Ovulation is a precisely timed process by which a mature oocyte is released from an ovarian follicle. This process is initiated by the pituitary surge of luteinizing hormone (LH), is temporally associated with transcriptional regulation of numerous genes, and is presumed to involve the synthesis and͞or activation of specific proteases that degrade the follicle wall. The progesterone receptor (PR), a nuclear receptor transcription factor, is induced in granulosa cells of preovulatory follicles in response to the LH surge and has been shown to be essential for ovulation, because mice lacking PR fail to ovulate and are infertile. Using these mice as a model in which to elucidate PR-regulated genes in the ovulation process, we show that the matrix metalloproteinases MMP-2 and MMP-9 are not targets of PR during ovulation. In contrast, two other proteases, ADAMTS-1 (A disintegrin and metalloproteinase with thrombospondin-like motifs) and cathepsin L (a lysosomal cysteine protease), are transcriptional targets of PR action. ADAMTS-1 is induced after LH stimulation in granulosa cells of preovulatory follicles and depends on PR. Cathepsin L is induced in granulosa cells of growing follicles by follicle-stimulating hormone, but the highest levels of cathepsin L mRNA occur in preovulatory follicles in response to LH in a PR-dependent manner. The identification of two regulated proteases in the ovary, together with their abnormal expression in anovulatory PR knockout mice, suggests that each plays a critical role in follicular rupture and represents a major advance in our understanding of the proteolytic events that control ovulation.

Oocyte and embryo metabolism are closely linked with their subsequent developmental capacity. Lipids are a potent source of cellular energy, yet little is known about lipid metabolism during oocyte maturation and early embryo development. Generation of ATP from lipids occurs within mitochondria via beta-oxidation of fatty acids, with the rate-limiting step catalyzed by carnitine palmitoyl transferase I (CPT1B), a process also requiring carnitine. We sought to investigate the regulation and role of beta-oxidation during oocyte maturation and preimplantation development. Expression of Cpt1b mRNA, assessed by real-time RT-PCR in murine cumulus-oocyte complexes (COCs), increased following hormonal induction of oocyte maturation and ovulation in vivo with human chorionic gonadotropin (5 IU) and in embryos reaching the blastocyst stage. Beta-oxidation, measured by the production of (3)H(2)O from [(3)H]palmitic acid, was significantly increased over that in immature COCs following induction of maturation in vitro with epidermal growth factor (3 ng/ml) and follicle-stimulating hormone (50 mIU/ml). The importance of lipid metabolism for oocyte developmental competence and early embryo development was demonstrated by assessing the rate of embryo development following inhibition or upregulation of beta-oxidation with etomoxir (an inhibitor of CPT1B) or L-carnitine, respectively. Inhibition of beta-oxidation during oocyte maturation or zygote cleavage impaired subsequent blastocyst development. In contrast, L-carnitine supplementation during oocyte maturation significantly increased beta-oxidation, improved developmental competence, and in the absence of a carbohydrate energy supply, significantly increased 2-cell cleavage. Thus, carnitine is an important cofactor for developing oocytes, and fatty acids are an important energy source for oocyte and embryo development.

In obesity, accumulation of lipid in nonadipose tissues, or lipotoxicity, is associated with endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and ultimately apoptosis. We have previously shown that obese women have increased triglycerides in follicular fluid; thus, the present study examined whether high-fat diet-induced obesity causes lipotoxicity in granulosa cells and the cumulus-oocyte complex (COC). Oocytes of mice fed a high-fat diet had dramatically increased lipid content and reduced mitochondrial membrane potential compared to those of mice fed a control diet. COCs from mice fed a high-fat diet had increased expression of ER stress marker genes ATF4 and GRP78. Apoptosis was increased in granulosa and cumulus cells of mice fed a high-fat diet. Mice fed a high-fat diet also exhibited increased anovulation and decreased in vivo fertilization rates. Thus, lipid accumulation, ER stress, mitochondrial dysfunction, and apoptosis are markedly increased in ovarian cells of mice fed a high-fat diet. ER stress markers were also analyzed in granulosa cells and follicular fluid from women with varying body mass indices (BMI). ATF4 was increased in granulosa cells and [Ca(2+)] in follicular fluid from obese women compared to nonobese women. These results indicate that lipotoxicity may be occurring in ovarian cells of obese women and may contribute to the reduced pregnancy rates observed in response to obesity.

Successful ovulation requires that developmentally competent oocytes are released with appropriate timing from the ovarian follicle. Somatic cells of the follicle sense the ovulatory stimulus and guide resumption of meiosis and release of the oocyte, as well as structural remodelling and luteinization of the follicle. Complex intercellular communication co-ordinates critical stages of oocyte maturation and links this process with release from the follicle. To achieve these outcomes, ovulation is controlled through multiple inputs, including endocrine hormones, immune and metabolic signals, as well as intrafollicular paracrine factors from the theca, mural and cumulus granulosa cells and the oocyte itself. This review focuses on the recent advances in understanding of molecular mechanisms that commence after the gonadotrophin surge and culminate with release of the oocyte. These mechanisms include intracellular signalling, gene regulation and remodelling of tissue structure in each of the distinct ovarian compartments. Most critical ovulatory mediators exert effects through the cumulus cell complex that surrounds and connects with the oocyte. The convergence of ovulatory signals through the cumulus complex co-ordinates the key mechanistic processes that mediate and control oocyte maturation and ovulation.

Ovulation is a complex process that is initiated by the lutenizing hormone surge and is controlled by the temporal and spatial expression of specific genes. This review focuses on recent endocrine, biochemical, and genetic information that has been derived largely from the identification of new genes that are expressed in the ovary, and from knowledge gained by the targeted deletion of genes that appear to impact the ovulation process. Two main areas are described in most detail. First, because mutant mouse models indicate that appropriate formation of the cumulus matrix is essential for successful ovulation, genes expressed in the cumulus cells and those that control cumulus expansion are discussed. Second, because mice null for the progesterone receptor fail to ovulate and are ideal models for dissecting the critical events downstream of progesterone receptor, genes expressed in mural granulosa cells that regulate the expression of novel proteases are described.

Metabolism and ATP levels within the oocyte and adjacent cumulus cells are associated with quality of oocyte and optimal development of a healthy embryo. Lipid metabolism provides a potent source of energy and its importance during oocyte maturation is being increasingly recognised. The triglyceride and fatty acid composition of ovarian follicular fluid has been characterised for many species and is influenced by nutritional status (i.e. dietary fat, fasting, obesity and season) as well as lactation in cows. Lipid in oocytes is a primarily triglyceride of specific fatty acids which differ by species, stored in distinct droplet organelles that re-localise during oocyte maturation. The presence of lipids, particularly saturated vs unsaturated fatty acids, in in vitro maturation systems affects oocyte lipid content as well as developmental competence. Triglycerides are metabolised by lipases that have been localised to cumulus cells as well as oocytes. Fatty acids generated by lipolysis are further metabolised by b-oxidation in mitochondria for the production of ATP. b-oxidation is induced in cumulus-oocyte complexes (COCs) by the LH surge, and pharmacological inhibition of b-oxidation impairs oocyte maturation and embryo development. Promoting b-oxidation with L-carnitine improves embryo development in many species. Thus, fatty acid metabolism in the mammalian COC is regulated by maternal physiological and in vitro environmental conditions; and is important for oocyte developmental competence.

Obese women exhibit an altered ovarian follicular environment, particularly increased metabolite, C-reactive protein, and androgen activity levels, which may be associated with poorer reproductive outcomes typically observed in these patients.

ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs-1) is a member of the ADAMTS family of metalloproteases which, together with AD-AMTS-4 and ADAMTS-5, has been shown to degrade members of the lectican family of proteoglycans. AD-AMTS-1 mRNA is induced in granulosa cells of periovulatory follicles by the luteinizing hormone surge through a progesterone receptor-dependent mechanism. Female progesterone receptor knockout (PRKO) mice are infertile primarily due to ovulatory failure and lack the normal periovulatory induction of ADAMTS-1 mRNA. We therefore investigated the protein localization and function of ADAMTS-1 in ovulating ovaries. Antibodies against two specific peptide regions, the prodomain and the metalloprotease domain of ADAMTS-1, were generated. Pro-ADAMTS-1 of 110 kDa was identified in mural granulosa cells and appears localized to cytoplasmic secretory vesicles. The mature (85-kDa prodomain truncated) form accumulated in the extracellular matrix of the cumulus oocyte complex (COC) during the process of matrix expansion. Each form of AD-AMTS-1 protein increased >10-fold after the ovulatory luteinizing hormone surge in wild-type but not PRKO mice. Versican is also localized selectively to the ovulating COC matrix and was found to be cleaved yielding a 70-kDa N-terminal fragment immunopositive for the neoepitope DPEAAE generated by ADAMTS-1 and AD-AMTS-4 protease activity. This extracellular processing of versican was reduced in ADAMTS-1-deficient PRKO mouse ovaries. These observations suggest that one function of ADAMTS-1 in ovulation is to cleave versican in the expanded COC matrix and that the anovulatory phenotype of PRKO mice is at least partially due to loss of this function. ADAMTS-11 (a disintegrin and metalloprotease with thrombospondin-like repeats-1) is a multidomain, multifunctional metalloprotease first cloned from a colon carcinoma cell line (1). Subsequently, ADAMTS-1 has been shown to be induced by luteinizing hormone (LH) in periovulatory follicles of mouse (2), rat (3), and mare.2 Expression of ADAMTS-1 is dependent on LH-induced expression of progesterone and progesterone receptor (PR) in granulosa cells (2, 3). Progesterone receptor null (PRKO) mice fail to ovulate but differentiate normally to form corpora lutea that contain entrapped oocytes (2, 4, 5). AD-AMTS-1 null mice also display impaired ovulation with morphologically abnormal ovaries (6). These observations have indicated that ADAMTS-1 may play a key role in the ovulation process.Ovulation in the mammalian ovary occurs in response to the LH surge that activates several extracellular matrix (ECM) remodeling processes. Dissolution of the connective matrix and cellular layers at the follicular apex and ovarian surface are necessary for release of the oocyte. Based on the anovulatory phenotypes of mice with null mutations of cyclooxygenase-2 (COX-2) (7, 8), prostaglandin E 2 receptor (9, 10), and bikunin (11), it is becoming evident that expansion of the matrix surrounding the cumulus cells and oocyte is ess...