Schramm R D, Bavister B D
Wisconsin Regional Primate Research Center, University of Wisconsin, Madison 53715, USA.
Hum Reprod. 1996 Aug;11(8):1698-702. doi: 10.1093/oxfordjournals.humrep.a019472.
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 development capacity of oocytes from non-stimulated macaque monkeys and confers on them competence to develop into blastocysts in vitro. Antral follicles > or = 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 10(6) live cells/ml) from non-stimulated monkeys, or (iii) FSH-primed granulosa cells (5 x 10(6) live cells/ml) from monkeys primed with purified FSH for 8.5 days. All treatments contained gonadotrophins (5 micrograms/ml FSH; 10 micrograms/ml luteinizing hormone). Oocytes were cultured for 36-41 h in 25 microliters 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 < or = 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 < or = 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 < or = 0.05) competent to develop into blastocysts (6.5%) than controls (0.5%). 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.
本研究的具体目的是确定在体外成熟（IVM）过程中，将从经促卵泡激素（FSH）预处理与未经刺激的猴子中新鲜获取的颗粒细胞共同培养，是否能提高未经刺激的猕猴卵母细胞的减数分裂和发育能力，并赋予它们在体外发育成囊胚的能力。从11只未经刺激的恒河猴切除的卵巢中解剖出直径≥1mm的窦卵泡。将卵丘包裹的生发泡期卵母细胞（n = 282）随机分为三种IVM处理组进行培养：（i）对照组（无颗粒细胞），（ii）来自未经刺激猴子的未刺激颗粒细胞（5×10⁶活细胞/ml），或（iii）用纯化FSH预处理8.5天的猴子的FSH预处理颗粒细胞（5×10⁶活细胞/ml）。所有处理均含有促性腺激素（5μg/ml FSH；10μg/ml黄体生成素）。卵母细胞在含20%小牛血清的改良CMRL - 1066培养基的25μl液滴（每滴1至10个卵母细胞）中培养36 - 41小时，然后进行授精，并在相同培养基（不含促性腺激素）中培养至发育停滞或透明带脱出。三种处理方法在核成熟、受精和卵裂至8细胞阶段方面无差异。然而，与对照组相比，用未刺激的颗粒细胞或FSH预处理的颗粒细胞共同培养可提高（P≤0.01）发育至9至15细胞阶段的比例。与对照组相比（分别为23%、11%和5%），用FSH预处理的颗粒细胞而非未刺激的颗粒细胞培养卵母细胞可提高（P≤0.05）桑椹胚阶段的发育比例。用FSH预处理的颗粒细胞培养的卵母细胞发育成囊胚的能力（6.5%）比对照组（0.5%）更强（P≤0.05）。总之，用从FSH预处理的猴子中回收的新鲜颗粒细胞培养未经刺激猴子的未成熟卵母细胞，可提高其在体外发育成桑椹胚和囊胚的能力，并首次从未经刺激的猕猴体外成熟/体外受精的卵母细胞中产生了体外囊胚。
