Comparative study of cytology of cynomolgus monkey oocytes matured in vivo and in vitro

Abstract

This study was conducted to compare 1) the capacity for maturation of oocytes from cynomolgus monkeys cultured in vitro before or after injection of r-hCG, 2) spindle integrity, and 3) the distribution of mitochondria in oocytes. In total, 31 stimulation cycles from 14 animals were performed. The oocytes were analyzed before or after hCG treatment and maturation in vitro. Beginning within 3 days after initiation of menstruation, adult monkeys were given 60 IU r-hFSH for 6 days, followed by 60 IU r-hFSH and 60 IU r-hLH for 2 days to stimulate multiple follicle growth. The GnRH antagonist, Antide, was administered daily (0.5 mg/kg body weight) to prevent an endogenous LH surge. Follicle development was monitored by serum estradiol concentrations and ultrasonography. Follicle aspiration was performed on anesthesized monkeys without hCG or 12, 24, or 36 h after receiving 1000 IU r-hCG. The oocytes were cultured in M199 medium containing 10% FBS and 75 μIU/ml FSH and LH for a total (in vivo and in vitro) of 40–44 h post-hCG. The maturity of oocytes was indicated by cumulus expansion and the emission of polar bodies. After removing the cumulus cells, oocytes were fixed in 3.7% formaldehyde. The spindles were visualized by using an α-tubulin antibody. Oocytes possessing a bipolar-shaped spindle with well-aligned chromosomes at the equator were considered to be normal. Finally the distribution of mitochondria was studied using MitoTracker® Red 580 and confocal microscopy. Oocytes cultured without in vivo exposure to hCG had a maturation rate of 79.6% (82/111 GV). In comparison, 82.0% (41/51 GV) and 93.8% (60/64 GVBD) of oocytes retrieved at 12h or 24 h after hCG had matured, respectively. In total, 84.5% of oocytes (82/97) retrieved at 36h after hCG had reached MII at the time of aspiration. There was no significant difference in maturation of oocytes that had been matured longer in vivo (p<0.05). The majority of oocytes in all four groups possessed abnormal spindles with asymmetric, tripolar and multipolar spindles, as well as irregular bipolar formations and unaligned chromosomes (Table 1). Spindle abnormalities were generally associated with chromosome displacement (97.9%: 142 with chromosome displacement /145 with abnormal spindles). Mitochondria were distributed mainly around the cortical region in MII oocytes, whereas in GV and GVBD oocytes they were throughout the ooplasm. The duration of culture did not significantly affect the proportion of oocytes undergoing maturation, nor did it affect the incidence of cytological anomalies. The high incidence of defects of spindle formation and chromosome alignment indicates a poor prognosis for post-fertilization development, which is comparable to human oocytes. This non-human primate appears to be a good model for studying oogenesis and embryogenesis in humans.