Granulosa cells from follicle stimulating hormone-primed monkeys enhance the developmental competence of in-vitro-matured oocytes from non-stimulated rhesus monkeys

Abstract

The specific aims of this study were to determine if culture with freshly recovered granulosa cells obtained from follicle stimulating hormone (FSH)-primed versus non-stimulated monkeys during in-vitro maturation (IVM) improves the meiotic and developmental capacity of oocytes from non-stimulated macaque monkeys and confers on them competence to develop into blastocysts in vitro. Antral follicles ≤ 1 mm in diameter were dissected from the excised ovaries of 11 non-stimulated rhesus monkeys. Cumulus-enclosed germinal vesicle-stage oocytes (n = 282) were randomly cultured in each of three IVM treatments: (i) control (no granulosa cells), (ii) non-stimulated granulosa cells (5 x 106 live cells/ml) from non-stimulated monkeys, or (iii) FSH-primed granulosa cells (5 x 106 live cells/ml) from monkeys primed with purified FSH for 8.5 days. All treatments contained gonadotrophins (5 μg/ml FSH:; 10 μg/ml luteinizing hormone). Oocytes were cultured for 36-41 h in 25 μl drops (one to 10 oocytes per drop) of modified CMRL-1066 medium containing 20% bovine calf serum, and then inseminated and cultured in the same medium (minus gonadotrophins) until developmental arrest or zona escape. Nuclear maturation, fertilization and cleavage to the 8-cell stage did not differ for the three treatment methods. However, development to the 9- to 15-cell stage was improved (P ≤ 0.01) by culture with either non-stimulated granulosa or FSH-primed granulosa cells compared with control. The culture of oocytes with FSH primed granulosa but not non-stimulated granulosa cells enhanced (P ≤ 0.05) development to the morula stage compared with control (23, 11 and 5% respectively). Oocytes cultured with FSH-primed granulosa cells were more (P ≤ 0.05) competent to develop into blastocysts (6.5%) than controls (0.5%) or those cultured with non-stimulated granulosa cells (0.6%). In conclusion, the culture of immature oocytes from non-stimulated monkeys with fresh granulosa cells recovered from FSH-primed monkeys enhanced their competence to develop into morulae and blastocysts in vitro, and resulted in the first blastocysts produced in vitro from in-vitro-matured/in-vitro-fertilized oocytes of non-stimulated macaques.