Effects Of AY9944A-7on Meiotic Maturation And Preimplantation Developmental Competence Of Ovine Oocytes Maturing In Vitro

Intermediates in the cholesterol biosynthetic pathway, follicular fluid meiosis-activating sterol (FF-MAS) and testis meiosis-activating sterol (T-MAS), have been confirmed to be closely related to the oocyte meiosis. FF-MAS is converted to T-MAS by a sterol Δ14-reductase. AY9944A-7is an inhibitor of Δ14-reductase. So AY9944A-7can promote accumulation of FF-MAS in ovary by inhibiting the conversion of FF-MAS to T-MAS and blocking metabolism of FF-MAS downstream. The activity of AY9944A-7to induce oocyte maturation has so far been reported in mouse, rat and porcine. The aim of the present study was to evaluate the effects of AY9944A-7on spontaneous meiotic maturation, inhibitor-arrested meiotic maturation and gonadotropin-induced meiotie maturation in ovine oocytes. In addition, the effect of AY9944A-7on the cytoplasmic maturation of sheep oocytes was investigated. Usually, the temporal and spatial redistribution of cortical granules (CGs) are used as indicators to evaluate cytoplasmic maturation in vitro. Finally, the present study was performed to investigate the competence of the parthenogenetic embryos derived from maturing oocytes treated by AY9944A-7to complete pre-implantation development indicated by cleavage rate, blastocyst rate and percentage of two-cell stage embryos that developed to the blastocyst stage. The results are as follow:Sheep cumulus oocyte complexes (COCs), split COCs and denuded oocytes (DO) were cultured in medium supplemented with hypoxanthine (Hx,4mM) or IBMX (250μM) to maintain the oocytes in germinal vesicle (CV) stage. The effects of AY9944A-7on meiotic resumption inhibited by Hx or IBMX in sheep COCs, DOs and split COCs were evaluated by AY9944A-7treatment of maturing oocytes. The results indicated that AY9944A-7only induced meiotic resumption inhibited by Hx or IBMX in sheep COCs. When the COCs were separated into cumulus cells and oocyte and co-cultured, AY9944A-7had no significant effect on stimulating resumption of meiosis. The optimal dose for AY9944A-7to overcome the meiotic inhibition and resume meiosis was20～30μM.40μM AY9944A-7markedly increased degeneration rate (P<0.01).When sheep fully grown oocytes were released from the follicle and cultured in vitro spontaneous maturation model for24h, about82.25%of COCs resumed maturation (oocytes with GVBD), and about72.46%emitted the PBI. The percentage of GVBD reached88.67%,91.29%and89.33%, and the percentage with polar bodies increased to85.94%,88.44%and85.27%respectively after10,20and30μM AY9944A-7treatment. AY9944A-7showed no significant stimulatory effect on resumption of meiosis on either DO or split COC. The optimal dose for AY9944A-7to promote spontaneous maturation in vitro was20μM and higher dose of AY9944A-7(30and40μM) was detrimental to the ability of oocytes to resume meiosis, resulting in increased degeneration rate.When spontaneous maturation of sheep COCs was arrested by Hx (4mM) or IBMX (250μM), the resumption of meiosis might be induced by FSH (10μg/ml) or LH (10μg/ml). The present study was performed to explore the possible role of AY9944A-7in FSH or LH-induced meiotic resumption of ovine oocytes maturing in vitro. FSH can overcome the meiotic inhibition by Hx or IBMX, and induce sheep COCs undergoing GVBD and extruding PBI (P<0.01), but LH had no effect on oocyte meiotic resumption. In both inhibitory agents used, AY9944A-7promoted FSH-induced GVBD and PBI extrusion in sheep COCs significantly (P<0.05或P<0.01).The temporal and spatial redistribution of cortical granules (CGs) as a indicator evaluating cytoplasmic maturation, the present study was performed to investigate the effect of AY9944A-7(0,10,20,30,40μM) on the cytoplasmic maturation of sheep oocytes. The results showed that the rate of oocytes with CGs transfering completely in cortex was72.29%and78.81%respectively in the treatment of10and20μM AY9944A-7and increased by10.02%and16.54%respectively compared with the control group. The results of the present study indicated that treatments of maturing oocytes with the proper concentration of AY9944A-7promoted temporal and spatial translocation or redistribution of CGs and improved the quality of cytoplasmic maturation of ovine oocytes in vitro. The sheep COCs were cultured in maturation medium supplemented with0,10,20,30and40μM AY9944A-7respectively for24h, after which those with an extruded first polar body were considered mature and selected for parthenogenetic activation, followed by in vitro embryo culture. Cleavage rate, blastocyst rate and percentage of two-cell stage embryos that developed to the blastocyst stage were counted. Effects of treatments with AY9944A-7during maturation on preimplantation developmental competence of parthenogenetic embryos were studied. The results showed that the cleavage rate of parthenogenetic embryos derived from maturing oocytes treated by different concentration of AY9944A-7was higher than that in control group, but the increase reached significance only in20μM AY9944A-7treatment of maturing oocytes (P<0.05). There was a significant increase in the percentage of blastocyst stage and two-cell stage embryos that developed to the blastocyst stage resulting from treatment with10and20μM AY9944A-7(17.17%å'Œ22.73%vs.13.24%, P<0.05;24.07%å'Œ29.53%vs.15.42%, P<0.05). The results indicated that10～20μM AY9944A-7improved oocyte quality and promoted the competence of the embryos to complete pre-implantation development.In conclusion, the addition of20μM AY9944A-7to the maturation medium resulted in the increase in frequency of maturation to Mâ…¡ caused the improvement of oocyte quality, and led to the promotion of competence of the embryos to complete pre-implantation development.