Glutathione synthesis during in vitro maturation of ovine oocytes: effect of cysteamine and β-mercaptoethanol

Sheep oocytes were collected either by vacuum aspiration with a 20-gauge needle or by slicing ovaries obtained from a local slaughterhouse. The oocytes were in vitro matured, fertilized and cultured as described previously (Thompson et al, Biol. Reprod. 1995, 53:1385-1391). The rate of embryo development was recorded in sheep oocytes matured in IVM medium supplemented with 0, 50, 100, 200 uM cysteamine (exp.1) or with 0, 50, 100, 200 uM l beta-mercaptoethanol (exp.2). After IVM, GSH levels were measured in oocytes matured with 200 pM cysteamine or 50 uM beta-mercaptoethanol with or without 5 mM buthionine sulfoximide (BSO), an inhibitor of GSH synthesis. Logarithmic transformed data were analyzed by ANOVA and Tukey's test. It was demonstrated that 200 uM cysteamine stimulates embryo development (50.2 vs 33.7%) whereas beta-mercaptoethanol does not (31.8 vs 38.1%). Furthermore, GSH synthesis was stimulated in IVM oocytes in the presence of either cysteamine (6.7 vs 4.2 picomol/oocyte) or beta-mercaptoethanol (8.8 vs 6.5 picomol/oocyte). Moreover, the results obtained in the control groups _+ BSO suggests that intracellular GSH synthesis occurs

Cryopreservation of in vitro-produced ovine embryos

The purpose of this study was to evaluate different cryopreservation protocols for in vitro-produced ovine embryos and assess the survival rate after cryopreservation. The experiment was also designed to examine whether this technique is feasible to apply in large-scale operations. In a first experiment, ovine embryos cryopreserved in ethylene glycol using 3 steps (E-3S) showed a higher hatching rate and nuclei number than freezing with glycerol in 3 steps (G-3S) (40.0% versus 20.0% and 135.4 ± 20.7 versus 118.5 ± 19.5, respectively). In a second experiment, vitrification with ethylene glycol + Ficoll 70 + sucrose (EFS) recorded a higher hatching rate and nuclei numbers than vitrification with propylene glycol + glycerol (Pg + Gly) (51.1% versus 31.1% and 133.8 ± 36.8 versus 113.5 ± 22.0, respectively). In a third phase, vitrification of embryos with ethylene glycol + glycerol (Eg + Gly) resulted in higher development and hatching rates and nuclei number than EFS (87.3% versus 65.4%; 76.4% versus 54.5% and 135.7 ± 34.5 versus 113.1 ± 14.1, respectively). In a fourth experiment, fresh in vivo embryos produced a higher lambing rate (67.8%) than the other methods, and E-3S in vitro resulted in lower lambing rates (23.0%). E-3S in vivo, Eg + Gly in vivo, fresh in vitro and Eg + Gly in vitro recorded similar lambing rates (42.8, 37.5, 37.5 and 26.6%, respectively). Ewes receiving in vitro-produced ovine embryos resulted in higher assisted births and perinatal losses than those receiving in vivo-produced embryos (3.6% versus 16.9% and 1.2% versus 10.1%, respectively). Results demonstrate that the transfer of in vitro-produced embryos vitrified with Eg + Gly could have wide application, if the negative side effects of the produced offspring can be managed.Fil: Martínez, A.G.. Centro de Investigaciones Reproductivas Perez Companc; ArgentinaFil: Valcárcel, A.. Centro de Investigaciones Reproductivas Perez Companc; ArgentinaFil: Furnus, Cecilia Cristina. Centro de Investigaciones Reproductivas Perez Companc; ArgentinaFil: de Matos, D.G.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina. Centro de Investigaciones Reproductivas Perez Companc; ArgentinaFil: Iorio, G.. Centro de Investigaciones Reproductivas Perez Companc; ArgentinaFil: de las Heras, M.A.. Centro de Investigaciones Reproductivas Perez Companc; Argentin

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus-oocyte complexes

Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus–oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 (ADAM10), ADAM17, amphiregulin (AREG), and epiregulin (EREG) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 (PTGS2), pentraxin 3 (PTX3) and tumor necrosis factor alpha-induced protein 6 (TNFAIP6 (TSG6)) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 (HAS2) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro-matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10, ADAM17, AREG, and EREG and the second on downstream genes, particularly PTGS2.Ester S Caixeta, Melanie L Sutton-McDowall, Robert B Gilchrist, Jeremy G Thompson, Christopher A Price, Mariana F Machado, Paula F Lima and José Buratin