[show abstract][hide abstract] ABSTRACT: Fatty acid oxidation is an important energy source for the oocyte; however, little is known about how this metabolic pathway is regulated in cumulus-oocyte complexes. Analysis of genes involved in fatty acid oxidation showed that many are regulated by the luteinizing hormone surge during in vivo maturation, including acyl-CoA synthetases, carnitine transporters, acyl-CoA dehydrogenases and acetyl-CoA transferase, but that many are dysregulated when cumulus-oocyte complexes are matured under in vitro maturation conditions using follicle stimulating hormone and epidermal growth factor. Fatty acid oxidation, measured as production of (3)H2O from [(3)H]palmitic acid, occurs in mouse cumulus-oocyte complexes in response to the luteinizing hormone surge but is significantly reduced in cumulus-oocyte complexes matured in vitro. Thus we sought to determine whether fatty acid oxidation in cumulus-oocyte complexes could be modulated during in vitro maturation by lipid metabolism regulators, namely peroxisome proliferator activated receptor (PPAR) agonists bezafibrate and rosiglitazone. Bezafibrate showed no effect with increasing dose, while rosiglitazone dose dependently inhibited fatty acid oxidation in cumulus-oocyte complexes during in vitro maturation. To determine the impact of rosiglitazone on oocyte developmental competence, cumulus-oocyte complexes were treated with rosiglitazone during in vitro maturation and gene expression, oocyte mitochondrial activity and embryo development following in vitro fertilization were assessed. Rosiglitazone restored Acsl1, Cpt1b and Acaa2 levels in cumulus-oocyte complexes and increased oocyte mitochondrial membrane potential yet resulted in significantly fewer embryos reaching the morula and hatching blastocyst stages. Thus fatty acid oxidation is increased in cumulus-oocyte complexes matured in vivo and deficient during in vitro maturation, a known model of poor oocyte quality. That rosiglitazone further decreased fatty acid oxidation during in vitro maturation and resulted in poor embryo development points to the developmental importance of fatty acid oxidation and the need for it to be optimized during in vitro maturation to improve this reproductive technology.
PLoS ONE 01/2014; 9(2):e87327. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Exposure of cumulus-oocyte complexes to the hyperglycaemia mimetic, glucosamine, during in vitro maturation impairs embryo development, potentially through upregulation of the hexosamine biosynthesis pathway. This study examined the effects of in vivo periconception glucosamine exposure on reproductive outcomes in young healthy mice, and further assessed the effects in overweight mice fed a high-fat diet. Eight-week-old mice received daily glucosamine injections (20 or 400mgkg) for 3-6 days before and 1 day after mating (periconception). Outcomes were assessed at Day 18 of gestation. Glucosamine treatment reduced litter size independent of dose. A high-fat diet (21% fat) for 11 weeks before and during pregnancy reduced fetal size. No additional effects of periconception glucosamine (20mgkg) on pregnancy outcomes were observed in fat-fed mice. In 16-week-old mice fed the control diet, glucosamine treatment reduced fetal weight and increased congenital abnormalities, but did not alter litter size. As differing effects of glucosamine were observed in 8-week-old and 16-week-old mice, maternal age effects were assessed. Periconception glucosamine at 8 weeks reduced litter size, whereas glucosamine at 16 weeks reduced fetal size. Thus, in vivo periconception glucosamine exposure perturbs reproductive outcomes in mice, with the nature of the outcomes dependent upon maternal age.
Reproduction Fertility and Development 02/2013; 25(2):405-16. · 2.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: Successful embryo and fetal development is dependent on the quality of the oocyte from which it was derived. Several studies to date have demonstrated the link between appropriate metabolism and sufficient ATP production with oocyte quality and preimplantation embryo development. Metabolism of fatty acids for the purpose of synthesizing ATP occurs within mitochondria via β-oxidation and entry of fatty acids into this organelle is the rate-limiting step in this process. Transport of activated fatty acids into mitochondria is catalyzed by carnitine palmitoyl transferase-I (CPTI) which also requires the metabolite carnitine. Once inside the mitochondrial matrix, fatty acids are broken down into acetyl CoA molecules which are further metabolized via the TCA cycle and electron transport chain to produce ATP. The potential to improve oocyte quality by modulating fatty acid metabolism and β-oxidation with carnitine in culture media formulations or via dietary supplementation has received little attention. This review summarizes studies to date investigating the developmental importance of β-oxidation through the use of metabolic inhibitors and whether regulation by carnitine, in vitro or in vivo, has beneficial effects on oocyte and embryo development. Overall, there is little evidence to date that dietary carnitine can improve oocyte quality or female fertility; however inclusion of l-carnitine to in vitro oocyte maturation and embryo growth media improves embryo outcomes, most likely by supplying the oocyte and embryo with an essential co-factor required to utilize fatty acids.
[show abstract][hide abstract] ABSTRACT: While formation of the expanded cumulus matrix and its importance for oocyte maturation and ovulation are well described, its function in these processes remains unknown. The degree of expansion and expression of cumulus matrix genes are positively correlated with oocyte quality, suggesting that this matrix plays a key role in oocyte maturation. Based on recognized filtration properties of analogous matrices, we investigated whether the cumulus matrix acts as a molecular filter by assessing diffusion of fluorescently labeled dextrans (neutral and negatively charged) and hydrophilic (glucose) and hydrophobic (cholesterol) metabolites in cumulus oocyte complexes (COCs). Expanded in vivo-matured COCs resisted absorption of glucose and cholesterol compared to unexpanded COCs. In vitro-matured (IVM) COCs have a pronounced deficiency in cumulus matrix proteins and have poor oocyte quality. Here we demonstrate that IVM cumulus matrix has deficient filtration properties, with dextran and glucose and cholesterol molecules diffusing more readily into IVM than in vivo-matured COCs. Taking the inverse approach, we found that prostaglandin E2 (PGE2), synthesized by cumulus cells, is retained within the matrix of in vivo-matured COCs but IVM COCs have reduced capacity to retain PGE2, secreting significantly more into the medium. This is the first demonstration of a biophysical property of the cumulus matrix. The ability to regulate metabolite supply from the surrounding environment while sequestering vital signaling factors, such as PGE2, is likely to impact oocyte maturation. Thus, IVM may reduce oocyte quality due to dysregulated control of metabolites and signaling molecules.
Biology of Reproduction 07/2012; 87(4):89. · 4.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the ovarian follicle, oocyte-secreted factors induce cumulus-specific genes and repress mural granulosa cell specific genes to establish these functionally distinct cell lineages. The mechanism establishing this precise morphogenic pattern of oocyte signaling within the follicle is unknown. The present study investigated a role for heparan sulphate proteoglycans (HSPG) as coreceptors mediating oocyte secreted factor signaling. In vitro maturation of cumulus oocyte complexes in the presence of exogenous heparin, which antagonizes HSPG signaling, prevented cumulus expansion and blocked the induction of cumulus-specific matrix genes, Has2 and Tnfaip6, whereas conversely, the mural granulosa-specific genes, Lhcgr and Cyp11a1, were strongly up-regulated. Heparin also blocked phosphorylation of SMAD2. Exogenous growth differentiation factor (GDF)-9 reversed these heparin effects; furthermore, GDF9 strongly bound to heparin sepharose. These observations indicate that heparin binds endogenous GDF9 and disrupts interaction with heparan sulphate proteoglycan coreceptor(s), important for GDF9 signaling. The expression of candidate HSPG coreceptors, Syndecan 1-4, Glypican 1-6, and Betaglycan, was examined. An ovulatory dose of human chorionic gonadotropin down-regulated Betaglycan in cumulus cells, and this regulation required GDF9 activity; conversely, Betaglycan was significantly increased in luteinizing mural granulosa cells. Human chorionic gonadotropin caused very strong induction of Syndecan 1 and Syndecan 4 in mural granulosa as well as cumulus cells. Glypican 1 was selectively induced in cumulus cells, and this expression appeared dependent on GDF9 action. These data suggest that HSPG play an essential role in GDF9 signaling and are involved in the patterning of oocyte signaling and cumulus cell function in the periovulatory follicle.
[show abstract][hide abstract] ABSTRACT: To determine whether the high lipid content of human follicular fluid influences oocyte maturation.
Mouse oocytes as substitutes for human oocytes were exposed to follicular fluids of differing lipid content with outcome monitoring.
Private infertility clinic and university laboratory.
Seventy-four women seeking assisted reproduction, and gonadotropin-stimulated mice.
Assay of follicular fluids for triglyceride and free fatty acids, and stimulation of mouse cumulus-oocyte complexes (COCs) to maturity in vitro in the presence of lipid-rich or lipid-poor follicular fluid.
Oocyte lipid content, expression of endoplasmic reticulum stress marker genes, and oocyte maturation assessed in mouse COCs exposed to lipid-rich follicular fluid were compared with complexes exposed to lipid-poor follicular fluid and complexes matured in vivo.
Follicular fluids were obtained from women of known body mass index undergoing oocyte aspiration at a private infertility clinic, and the follicular fluids were assayed for triglyceride and free fatty acids; those with the highest and lowest levels of these lipids were selected. The mouse COCs exposed to lipid-rich follicular fluid during their maturation had increased oocyte lipid content, induction of endoplasmic reticulum stress markers, and impaired oocyte nuclear maturation.
Increased body mass index is associated with elevated triglycerides and free fatty acids in ovarian follicular fluid. Maturation within this lipid-rich environment is detrimental to oocytes.
Fertility and sterility 03/2012; 97(6):1438-43. · 3.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: Fatty acids such as palmitic acid at high levels are known to induce endoplasmic reticulum (ER) stress and lipotoxicity in numerous cell types and thereby contribute to cellular dysfunctions in obesity. To understand the impact of high fatty acids on oocytes, ER stress and lipotoxicity were induced in mouse cumulus-oocyte complexes during in vitro maturation using the ER Ca(2+) channel blocker thapsigargin or high physiological levels of palmitic acid; both of which significantly induced ER stress marker genes (Atf4, Atf6, Xbp1s, and Hspa5) and inositol-requiring protein-1α phosphorylation, demonstrating an ER stress response that was reversible with the ER stress inhibitor salubrinal. Assessment of pentraxin-3, an extracellular matrix protein essential for fertilization, by immunocytochemistry and Western blotting showed dramatically impaired secretion concurrent with ER stress. Mitochondrial activity in oocytes was assessed by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide staining of inner mitochondrial membrane potential, and oocytes matured in thapsigargin or high-dose palmitic acid had significantly reduced mitochondrial activity, reduced in vitro fertilization rates, and were slower to develop to blastocysts. The deficiencies in protein secretion, mitochondrial activity, and oocyte developmental competence were each normalized by salubrinal, demonstrating that ER stress is a key mechanism mediating fatty acid-induced defects in oocyte developmental potential.
[show abstract][hide abstract] ABSTRACT: Although current embryo culture media are based on carbohydrate metabolism of embryos, little is known about metabolism of endogenous lipids. L-carnitine is a β-oxidation cofactor absent in most culture media. The objective was to investigate the influence of L-carnitine supplementation on bovine embryo development. Abattoir-derived bovine cumulus oocyte complexes were cultured and fertilized. Post-fertilization, presumptive zygotes were transferred into a basic cleavage medium ± carbohydrates (glucose, lactate and pyruvate) ± 5 mm L-carnitine and cultured for 4 days in vitro. In the absence of carbohydrates during culture, embryos arrested at the 2- and 4-cell stages. Remarkably, +L-carnitine increased development to the morula stage compared to +carbohydrates alone (P < 0.001). The beneficial effects of L-carnitine were further demonstrated by inclusion of carbohydrates, with 14-fold more embryos reaching the morula stage after culture in the +carbohydrates +L-carnitine group compared to the +carbohydrates group (P < 0.05). Whereas there was a trend for +L-carnitine to increase ATP (P = 0.09), ADP levels were higher and ATP: ADP ratio were 1.9-fold lower (main effect, P < 0.05) compared to embryos cultured in -L-carnitine. Therefore, we inferred that +L-carnitine embryos were more metabolically active, with higher rates of ATP-ADP conversion. In conclusion, L-carnitine supplementation supported precompaction embryo development and there was an additive effect of +L-carnitine +carbohydrates on early embryo development, most likely through increased β-oxidation within embryos.
[show abstract][hide abstract] ABSTRACT: Ovulation, the release of the oocyte from the ovarian follicle, is initiated by the luteinizing hormone surge. It is clear that highly controlled degradation of the follicle and ovarian wall is required for passage of the oocyte and accompanying cumulus cells from the follicle, but the mechanism has not yet been elucidated. Here we show that cumulus oocyte complexes (COCs) adopt transient adhesive, migratory, and matrix-invading capacities at the time of ovulation. We characterized cell adhesion, migration, and invasion in preovulatory and postovulatory mouse COCs collected over a time course post-human chorionic gonadotropin (hCG) administration. Adhesion of dispersed cumulus cells and intact COCs to extracellular matrix proteins present in the ovarian wall (collagens, laminin, and fibronectin) increased significantly after hCG treatment and declined immediately after ovulation. Cumulus cell migration was low in unexpanded, equine chorionic gonadotropin-only treated COCs, but increased 4, 8, and 10 h post-hCG, reaching a peak at 12 h post-hCG that coincided with ovulation. The ability of cumulus cells to migrate was rapidly diminished in COCs isolated from the oviduct within 2 h postovulation. Cell migration was cumulus cell specific and was not observed in granulosa cells. Invasion through three-dimensional collagen I and matrigel barriers by preovulatory expanded COCs was equivalent to that of a known invasive breast cancer cell line (MB-231). Cumulatively, these results demonstrate that cumulus cells in the expanded COC transition to an adhesive, motile, and invasive phenotype in the periovulatory period that may be required for successful release of the oocyte from the ovary at ovulation.
Biology of Reproduction 01/2012; 86(4):125. · 4.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Obesity can have detrimental effects on pregnancy rates in natural conceptions and also in women undergoing IVF or intracytoplasmic sperm injection (ICSI). This review summarises the most recent clinical literature investigating whether obesity impacts oocyte quality and early embryo growth. In other tissues, obesity leads to lipotoxicity responses including endoplasmic reticulum stress, mitochondrial dysfunction and apoptosis. Recent reports indicate that lipotoxicity is a mechanism by which obesity may impact oocyte quality.
Reproduction Fertility and Development 12/2011; 24(1):29-34. · 2.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: Current embryo culture media are based on the carbohydrate metabolism of embryos. However, little is known about the metabolism of endogenous lipids. This is surprising given the high intracellular lipid densities of embryos of some species and the potential for ATP production via β-oxidation. L-carnitine is a β-oxidation co-factor that is absent in most culture media. The aim of this study is to investigate the influence of carnitine supplementation±carbohydrates on bovine embryo development. Abattoir-derived cattle cumulus-oocyte complexes were cultured and fertilised (Sutton-McDowall et al. 2006 Biol. Reprod. 74, 881-888). Post-fertilisation (24h), presumptive zygotes were transferred into an amino acid-free cleavage media±carbohydrates (glucose, lactate and pyruvate) ±5mM carnitine and cultured for 4 days. The absence of carbohydrates during culture resulted in embryos arresting at the 2- and 4-cell stages. Remarkably, +carnitine significantly increased development to the morula stage compared with +carbohydrates alone (20.4±3% vs 4.7±2.5% morula development; P<0.001). The combination of carbohydrates and carnitine supplementation further improved embryo development, with 14-fold more embryos reaching the morula stage after culture in the +carbohydrates +carnitine group compared with the +carbohydrates group (+carbohydrates=3.1±1.9 vs +carbohydrates +carnitine=43.8±9.1% morula development; P<0.05). The beneficial effects of carnitine supplementation on embryo development were reversed when embryos were cultured in presence of etomoxir, a non-reversible inhibitor of the rate-limiting enzyme of β-oxidation (development to 8-cell stage; +carnitine=33.9±8% vs +carnitine +etomoxir=19.2±4.9%; P<0.05). Intracellular lipid content of embryos +carnitine was determined by culturing presumptive zygotes in media -carbohydrates±carnitine for 24h. Lipid content of embryos was determined by measuring BODIPY 493/503 dye fluorescence. Carnitine supplementation reduced fluorescence intensity 1.8-fold (P<0.001). Adenosine triphosphate and ATP:ADP levels were measured in embryos after 24h of culture±carbohydrates±carnitine. While there was a trend for +carnitine to increase ATP levels (P=0.09), ADP levels were higher and ATP:ADP ratio were 1.9-fold lower (main effect, P<0.05) compared with embryos cultured in -carnitine. This indicates +carnitine embryos were more metabolically active, with higher rates of ATP-ADP conversion. We have shown carnitine supplementation supports pre-compaction embryo development and there is an additive effect of +carnitine +carbohydrate on early embryo development. This is most likely through increased β-oxidation levels within embryos. Current disparities between in vivo and in vitro embryo production, in particular increased lipid content (Romek et al. 2010 Theriogenology 74, 265-276) and decreased developmental potential of in vitro-produced embryos, may be an artefact resulting from limited lipid oxidation in vitro.
Reproduction Fertility and Development 12/2011; 24(1):158-9. · 2.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: Oocyte developmental competence is acquired throughout folliculogenesis and is associated with appropriate differentiation and responsiveness to the luteinizing hormone (LH) surge. The recent development of a novel system for culturing ovarian follicles in a three-dimensional alginate matrix shows promise in phenocopying in vivo folliculogenesis. However, oocytes from follicles grown in vitro have a reduced capacity to complete nuclear maturation and be fertilized compared to oocytes matured in vivo. Oocyte metabolism is closely linked with oocyte quality, and we have recently shown that beta-oxidation of lipids is essential for oocyte developmental competence. Thus we investigated whether upregulation of beta-oxidation by treatment with the fatty acid transport cofactor l-carnitine could improve folliculogenesis and developmental competence of mouse follicles following three-dimensional culture. Ovarian hormones (androstenedione, estradiol, and progesterone) and the induction of cumulus matrix proteins (hyaluronan and ADAMTS1) were similar to in vivo follicles, indicating that appropriate differentiation of follicular cells occurs in cultured follicles after an LH/human chorionic gonadotropin (hCG) stimulus. l-carnitine did not alter survival, growth, or differentiation of follicles. However, l-carnitine supplementation significantly increased beta-oxidation, and markedly improved both fertilization rate and blastocyst development. Together, these results show that appropriate responsiveness of the follicle to the LH/hCG surge occurs following three-dimensional follicle culture but limitations on key metabolic requirements remain. l-carnitine supplementation during in vitro follicle culture increased lipid metabolism and improved oocyte developmental competence.
Biology of Reproduction 05/2011; 85(3):548-55. · 4.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: This review summarizes some of the recent advances in obesity research and describes how we and others have built upon these findings to better understand the impact of obesity on granulosa cells, cumulus cells and oocytes within the ovaries of obese females. Obesity is associated with lipid accumulation in non-adipose tissue cells and the induction of oxidative stress and endoplasmic reticulum stress responses that are tightly linked with systemic inflammation. Analysis of ovarian cells and fluid of obese women indicates that these same mechanisms are activated in the ovary in response to obesity. Studies in mice support this and allow further dissection of the pathways by which diet-induced obesity contributes to changes in mitochondria and the endoplasmic reticulum. These studies are in their infancy but cumulatively provide basic information about the cellular mechanisms that may lead to the impaired ovulation and reduced oocyte developmental potential that is observed in obese females.
Journal of Reproductive Immunology 02/2011; 88(2):142-8. · 2.34 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Biology of Reproduction 12/2010; 83(6):909-18. · 4.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: The lymphatic vasculature plays a number of essential physiological roles including maintaining fluid homeostasis, providing a network for the transport of immune cells, and facilitating the uptake of fat-soluble nutrients from the gastrointestinal tract. Although the critical importance and remodeling capacity of the blood vasculature has been well described within the ovary, just a few reports describe the lymphatic vasculature. Using histological and molecular techniques, we report the kinetics of ovarian lymphangiogenesis and the hormonal regulation of lymphangiogenic growth factors associated with key stages of ovarian follicle growth. We exploited the Adamts1-null mouse model, a model with a previously characterized lymphatic defect to further interrogate the mechanisms controlling ovarian lymphangiogenesis. The establishment and development of the ovarian lymphatic vascular network in postnatal developing ovaries was associated with the presence and hormonal regulation of the lymphangiogenic growth factors and their receptors, including Vegfc, Vegfd, and Vegfr3. We characterized the hormonally regulated remodeling of the ovarian lymphatic vasculature in response to FSH and estradiol. The lymphatic network was defective in the Adamts1-null ovary, clearly demonstrating both the involvement of FSH/estradiol and the Adamts1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) protease in ovarian lymphangiogenesis. This study provides the first evidence of a malleable lymphatic system responsive to hormonal changes of the female reproductive cycle, at least in the mouse ovary, suggesting a role for lymphatic vessel functions in normal folliculogenesis.
[show abstract][hide abstract] ABSTRACT: Lipid droplet proteins regulate the storage and utilisation of intracellular lipids. Evidence is emerging that oocyte lipid utilisation impacts embryo development, but lipid droplet proteins have not been studied in oocytes. The aim of the present study was to characterise the size and localisation of lipid droplets in mouse oocytes during the periovulatory period and to identify lipid droplet proteins as potential biomarkers of oocyte lipid content. Oocyte lipid droplets, visualised using a novel method of staining cumulus-oocyte complexes (COCs) with BODIPY 493/503, were small and diffuse in oocytes of preovulatory COCs, but larger and more centrally located after maturation in response to ovulatory human chorionic gonadotrophin (hCG) in vivo, or FSH + epidermal growth factor in vitro. Lipid droplet proteins perilipin, perilipin-2, cell death-inducing DNA fragmentation factor 45-like effector (CIDE)-A and CIDE-B were detected in the mouse ovary by immunohistochemistry, but only perilipin-2 was associated with lipid droplets in the oocyte. In COCs, perilipin-2 mRNA and protein increased in response to ovulatory hCG. IVM failed to induce perilipin-2 mRNA, yet oocyte lipid content was increased in this context, indicating that perilipin-2 is not necessarily reflective of relative oocyte lipid content. Thus, perilipin-2 is a lipid droplet protein in oocytes and its induction in the COC concurrent with dynamic reorganisation of lipid droplets suggests marked changes in lipid utilisation during oocyte maturation.
Reproduction Fertility and Development 10/2010; 22(8):1262-71. · 2.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: Remodeling of ovarian follicle extracellular matrix is essential for ovulation and vascularization of the corpus luteum (CL). Formation of the cumulus matrix around oocytes also plays an important role in ovulation and subsequent fertilization of oocytes. ADAMTS1 is an extracellular metalloprotease induced in ovarian follicles by ovulatory hormones and is required for fertility. In this study, we identified ADAMTS1-mediated structural and morphological changes in remodeling of the follicle and cumulus oocyte complex (COC). In Adamts1(-/-) mice, the ovulation rate was 77% reduced and fertilization of ovulated oocytes was reduced a further 63%, resulting in a reduced number of litters and pups per litter. Morphological assessment of peri-ovulatory ovaries revealed abnormal morphogenesis with a lack of thecal/vascular invagination in the basal region of follicles. Cleavage of the ADAMTS1 substrate, versican, at these invaginating regions was abundant in Adamts1(+/-) but undetectable in Adamts1(-/-) ovaries, indicating that processing of versican by ADAMTS1 is involved in ovulating follicle remodeling. Versican and hyaluronan localization was abnormal during COC matrix expansion, and versican persisted beyond the expected time of fertilization in Adamts1(-/-) but was catabolized and cleared from control COC. The results demonstrate that ADAMTS1 is critical in both ovulation and fertilization processes in vivo. The protease activity of ADAMTS1 mediates neomorphogenesis of the ovulating follicle wall and COC matrix necessary for successful ovulation and fertilization, as well as subsequent catabolism of versican required for degradation of COC matrix after fertilization.
Biology of Reproduction 10/2010; 83(4):549-57. · 4.03 Impact Factor