/2) in granulosa cells. The downstream targets of these kinases required for PGR induction are currently unknown. The distal region of the PGR promoter contains a CAAT box which binds NF-YB transcription factor, a GC-rich region which binds Sp1/3, and a GATA site which binds GATA4. The proximal promoter consists of an ERE1/2 site, two Sp1/Sp3binding sites and a series of ERE sites. Interestingly, the only essential regulatory sites are the Sp1/Sp3 sites, and even though estrogen receptors are abundant in granulosa cells, they do not bind the EREs. How the constitutively bound Sp1/Sp3 controls transcription at the PGR promoter is not known, but is thought to involve the recruitment of additional, yet to be identified, coregulatory factors. (Sequence numbering based on the murine PGR promoter and adapted from Sriraman et al., 2003).

Figure 2. Progesterone receptor (PGR) is expressed in the ovary in a temporal and cell-type specific manner, as demonstrated by PRlacZ mice, which express the lacZ reporter (blue) under the control of the endogenous PGR promoter.

Top panels: Ovaries from mice treated with PMSG do not express PGR, while the oviduct expresses high levels. Middle panels: In response to hCG to mimic the LH surge, lacZ staining is detected within 8h, specifically in the granulosa cells of preovulatory follicles. Lower panel: In preovulatory follicles, lacZ is detected in mural granulosa cells (blue arrow), but not cumulus cells (black arrow) or the oocyte (O). (Reprinted with permission from Ismail et al., 2002). Copyright 2002, The Endocrine Society.

Figure 3. Mice null for PGR (PRKO) demonstrate that PGR is essential for ovulation, particularly follicular rupture, but not follicle growth or luteinization.

Upper panels show ovary sections from mice heterozygous or null for PGR that were treated with PMSG to stimulate preovulatory follicle growth. Lower panels show ovary sections from mice treated with PMSG followed by hCG for 48h to stimulate ovulation and luteinization. Lower right panel shows that in PRKO mice, oocytes remain trapped within luteinized follicles. (Reprinted with permission from Robker et al., 2000).

Figure 4. A working model of PGR-dependent pathways that are essential for ovulatory events.

The LH surge induces the expression of PGR specifically in granulosa cells, as well as luteinization-specific genes including steroidogenic enzymes that synthesize progesterone (StAR, P450scc), as well as PGR. Locally secreted progesterone (P4) activates PGR, which then transactivates intermediary genes including Hif1α (which is presumably also dependent on hypoxic conditions), which acts in concert with PGR to induce Adamts1 and VegfA. PGR regulation of EGF-like ligands Areg and Ereg would stimulate EGF-R, including that present on cumulus cells. PGR-mediated PPARγ induction acts upon ligand activation, perhaps supplied by COX-2 generated products such as PGJ2, to transactivate genes including Edn2. Edn2 receptors Ednrb are present on cumulus cells, as well as on ovarian smooth muscle cells. Oocyte-secreted factors (OSFs) regulate the induction of COX-2 and prostaglandin PGE, which acts on EP2 receptors, specifically in cumulus cells. Through this sequential cascade of PGR-initiated gene induction, integrated functional events including protease activation, cumulus expansion, smooth muscle contraction, vascular permeability and angiogenesis all contribute to follicle rupture and oocyte release.

Figure 5. Regulatory elements in the ADAMTS1 promoter, a well-characterized PGR-responsive gene in granulosa cells.

The LH surge induces a suite of transcription factors including C/EBPβ, Egr-1 and PGR. C/EBPβ and Egr-1 have been demonstrated to directly bind to the Adamts1 promoter at sites that are essential for induction in response to ovulatory cAMP (Doyle et al., 2004). In contrast, there are no PREs in the promoter and PGR-dependent transcription is proposed to occur via PGR acting as an inducible coregulator with Sp1/Sp3 transcription factors, which are constitutively bound to a series of GC-rich regions (I, II, and III). BTM = basal transcriptional machinery.