Effect of Follicular Aging on the Nuclear Maturation and Distribution of Lipid Droplets in Bovine Oocytes.

Success of embryo transfer programs depends on optimal superstimulation and effects of the treatment on oocyte quality. Assessment of oocyte quality is based on resumption of meiosis (nuclear maturation) and redistribution of organelles within the ooplasm. Lipids act as important source for ATP generation through β-oxidation and poor quality oocytes tend to accumulate lipid droplets within ooplasm. Our objective was to study the effect of superstimulation protocols on nuclear maturation and distribution of lipid droplets in the ooplasm. We hypothesized that follicular aging after FSH starvation will result in maturation failure with accumulation of larger lipid droplets as compared to superstimulation with continued FSH support. Hereford heifers (n=12) were given prostaglandin F2a (PGF) to induce ovulation. Five days later, a progesterone releasing device (CIDR) was placed into vagina and transvaginal ultrasound-guided ablation of follicles was done to synchronize emergence of a new follicular wave. Next day (expected day of emergence of a new wave, Day 0), heifers were randomly allocated to three groups (n=4 each group). Heifers in standard FSH and FSH starvation groups were given 8 im injections of FSH (12-hour intervals) over 4 days, while those in long FSH group were given 14 im injections over 7 days. Two injections of PGF (at 12 hour interval) were given on Day 3 to the standard FSH group and on Day 6 to FSH starvation and long FSH groups. In all groups, CIDR was removed at second PGF and LH was given 24 hours later. Cumulus-Oocyte-Complexes were collected 18-20 hours post-LH by follicular aspiration. Oocytes were fixed and stained for nuclear maturation (Lamin/DAPI) and lipid droplets (Nile Red, 5 μg/ml). Oocytes were imaged with Zeiss LSM 710 confocal microscope. Oocyte volume sets were deconvoluted using Autoquant X2 and lipid droplets segmented using Imaris Pro 7.4.The proportion of mature oocytes were compared using Mantel-Haenszel Chi square test and lipid droplets data were compared using ANOVA. Long FSH group had greater proportion (59/100, 59%, P<0.01) of mature oocytes compared to standard FSH and FSH starvation groups (5/23, 21.7% and 2/25, 8.0%, respectively). The proportions of good (Grades 1 and 2) versus bad (Grades 3 and 4) oocytes in FSH starvation and long FSH treatments were not different (P=0.28) from that of standard FSH treatment. The FSH starvation and long FSH treatments did not cause significant changes in the total number (P=0.39) and total volume (P=0.58) of lipid droplets compared to standard FSH treatment. The percentage of lipid droplets in the peripheral ooplasm (10 μm distance from the ooplasmic membrane) of oocytes from FSH starvation and long FSH treatments did not differ from standard FSH treatment (50.1±2.7% and 40.4±3.8% vs 49.1±4.0%, P=0.66). Average volume of lipid droplets in oocytes was higher in FSH starvation group (11.5±1.5μm³, P=0.03) compared to standard FSH and long FSH groups (7.2±0.6μm³ and 8.0±0.8μm³). Average surface area of lipid droplets in oocytes was higher in FSH starvation group (20.3±1.5 μm² P=0.04) compared to standard FSH group (14.6±1.1μm²) and was not different from long FSH group (17.1±1.1μm²). In conclusion, our hypothesis was supported in that FSH starvation caused maturation failure and accumulated larger lipid droplets in the ooplasm. Natural Sciences and Engineering Research Council of Canada funded