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:Despite great improvements in assisted reproductive technology (ARTs), the success of in vitro embryo production remains relatively low. Most of the oocytes used to produce in vitro embryos are recovered from smaller follicles, forming a heterogeneous population that must be matured in vitro. Since the original in vitro maturation (IVM ) experiments (Heilbrunn et al. , 1939) the process by which the most competent oocytes are selected to produce blastocysts remains similar and is still based on morphological aspects of the oocyte cytoplasm and the number of layers and compaction of cumulus cells attached to the oocyte surface (Armstrong, 2001, Coticchio et al. , 2004, Krisher, 2003, Lonergan et al. , 2003). Ovaries from crossbred cows (Bos indicus x Bos taurus) were collected immediately after slaughter and transported to the laboratory in saline solution (0.9% NaCl) supplemented with penicillin G (100 IU/mL) and streptomycin sulfate (100 mg/mL) at 35-37C. The follicles were dissected from the ovarian cortex using scissors, scalpels and tweezers in TCM-199 medium supplemented with Hank’s salts and 10% fetal calf serum (FCS; GIBCO BRL) at 36C. Follicles were measured using a graduated eyepiece (OSM-4; Olympus) and then classified morphologically into two groups according to their diameter: 1.0-3.0 mm or ≥8.0 mm. The criteria used for follicle selection included the presence of extensive and fine vascularization and a shiny and translucent appearance. After follicular rupture, the presence of granulosa cells with a regular and healthy appearance and no free-floating particles in the follicular fluid were also used as selection criteria. Only COCs with a homogeneous granulated cytoplasm and at least three layers of compact of cumulus cells were used in the present study. The selected COCs were transferred to a 50 μL droplet of phosphate-buffered saline (PBS), and the cumulus cells were mechanically removed by repeated pipetting. After cumulus collection, they were transferred to a 0.2-mL tube and centrifuged twice for 2 min at 700 g. The supernatant was removed, and 2μL of RNAlater (Applied Biosystems, Foster City, CA, USA) was added to the pellet, which was then stored at -20C until RNA extraction. For each follicle size group, three replicas of pooled cumulus cells corresponding to 30 oocytes were stored for RNA extraction and subsequent microarray assays, and four independent replicates corresponding to 20 oocytes were stored for RNA extraction for subsequent qPCR assays. During oogenesis, the somatic cells that surround the oocyte proliferate and differentiate into cumulus cells (CCs), which are metabolically coupled to the oocyte, forming the cumulus-oocyte complexes (COCs) (van den Hurk and Zhao, 2005). The CCs remain in strong contact with the oocyte, maintained by cytoplasmic bridges called gap-junctions, as well as through justacrine and paracrine signaling networks (Albertini et al. , 2001, Gilchrist et al. , 2004, Tanghe et al. , 2002, Vozzi et al. , 2001, Webb et al. , 2002). This intense bidirectional communication between the CCs and the oocyte is maintained during all phases of folliculogenesis and is essential for the acquisition of oocyte competence that is required for blastocyst development (Assidi et al., 2008, Fair, 2003, Gilchrist et al., 2004). An informative model to access the level of oocyte competence that has been used by many research groups is follicle size (Donnison and Pfeffer, 2004, Franco et al. , 2013, Lequarre et al. , 2005, Mourot et al., 2006). In a previous study, we showed that oocytes obtained from follicles 1-3 mm in diameter are significantly less competent in producing blastocysts than oocytes obtained from follicles ≥8 mm in diameter (Franco et al., 2013). In the present study, we use the same model to compare the gene expression profile of more than 23,000 bovine transcripts between the CCs obtained from incompetent COCs (1-3 mm) and competent COCs (≥8 mm). We have found substantial differences in the expression of several gene clusters representing distinct metabolic pathways such as energy metabolism, cell signaling, cell cycle, DNA repair, meiosis, and inflammation.

Project description:Despite great improvements in assisted reproductive technology (ARTs), the success of in vitro embryo production remains relatively low. Most of the oocytes used to produce in vitro embryos are recovered from smaller follicles, forming a heterogeneous population that must be matured in vitro. Since the original in vitro maturation (IVM ) experiments (Heilbrunn et al. , 1939) the process by which the most competent oocytes are selected to produce blastocysts remains similar and is still based on morphological aspects of the oocyte cytoplasm and the number of layers and compaction of cumulus cells attached to the oocyte surface (Armstrong, 2001, Coticchio et al. , 2004, Krisher, 2003, Lonergan et al. , 2003). Ovaries from crossbred cows (Bos indicus x Bos taurus) were collected immediately after slaughter and transported to the laboratory in saline solution (0.9% NaCl) supplemented with penicillin G (100 IU/mL) and streptomycin sulfate (100 mg/mL) at 35-37C. The follicles were dissected from the ovarian cortex using scissors, scalpels and tweezers in TCM-199 medium supplemented with Hank’s salts and 10% fetal calf serum (FCS; GIBCO BRL) at 36C. Follicles were measured using a graduated eyepiece (OSM-4; Olympus) and then classified morphologically into two groups according to their diameter: 1.0-3.0 mm or ≥8.0 mm. The criteria used for follicle selection included the presence of extensive and fine vascularization and a shiny and translucent appearance. After follicular rupture, the presence of granulosa cells with a regular and healthy appearance and no free-floating particles in the follicular fluid were also used as selection criteria. Only COCs with a homogeneous granulated cytoplasm and at least three layers of compact of cumulus cells were used in the present study. The selected COCs were transferred to a 50 μL droplet of phosphate-buffered saline (PBS), and the cumulus cells were mechanically removed by repeated pipetting. After cumulus collection, they were transferred to a 0.2-mL tube and centrifuged twice for 2 min at 700 g. The supernatant was removed, and 2μL of RNAlater (Applied Biosystems, Foster City, CA, USA) was added to the pellet, which was then stored at -20C until RNA extraction. For each follicle size group, three replicas of pooled cumulus cells corresponding to 30 oocytes were stored for RNA extraction and subsequent microarray assays, and four independent replicates corresponding to 20 oocytes were stored for RNA extraction for subsequent qPCR assays.

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Influence Of Insulin On Oocyte Maturation In Invitro Produced Bovine Embryos

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