Abstract
BackgroundThe competence for embryonic development after IVF is low in the yak, therefore, we investigated the effects of supplementation of FSH, LH and the proteasome inhibitor MG132 in IVM media on yak oocyte competence for development after IVF.MethodsIn Experiment 1, yak cumulus-oocyte complexes (COCs) were in vitro matured (IVM) in TCM-199 with 20% fetal calf serum (FCS), 1 microg/mL estradiol-17beta, and different combinations of LH (50 or 100 IU/mL) and FSH (0, 1, 5, 10 microg/mL) at 38.6 degrees C, 5% CO2 in air for 24 h. Matured oocytes were exposed to frozen–thawed, heparin-capacitated yak sperm. Presumptive zygotes were cultured in SOF medium containing 6 mg/ml BSA, 0.5 mg/mL myoinositol, 3% (v/v) essential amino acids, 1% nonessential amino acids and 100 μg/mL L-glutamine (48 h, 38.5 degrees C, 5% CO2, 5% O2, and 90% N2). In Experiment 2, cumulus cells were collected at the end of IVM to determine FSHR and LHR mRNA expression by real-time PCR. In Experiment 3 and 4, COCs were cultured in the presence or absence of the proteasomal inhibitor MG132 from either 0–6 h or 18–24 h after initiation of maturation.ResultsThe optimum concentration of FSH and LH in IVM media was 5 microg/mL FSH and 50 IU/mL LH which resulted in the greatest cleavage (79.1%) and blastocyst rates (16.1%). Both FSHR and LHR mRNA were detected in yak cumulus cells after IVM. Treatment with MG132 early in maturation reduced (P < 0.05) cleavage and blastocyst rates. Conversely, treatment with MG132 late in maturation improved (P < 0.05) blastocyst rate. Optimal results with MG132 were achieved at a concentration of 10 microM.ConclusionsAn optimum concentration of FSH and LH in IVM medium, and treatment with MG132 late in maturation can improve yak oocytes competence for development after IVF.
Highlights
The competence for embryonic development after IVF is low in the yak, we investigated the effects of supplementation of FSH, LH and the proteasome inhibitor MG132 in in vitro maturation (IVM) media on yak oocyte competence for development after IVF
In vitro culture systems of most species often occur in in vitro maturation (IVM) media supplemented with gonadotrophins such as FSH and LH to induce cumulus cell expansion, nuclear maturation, cytoplasmic maturation, and to improve embryonic development in mice [2,3], pig [4], cow [5], equine [6] and domestic dog [7]
There was a tendency for high LH concentration (100 IU/ml) in IVM medium to decrease the subsequent cleavage rate
Summary
The competence for embryonic development after IVF is low in the yak, we investigated the effects of supplementation of FSH, LH and the proteasome inhibitor MG132 in IVM media on yak oocyte competence for development after IVF. Throughout follicular development, the oocytes undergo a series of profound changes involving both nuclear and cytoplasmic maturation induced by the pre-ovulatory surge of gonadotropins during this period [1]. These changes are essential for the formation of oocytes having the capacity for fertilization and embryonic development. In vitro culture systems of most species often occur in in vitro maturation (IVM) media supplemented with gonadotrophins such as FSH and LH to induce cumulus cell expansion, nuclear maturation, cytoplasmic maturation, and to improve embryonic development in mice [2,3], pig [4], cow [5], equine [6] and domestic dog [7]. Treatment of oocytes with MG132 late in maturation increased abundance of specific transcripts and improved developmental competence of parthenogenetically-activated oocytes in the pig [10] and in vitro matured oocytes in cattle [11]
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