Studies using microarrays in the pig, cow, and mouse have revealed several genes in granulosa cells of the preovulatory follicles that are associated with ovulation (Jiang et al., 2004; Friedmann et al., 2005; Richards, 2006). Before LH surge, porcine granulosa cells have been shown to express genes related to the proliferation, active metabolism and oxidative stress responses, whereas, after LH surge, these cells expressed genes related to non-proliferation, migration and angiogenesis (Agca et al., 2006). A comprehensive list of ovulation specific genes in follicular cells of cow, mouse, rat, human, monkey and pig has been provided elsewhere (Richards, 2006); however, the gene list needs to be updated.
The expression of genes in granulosa cells changes within minutes to hours after LH surge. In mouse preovulatory follicle, granulosa cells express genes within 1 h after LH surge
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and known as early response genes (Carletti & Christenson, 2009). The expression of 57 early response genes up-regulated and some of these genes were related to EGF-like ligands
(amphiregulin, epiregulin, and epigen), transcription factors (B-cell translocation 1 (BTG-1, and -2), early growth response-1 (EGR-1), nuclear receptor subfamily 4 group A member 1 (NR4A1 and NR4A2), hairy enhancer of split-related with YRPW motif 1 (HEY-1) and GADD45B. Also, steroidogenic (CYP19A1), angiogenic (coagulation factor F3) and inflammatory genes (PTGS2 or COX2) up-regulated after LH within few minutes (Carletti & Christenson, 2009). Analyses of the genes expressed at later time points after LH have been documented in rat and mouse by other researchers (Espey & Richards, 2002; Hennebold, 2004; McRae et al., 2005).
Considering that the ovulation is controlled by genes that are temporally and transiently expressed after LH surge, microarrays were used by researchers to identify those genes in granulosa cells. In cattle, granulosa cells of preovulatory follicles were compared between three time points, i.e. 2 h before LH surge, 6 h and 22 h after LH surge to identify early (6 h) and late (22 h ) response genes (Gilbert et al., 2011). Early response to LH was associated with the preparation of ovulation such as response to a gonadotropin (LH), vascularization, and lipid synthesis. Late response of granulosa cells to LH was associated with luteinization and secretion such as protein localization, and intracellular transport (Gilbert et al., 2011). In another study, the impact of LH surge on gene expression of granulosa cells was determined to assess markers of oocyte competence (Gilbert et al., 2012). The authors concluded that the gene expression in granulosa cells before LH surge can assess bovine oocyte competence accurately compared to those after LH surge (Gilbert et al., 2012). In granulosa cells of bovine preovulatory follicle, the expression of novel genes such as ADP-ribosylation factor GTPase-activating protein 3
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complex-1 (PRC1), serglycin (SRGN), replication protein A2 (RPA2), SCD, and the TRIB2 down-regulated 23 h after hCG treatment (Ndiaye et al., 2005). Most of those genes in bovine granulosa cells were related to follicular growth, ovulation and luteinization (Ndiaye et al., 2005). There are certain granulosa cell genes that are classical in the expression after LH surge and down-regulate (CYP19A1, FSH-R, CYP11A1, LRP8 and SERPINE2) during the bovine periovulatory period (Ndiaye et al., 2005).
In human, gonadotropin-induced genes were in granulosa cells were related to cell signaling such as adenylyl cyclase-7 and -9 (ADCY7/9), cAMP dependent phosphodiestrase (PDE) and a negative regulator of G-protein cell signaling (RGS16), cytoskeleton, and steroidogenesis (Sasson et al., 2004). Biomarkers for oocyte competence and a successful
pregnancy in women have been studied in granulosa cells and cumulus cells (Hamel et al., 2008; Hamel et al., 2010; Assidi et al., 2011). The best predictors of pregnancy were UDP-glucose pyrophosphorylase-2 (UGP2) and pleckstrin homology-like domain, family A, member 1 (PHLDA1) (Hamel et al., 2010). Increased expression of UGP2 indirectly regulates synthesis of glycosaminoglycans like hyaluronan by the granulosa cells in response to LH (Magee et al., 2001). Hyaluronan-treated bovine embryos have high post-transfer survival rates in receipiet cows (Block et al., 2009). Increased expression of the PHLDA1indicates early leutinization of human granulosa cells associated with competent oocyte to prevent apoptosis (Hamel et al., 2008)
Summary
The acquisition of FSH receptors by granulosa cells allows the follicles to respond to the cyclic rise of FSH and emerge from the pool of ovarian follicles. Certain autocrine and paracrine factors such as IGF(s), EGF, TGF, FGF and estrogen synthesizing enzymes (CYP11A1 and CYP19A1) are also involved in the emergence of follicular wave. The selection of a
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dominant follicle occurs when a drop in plasma FSH occurs due to the action of follicular products (inhibins and follistatin). Also the dominant follicle has acquired LHR by this time, so can continue its growth while subordinates regress. Apoptosis and atresia of the follicles result in 1) decreased intrafollicular estradiol concentrations, 2) high amounts of androgens from theca cells, 3) decrease in estradiol receptors on granulosa cells, and 4) expression of Fas antigen for FasL.
Granulosa cells of the growing dominant follicle before LH surge 1) acquire the maximum steroidogenic capacity to to process lipid and cholesterol and to produce estradiol and, 2) proliferate quickly under the influence of growth factors (e.g. IGF, and activins), gonadotropins (via the cAMP-PKA pathway), and estradiol. This results in increased DNA replication and mRNA expression of PCNA in granulosa cells. After the LH surge, the granulosa cells 1) rapidly but transiently inhibit cell cycle by inhibiting the transcription of cyclin D2 and E2 through the expression of cdK inhibitors, 2) synthesizes progesterone rather than estradiol and express high mRNA levels of STAR, HS3DB and CYP11A1, and 3) express early and late response genes that are critical for differentiation and luteinization. Finally, the process of ovulation in granulosa cells is governed by three P’s i.e. progesterone, prostaglandin and proteases.