Project description:BACKGROUND: Ovarian follicle development is a complex process. Paracrine interactions between somatic and germ cells are critical for normal follicular development and oocyte maturation. Studies have suggested that the health and function of the granulosa and cumulus cells may be reflective of the health status of the enclosed oocyte. The objective of the present study is to assess, using an in vivo immature rat model, gene expression profile in granulosa cells, which may be linked to the developmental competence of the oocyte. We hypothesized that expression of specific genes in granulosa cells may be correlated with the developmental competence of the oocyte. METHODS: Immature rats were injected with eCG and 24 h thereafter with anti-eCG antibody to induce follicular atresia or with pre-immune serum to stimulate follicle development. A high percentage (30-50%, normal developmental competence, NDC) of oocytes from eCG/pre-immune serum group developed to term after embryo transfer compared to those from eCG/anti-eCG (0%, poor developmental competence, PDC). Gene expression profiles of mural granulosa cells from the above oocyte-collected follicles were assessed by Affymetrix rat whole genome array. RESULTS: The result showed that twelve genes were up-regulated, while one gene was down-regulated more than 1.5 folds in the NDC group compared with those in the PDC group. Gene ontology classification showed that the up-regulated genes included lysyl oxidase (Lox) and nerve growth factor receptor associated protein 1 (Ngfrap1), which are important in the regulation of protein-lysine 6-oxidase activity, and in apoptosis induction, respectively. The down-regulated genes included glycoprotein-4-beta galactosyltransferase 2 (Ggbt2), which is involved in the regulation of extracellular matrix organization and biogenesis. CONCLUSIONS: The data in the present study demonstrate a close association between specific gene expression in mural granulosa cells and the developmental competence of oocytes. This finding suggests that the most differentially expressed gene, lysyl oxidase, may be a candidate biomarker of oocyte health and useful for the selection of good quality oocytes for assisted reproduction.

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.

Project description:BackgroundOocyte maturation and preimplantation embryo development are controlled by array of genes that are post-transcriptionally regulated by microRNAs. With respect to this, previously, we identified altered expression of microRNA-130b (miR-130b) during oocyte maturation. Here, we aimed to investigate the role of miR-130b in bovine granulosa and cumulus cell function, oocyte maturation and preimplantation embryo development using gain- and loss-of- function approach.MethodsFor this study, the granulosa cells, cumulus cells and the oocytes were collected from ovaries obtained from slaughterhouse. The genes targeted by miR-130b were identified using dual-luciferase reporter assay. The role of miR-130b in granulosa and cumulus cell function was investigated by increasing and inhibiting its expression in in vitro cultured cells using miR-130b precursor and inhibitor, respectively while the role of miR-130b on oocyte development, immature oocytes were microinjected with miR-130b precursor and inhibitor and the polar body extrusion, the proportion of oocytes reaching to metaphase II stage and the mitochondrial were determined in each oocyte group 22 h after microinjection. Moreover, to investigate the role of miR-130b during preimplantation embryo development, zygote stage embryos were microinjected with miR-130b precursor or inhibitor and the cleavage rate, morula and blastocyst formation was analyzed in embryos derived from each zygote group after in vitro culture.ResultsThe luciferase assay showed that SMAD5 and MSK1 genes were identified as the direct targets of miR-130b. Overexpression of miR-130b increased the granulosa and cumulus cell proliferation, while inhibition showed the opposite phenotype. Apart from these, modulation of miR-130b altered the lactate production and cholesterol biosynthesis in cumulus cells. Furthermore, inhibition of miR-130b expression during oocyte in vitro maturation reduced the first polar body extrusion, the proportion of oocytes reaching to metaphase II stage and the mitochondrial activity, while inhibition of miR-130b during preimplantation embryo development significantly reduced morula and blastocyst formation.ConclusionThis study demonstrated that in vitro functional modulation of miR-130b affected granulosa and cumulus cell proliferation and survival, oocyte maturation, morula and blastocyst formation suggesting that miR-130b is involved in bovine oocyte maturation and preimplantation embryo development.

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Effects of Dietary Fatty Acids on Bovine Oocyte Competence and Granulosa Cells.

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Effects of Dietary Fatty Acids on Bovine Oocyte Competence and Granulosa Cells.

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