L-Cysteine improves bovine oocyte developmental competence in vitro via activation of oocyte-derived growth factors BMP-15 and GDF-9.

This study was designed to investigate the effect of different concentrations of L-cysteine supplementation into the maturation medium on the oocyte nuclear maturation, cumulus cell expansion, ultrastructure of the oocytes and the expression of oocyte-derived growth factors BMP-15, GDF-9 and CB-1 genes. Cumulus oocyte complexes (COCs) were collected from cow's ovaries obtained from abattoir and incubated at 38.5°C in maturation media supplemented with 0, 0.6, 0.8 or 1 mM L-cysteine in 5% CO2 under humidified air for 24 hr. We found that a significantly higher percentage of oocytes progressed to metaphase II stage in the in vitro maturation (IVM) medium supplemented with L-cysteine, particularly 0.8 mM group, compared with untreated control oocytes. Additionally, L-cysteine treatment significantly increased the number of expanded COCs and the degree of expansion of individual COCs. Results of RT-qPCR showed significant increase in expression levels of BMP-15 and GDF-9 in L-cysteine-treated groups compared with control one. Electron microgram showed improvement of cytoplasmic maturation regarding ultrastructure of the oocytes and oocyte-cumulus cell gap junction communication in all L-cysteine-treated groups especially 0.8 mM L-cysteine-treated one. In conclusion, supplementation of IVM medium with a potential anti-oxidant, L-cysteine can effectively improve in vitro oocytes cytoplasmic and nuclear maturation via activation of oocyte maturation related BMP-15 and GDF-9 genes in bovine oocytes, benefiting the extended researches about the potential applications of L-cysteine in mammalian breeding technologies.

Growth differentiation factor-9 improves development, mitochondrial activity and meiotic resumption of sheep oocytes after in vitro culture of secondary follicles.

This study analysed the effect of growth differentiation factor-9 (GDF-9) on the in vitro culture of isolated ovine secondary follicles. The follicles were cultured in α-MEM supplemented with BSA, insulin, glutamine, hypoxanthine, transferrin, selenium, ascorbic acid and FSH (α-MEM+ -control medium) or α-MEM+ supplemented with 1, 10, 50 or 100 ng/ml GDF-9. Next, the oocytes were destined to in vitro maturation (IVM). After 12 days of culture, there were no differences regarding the percentage of normal follicles, antrum formation and follicle diameter between the treatments (p > 0.05). The rates of fully grown oocytes (≥110 µm) were higher (p < 0.05) in 100 ng/ml GDF-9 than other treatments, except for 10 ng/ml of GDF-9 (p > 0.05). Treatment containing 100 ng/ml GDF-9 showed higher (p < 0.05) mitochondrial activity than the control group. Moreover, 100 ng/ml GDF-9 showed more oocytes in MI than α-MEM+ , 1 or 50 ng/ml GDF-9 (p < 0.05). In conclusion, 100 ng/ml GDF-9 increased the growth, mitochondrial function and meiotic resumption of oocytes from in vitro grown sheep secondary follicles.

Temporal effects of exogenous oocyte-secreted factors on bovine oocyte developmental competence during IVM.

We investigated whether paracrine signalling between the bovine oocyte and cumulus cells is altered during the course of in vitro maturation (IVM). Bovine COCs were cocultured with denuded oocytes or treated with specific oocyte-secreted factors, namely recombinant bone morphogenetic protein (BMP)-15 or growth differentiation factor (GDF)-9, beginning from 0 or 9h IVM. To generate a 9-h denuded oocyte (DO) group, COCs were cultured intact for the first 9h of IVM and then denuded. Coculturing intact COCs with DOs denuded immediately after collection or following 9h of maturation did not affect cleavage rate, but improved blastocyst yield (P<0.05) on Day 8 (51 and 61%, respectively; P<0.05) and cell number compared with COCs cultured alone (41%). Significantly, we observed higher levels of endogenous GDF-9 and BMP-15 protein in oocytes of COCs matured for 9h compared with no incubation. The addition of 175 ng mL(-1) GDF-9 or 10%v/v BMP-15 from partially purified transfected 293H cell supernatant for 24h IVM significantly enhanced development to the blastocyst stage from 40% (control) to 51 and 47%, respectively (P<0.05). However, treatment of COCs with GDF-9 or BMP-15 between 9 and 24h of IVM did not increase blastocyst yield. These results provide evidence of quantitative and possibly qualitative temporal changes in oocyte paracrine factor production during IVM.