Ovulation is stimulated by the preovulatory surge of the pituitary luteinizing hormone (LH). Because the ovulatory response is commonly identified with inflammation, we explored the involvement of reactive oxygen species (ROS) in this process. Our experiments show that administration of broad-range scavengers of oxidative species into the ovarian bursa of mice, hormonally induced to ovulate, significantly reduced the rate of ovulation. LH-induced cumulus mucification/expansion, a necessary requirement for ovulation, was prevented by antioxidants both in vivo and in an ex vivo system of isolated intact ovarian follicles. Along this line, H 2 O 2 fully mimicked the effect of LH, bringing about an extensive mucification/expansion of the follicle-enclosed cumulusoocyte complexes. Impaired progesterone production was observed in isolated follicles incubated with LH in the presence of the antioxidant agents. Furthermore, LH-stimulated up-regulation of genes, the expression of which is crucial for ovulation, was substantially attenuated upon ROS ablation. This system was also used for demonstrating the role of ROS in phosphorylation and activation of the EGF receptor as well as its downstream effector, p42/44 MAPK. Together, our results provide evidence that ovarian production of ROS is an essential preovulatory signaling event, most probably transiently triggered by LH.ovary | cumulus expansion O vulation is an essential prelude for successful reproduction. The ovulatory process is initiated by the midcycle surge of the pituitary luteinizing hormone (LH) that induces substantial biochemical, molecular, and cellular changes, culminating in the release of a mature ovum surrounded by the cumulus cells (1). Before ovulation, the cumulus cells produce an extracellular, hyaluronan-rich matrix that brings about cumulus mucification and its expansion. This response to LH that is essential for ovulation (2) is subsequent to the expression of a set of specific genes (3) such as prostaglandin synthase 2 (Ptgs2) (4), hyaluronan synthase 2 (Has2) (5), tumor necrosis-stimulated gene 6 (Tnfaip6) (6), and CCAAT/enhancer-binding protein β (Cebpb), a downstream target of p42/44 MAPK (Erk1/2) (7). Also essential for ovulation is LH-induced progesterone receptor (Pgr) expression as well as progesterone production (8).Numerous genes related to inflammation are induced in preovulatory follicles by the LH surge (9-12). Moreover, ovulation is suppressed by agents that inhibit acute inflammatory reactions (13). The analogy of ovulation with an acute inflammation (9, 14) may suggest a role for reactive oxygen species (ROS) along this process. ROS originate from inflammatory cells, such as macrophages and neutrophils, which are massively recruited after the LH surge to the ovary (15), and their depletion impairs ovulation (16, 17). Cyclooxygenase 2 (Ptgs2), the expression of which is a hallmark for LHinduced ovulation, is associated with inflammation and generates ROS (18). ROS are also byproducts of monooxygenase reactions mediated by the P450...

In the present work, we employed bioinformatics search tools to select ovulation-associated cDNA clones with a preference for those representing putative novel genes. Detailed characterization of one of these transcripts, 6C3, by real-time PCR and RACE analyses led to identification of a novel ovulation-associated gene, designated Ncoa7B. This gene was found to exhibit a significant homology to the Ncoa7 gene that encodes a conserved tissue-specific nuclear receptor coactivator. Unlike Ncoa7, Ncoa7B possesses a unique and highly conserved exon at the 5 0 end and encodes a protein with a unique N-terminal sequence. Extensive bioinformatics analysis has revealed that Ncoa7B has one identifiable domain, TLDc, which has recently been suggested to be involved in protection from oxidative DNA damage. An alignment of TLDc domain containing proteins was performed, and the closest relative identified was OXR1, which also has a corresponding, highly related short isoform, with just a TLDc domain. Moreover, Ncoa7B expression, as seen to date, seems to be restricted to mammals, while other TLDc family members have no such restriction. Multiple tissue analysis revealed that unlike Ncoa7, which was abundant in a variety of tissues with the highest expression in the brain, Ncoa7B mRNA expression is restricted to the reproductive system organs, particularly the uterus and the ovary. The ovarian expression of Ncoa7B was stimulated by human chorionic gonadotropin. Additionally, using real-time PCR, we demonstrated the involvement of multiple signaling pathways for Ncoa7B expression on preovulatory follicles.

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