Bovine oocyte ability to embryonic development when maturing in different two-phase culture systems

Existing approaches to a two-phase method of culture of cattle oocytes do not take into account their specific demands during maturation from metaphase I to metaphase II including the need for normalization of profiles of sex steroid hormones in a culture medium. The aim of the presented research was to compare the developmental competence of bovine ( Bos taurus taurus ) oocytes matured in conventional single-phase and different two-phase systems. We have studied for the first time the ovum ability to develop to the blastocyst stage and the quality of embryos produced when replacing the standard medium at the second stage of culture with a medium free of follicle-stimulating hormone (FSH) as well as by a granulosa cell culture. When using the single-phase system, cumulus-oocyte complexes (COCs) were cultured for 24 h in the medium TCM 199 containing 10 % fetal bovine serum (FBS), 10 µg/ml FSH, and 10 µg/ml luteinizing hormone (LH). In the two-phase system, oocytes matured in the same conditions for first 12 h. Then COCs were transferred to a new medium (TCM 199 containing 10% FBS or the same medium supplemented with 5 µg/ml LH) and cultured for 12 h in the presence or in the absence of granulosa cells. After 24 h of maturation, oocyte fertilization was performed. In embryos that developed to the stages of late morula and blastocyst on day 7 of culture, the degree of apoptosis and the total number of nuclei were assessed (by the TUNEL method and DAPI staining, respectively). In media collected after oocyte maturation, levels of progesterone and 17b-estradiol were determined by enzyme immunoassay. The proportions of oocytes entered the first cleavage division (57.6-68.1 %) or developed to the stages of late morula/blastocyst (16.7-20.7 %) were not associated with the culture method or medium. The transfer of oocytes after 12 h of maturation to the medium free of gonadotropic hormones resulted in 1.3-1.4-fold increase in the total number of nuclei in the late morulae/blastocysts both in the absence and in the presence of granulosa cells, whereas this number decreased to the level of single-phase control on addition of LH. At the same time the proportion of apoptotic nuclei (2.3-6.9 %) did not depend on the system of oocyte maturation or the effect of LH. In the two-phase system, the level of 17b-estradiol in the maturation medium (with and without LH) was 1.3-1.4-fold lower (p < 0.01) as compared to the single-phase system. On the contrary, co-culture of COCs with granulosa cells led to a rise in the concentration of 17b-estradiol in the culture medium containing LH (from 418±16 to 496±26 pg/ml, p < 0.05). Meanwhile, no differences in the progesterone level in the culture medium of COCs were found between all the systems studied. The findings suggest that the two-phase system of maturation of bovine oocytes may be used as an alternative to the conventional IVM protocol. The transfer of oocytes after 12 h of culture to the medium free of gonadotropic hormones causes an increase in the quality of the late morulae/blastocysts produced as well as leads to normalization of the 17β-estradiol profile in the culture medium at the terminal step of the ovum maturation.