Project description:Oocyte competence for early embryo development relies on intercellular communication between the maturing oocyte and preovulatory follicle. Preovulatory follicle maturity, as indicated by serum estradiol concentration or follicle diameter, has previously been linked to pregnancy, follicular fluid metabolites, cumulus-oocyte metabolism, and oocyte competency for embryo development. Such relationships indicate metabolic and developmental programming of the oocyte based on the preovulatory follicle’s physiological status, but downstream impacts on the molecular signature of blastocysts have not been examined. We hypothesized that supplementing maturing oocytes with follicular fluid originating from preovulatory follicles of greater or lesser maturity would impact the transcriptome of resulting blastocysts and indicate metabolic programming of the embryo that originated from the oocyte’s maturation environment. The objective was to investigate the effect of follicle maturity on metabolic preparation of the oocyte by examining the functional genome of blastocysts originating from oocytes matured in the presence of follicular fluid from preovulatory follicles of greater or lesser maturity. In vitro maturing oocytes were supplemented with follicular fluid collected from preovulatory follicles of greater or lesser maturity. Following identical embryo culture procedures, RNA-sequencing was performed on pools of 2 blastocysts (Greater, n = 12; Lesser, n = 15; all with stage code = 7 and quality code = 1). A total of 12,310 gene transcripts were identified in blastocysts after filtering to remove lowly abundant transcripts. There were 113 transcripts that differed in abundance between blastocysts originating from oocytes matured in greater versus lesser maturity follicular fluid (eFDR < 0.01). Although no pathways were significantly enriched with differentially abundant transcripts, transcriptome profiles suggested improved Wnt/β-catenin signaling, metabolism, and protection from oxidative stress in blastocysts derived from oocytes matured in greater maturity follicular fluid, while unregulated cell growth presented in blastocysts resulting from the lesser follicle maturity treatment.
Project description:Oocyte quality is a well- established determinant of embryonic fate. However, the molecular participants and biological markers that affect and predict adequate embryonic development are largely elusive. We have previously reported that oocyte- directed Connexin 43 (Cx43) depletion leads to embryo implantation defects, although both the morphology of the oocyte and processes presiding embryo implantation appear to undergo normally. In the context of previous data determining Cx43 indispensability to oocyte and embryonic development, we show here that the timing of Cx43 depletion from the oocyte and the ovarian follicle is crucial in determining the severity of subsequent embryonic defects. Specifically, we show that the implantation defects of blastocysts resulting from oocyte- directed Cx43- depleted follicles (depletion occurs at day 3 postnatal), is not due to maternal luteal insufficiency but rather depends solely on the defective blastocysts. Gene expression microarray analysis revealed global defects in the expression of ribosomal proteins, translation initiation factors and other genes associated with cellular biosynthetic and metabolic processes in these defective oocytes and specifically blastocysts. We therefore propose that timely expression of Cx43 in the oocyte and ovarian follicles is a major determinant of oocyte developmental competence, by determining the ability of the resulting blastocyst to facilitate biomass expansion and undergo adequate embryo implantation
Project description:Oocyte quality is a well- established determinant of embryonic fate. However, the molecular participants and biological markers that affect and predict adequate embryonic development are largely elusive. We have previously reported that oocyte- directed Connexin 43 (Cx43) depletion leads to embryo implantation defects, although both the morphology of the oocyte and processes presiding embryo implantation appear to undergo normally. In the context of previous data determining Cx43 indispensability to oocyte and embryonic development, we show here that the timing of Cx43 depletion from the oocyte and the ovarian follicle is crucial in determining the severity of subsequent embryonic defects. Specifically, we show that the implantation defects of blastocysts resulting from oocyte- directed Cx43- depleted follicles (depletion occurs at day 3 postnatal), is not due to maternal luteal insufficiency but rather depends solely on the defective blastocysts. Gene expression microarray analysis revealed global defects in the expression of ribosomal proteins, translation initiation factors and other genes associated with cellular biosynthetic and metabolic processes in these defective oocytes and specifically blastocysts. We therefore propose that timely expression of Cx43 in the oocyte and ovarian follicles is a major determinant of oocyte developmental competence, by determining the ability of the resulting blastocyst to facilitate biomass expansion and undergo adequate embryo implantation To study the effect of CX43 on the transcriptom of the pre implantation stages, we compared CX43 KO oocytes to the WT oocytes in three different stages of the very early development. First comparison MII oocytes, second comparison blastocysts, third comparison implantation site.
Project description:Mono(2-ethylhexyl) phthalate (MEHP), the main di(2-ethylhexyl) phthalate (DEHP) metabolite, is a known reproductive toxicant. Residual levels of 20 nM MEHP have been found in follicular fluid aspirated from IVF-treated women and DEHP-treated animals. It is not yet clear whether these residual MEHP levels have any effect on the follicle-enclosed oocyte or developing embryo. To clarify this point, bovine oocytes were matured with or without 20 nM MEHP for 22 h. Microarray analysis was performed for both mature oocytes and 7-day blastocysts. A feasibility examination was performed on mature oocytes (n = 200/group) to reveal a possible direct effect on the oocyte proteomic profile. Transcriptome analysis revealed MEHP-induced alterations in the expression of 456 and 290 genes in oocytes and blastocysts, respectively. The differentially expressed genes are known to be involved in various biological pathways, such as transcription process, cytoskeleton regulation and metabolic pathway. Among these, the expression of 9 genes was impaired in both oocytes exposed to MEHP (i.e., direct effect) and blastocysts developed from those oocytes (i.e., carryover effect). In addition, 191 proteins were found to be affected by MEHP in mature oocytes. The study explores, for the first time, the risk associated with exposing oocytes to physiologically relevant MEHP concentrations to the maternal transcripts. Although it was the oocytes that were exposed to MEHP, alterations carried over to the blastocyst stage, following embryonic genome activation, implying that these embryos are of low quality.
Project description:Egg quality dictates fertility outcomes, and although there is a well-documented decline with advanced reproductive age, how it changes during puberty is less understood. Such knowledge is critical, since advances in Assisted Reproductive Technologies are enabling pre- and peri-pubertal patients to preserve fertility in the medical setting. Therefore, we investigated egg quality parameters in a mouse model of the pubertal transition or juvenescence (postnatal day; PND 11-40). Animal weight, vaginal opening, serum inhibin B levels, oocyte yield, oocyte diameter, and zona pellucida thickness increased with age. After PND 15, there was an age-associated ability of oocytes to resume meiosis and reach metaphase of meiosis II (MII) following in vitro maturation (IVM). However, eggs from the younger cohort (PND 16-20) had significantly more chromosome configuration abnormalities relative to the older cohorts and many were at telophase I instead of MII, indicative of a cell cycle delay. Oocytes from the youngest mouse cohorts originated from the smallest antral follicles with the fewest cumulus layers per oocyte, suggesting a more developmentally immature state. RNA Seq analysis of oocytes from mice at distinct ages revealed that the genes involved in cellular growth signaling pathways (PI3K, mTOR, and Hippo) were consistently repressed with meiotic competence, whereas genes involved in cellular communication were upregulated in oocytes with age. Taken together, these data demonstrate that gametes harvested during the pubertal transition have low meiotic maturation potential and derive from immature follicular origins.
Project description:<p>The metabolic profile of follicular fluid (FF) has been investigated to look for biomarkers for oocyte quality. Resolvin E1 (RvE1), a potent pro-resolving mediator, was reported to have protective action in cell function. The study aimed to examine the predictive value of RvE1 for oocyte quality and to explore the cellular mechanism of RvE1 in improving oocyte competence. Metabolic profiles of 80 FF samples showed a higher level of RvE1 in group A (blastocysts scored ≥ B3BC and B3CB according to Gardner's blastocyst scoring system, N=36) than that of group B (blastocysts scored < B3BC and B3CB, N=44, P=.0018). The receiver operating characteristic (ROC) curve analysis showed that RvE1 level in FF below 8.96 pg/ml (AUC:0.75; 95%CI: 0.64 – 0.86; P=.00012) could predict poor oocyte quality with specificity of 97.22%, suggesting RvE1 as a potential biomarker to exclude inferior oocytes. Besides, the level of RvE1 was found to be significantly lower in FF than in serum (57.49 to 17.62 pg/ml; P=.0037) and was gradually accumulated in the culture medium of cumulus cells (CCs) during cell culture, which indicated that RvE1 came from both blood exudates and local secretion. The in vitro experiment revealed the cellular mechanism of RvE1 in improving oocyte quality by decreasing the cumulus cell apoptotic rate and increasing cell viability and proliferation. It is the first time that the role of RvE1 in reproduction is explored. In conclusion, RvE1 is valuable as a potential exclusive biomarker for oocyte selection and plays a role in improving oocyte quality.</p>
Project description:Inhibitors of glucose (IO+DHEA group) or fatty acids (ETOMOXIR group) metabolism were applied during bovine oocyte in vitro maturation (IVM). Control group was conducted in standard maturation conditions. In vitro fertilization and embryo culture were applied. Obtained blastocysts were analysed with regard to lipidome, metabolome (mass spectrometry), transcriptome (RNA Seq) and lipid droplets staining (BODIPY).