Abstract
ABSTRACT Prostaglandin E2 (PGE2) is a key paracrine mediator of ovulation. Few specific PGE2-regulated gene products have been identified, so we hypothesized that PGE2 may regulate the expression and/or activity of a network of proteins to promote ovulation. To test this concept, Ingenuity Pathway Analysis (IPA) was used to predict PGE2-regulated functionalities in the primate ovulatory follicle. Cynomolgus macaques underwent ovarian stimulation. Follicular granulosa cells were obtained before (0 h) or 36 h after an ovulatory dose of human chorionic gonadotropin (hCG), with ovulation anticipated 37–40 h after hCG. Granulosa cells were obtained from additional monkeys 36 h after treatment with hCG and the PTGS2 inhibitor celecoxib, which significantly reduced hCG-stimulated follicular prostaglandin synthesis. Granulosa cell RNA expression was determined by microarray and analyzed using IPA. No granulosa cell mRNAs were identified as being significantly up-regulated or down-regulated by hCG + celecoxib compared with hCG only. However, IPA predicted that prostaglandin depletion significantly regulated several functional pathways. Cell cycle/cell proliferation was selected for further study because decreased granulosa cell proliferation is known to be necessary for ovulation and formation of a fully-functional corpus luteum. Prospective in vivo and in vitro experiments confirmed the prediction that hCG-stimulated cessation of granulosa cell proliferation is mediated via PGE2. Our studies indicate that PGE2 provides critical regulation of granulosa cell proliferation through mechanisms that do not involve significant regulation of mRNA levels of key cell cycle regulators. Pathway analysis correctly predicted that PGE2 serves as a paracrine mediator of this important transition in ovarian structure and function.
Highlights
The midcycle surge of luteinizing hormone (LH) initiates structural and functional changes within the ovulatory follicle which culminate in release of the oocyte and formation of the corpus luteum (Duffy et al 2019)
Granulosa cell mRNA levels present after ovarian stimulation and 36 h of exposure to human chorionic gonadotropin (hCG) (36 h hCG) were compared with granulosa cell mRNA levels present after ovarian stimulation in the absence of hCG (0 h hCG). This comparison yielded 31 mRNAs significantly increased by hCG treatment and 1 mRNA significantly decreased by hCG treatment (Table 2)
To determine if a subset of all hCG-induced changes are due to elevated follicular prostaglandins, additional monkeys underwent ovarian stimulation followed by 36 h treatment with hCG and the PTGS2 inhibitor celecoxib, which has previously been shown to reduce hCGstimulated prostaglandin E2 (PGE2) (Seachord et al 2005)
Summary
The midcycle surge of luteinizing hormone (LH) initiates structural and functional changes within the ovulatory follicle which culminate in release of the oocyte and formation of the corpus luteum (Duffy et al 2019). The LH surge initiates follicular synthesis of prostaglandins and, in particular, prostaglandin E2 (PGE2) (Sirois 1994; Sirois and Dore 1997; Mikuni et al 1998; Duffy and Stouffer 2001). Blockade of PGE2 synthesis or action results in unruptured follicles with retained oocytes (Hizaki et al 1999; Tilley et al 1999; Duffy and Stouffer 2002). Since the LH surge initiates synthesis of ovulatory prostaglandins, we might expect that prostaglandin-regulated genes should be a subset of all LH-regulated genes. Additional prostaglandin-regulated gene products have been identified after ovulatory dysfunctions were noted in mice lacking expression of the key prostaglandin synthesis enzyme PTGS2 (Hizaki et al 1999; Tilley et al 1999).
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