Effect of phosphodiesterase type 3 inhibitor on nuclear maturation and in vitro development of ovine oocytes

Abstract

The present study aims to investigate if prematuration culture (PMC) of ovine oocytes in the presence of a phosphodiesterase type 3 (PDE3) inhibitor cilostamide can improve the shortcomings of conventional in vitro maturation (IVM) system. Therefore, a two-step culture system consisting of 22 hours culture in the presence of 1, 10, and 20 μM cilostamide (PMC medium), followed by 22 hours culture in maturation medium, was designed. The effect of cilostamide on gap junction communications and nuclear status was studied. The variables assessed were chromosome organization, spindle pattern, polar body extrusion, and embryonic development. According to the results, inhibition of PDE3 could not permanently block nuclear maturation in ovine oocytes but it delayed the process of nuclear maturation. Elevation of intra-oocyte cAMP concentration could inhibit cumulus cells expansion and maintain gap junction communications between oocyte and cumulus cells. Deletion of cilostamide and refreshing maturation medium after 22 hours culture revealed that cumulus cells were completely expanded. The inhibitory effect induced by 1 μM cilostamide was reversible, and it increased the number of mature oocytes with aligned chromosomes and normal spindle. However, the inhibitory effects of 10 and 20 μM cilostamide was not fully reversible and was associated with deleterious effects on chromosome organization and spindle pattern. Investigation of embryonic development via parthenogenetic activation and in vitro fertilization revealed that the blastocyst rate of oocytes that were prematured with 1 μM cilostamide was not significantly different from oocytes that underwent conventional IVM but it was significantly reduced in oocytes that were prematured with 10 and 20 μM cilostamide. Our results provide the evidence that reduced cAMP via PDE3 is not the only mechanism that controls the progress of nuclear maturation in sheep oocytes, and that alternative or additional mechanisms may also exist.