Gilchrist R B, Ritter L J, Armstrong D T
The Reproductive Medicine Unit, Adelaide University, Adelaide, 5011, Australia.
Dev Biol. 2001 Dec 1;240(1):289-98. doi: 10.1006/dbio.2001.0451.
Oocytes secrete soluble factors that regulate the growth and differentiation of follicular cells, including maintenance of the distinctive cumulus cell phenotype. This study determines whether the mitogenic activity of oocytes is developmentally regulated and examines the responsiveness of follicular cells to oocytes at different stages of follicular development. Prepubertal SV129 mice of varying ages were primed with 5 IU equine chorionic gonadotropin (eCG) and oocytes/zygotes collected either 46 h post-eCG (immature oocytes), 12 h after administration of 5 IU human CG (hCG; ovulated ova), or 12 h post-hCG and mating (zygotes). Mural granulosa cells (MGC) from antral follicles and GC from preantral follicles were cultured +/- denuded oocytes (DO) for 18 h, followed by a 6-h pulse of [(3)H]thymidine as an indicator of cellular DNA synthesis. Coculturing MGC with meiotically maturing oocytes led to a dose-dependent increase in [(3)H]thymidine incorporation (20-fold above control levels at 0.5 DO/microl). However, [(3)H] counts remained unchanged from control levels when cultured with meiotically incompetent DO from 11- to 15-day-old mice (3% germinal vesicle breakdown; GVB), irrespective of dose of DO or developmental status of GC (MGC or preantral GC). In some treatments, spontaneous meiotic resumption of competent oocytes was prevented by culturing with 5 microM milrinone, a selective inhibitor of oocyte-specific cyclic nucleotide phosphodiesterase. The mitogenic capacity of oocytes was found to decline during and after oocyte maturation. [(3)H]Thymidine incorporation in MGC was highest (11-fold above controls) when cultured with meiotically inhibited (milrinone-treated) GV DO, stimulated 5.5-fold by culture with maturing oocytes, 3-fold with ovulated ova, and unstimulated by zygotes. [(3)H]Thymidine incorporation in MGC was not altered by the dose of milrinone, either in the presence or absence of DO. Metaphase I marked the beginning of the decline in the capacity of oocytes to promote MGC DNA synthesis. These results demonstrate that the capacity of oocytes to promote proliferation of granulosa cells follows a developmental program, closely linked to oocyte meiotic status, increasing with the acquisition of meiotic competence and declining during and after oocyte maturation.
卵母细胞分泌可溶性因子，调节卵泡细胞的生长和分化，包括维持独特的卵丘细胞表型。本研究确定卵母细胞的促有丝分裂活性是否受发育调控，并研究在卵泡发育的不同阶段卵泡细胞对卵母细胞的反应性。用5国际单位马绒毛膜促性腺激素（eCG）对不同年龄的青春期前SV129小鼠进行预处理，在注射eCG后46小时（未成熟卵母细胞）、注射5国际单位人绒毛膜促性腺激素（hCG；排卵的卵母细胞）后12小时或注射hCG并交配后12小时（合子）收集卵母细胞/受精卵。将来自窦状卵泡的壁颗粒细胞（MGC）和来自窦前卵泡的颗粒细胞（GC）与去透明带卵母细胞（DO）一起培养或不与去透明带卵母细胞一起培养18小时，然后用[³H]胸苷进行6小时脉冲处理，作为细胞DNA合成的指标。将MGC与减数分裂成熟的卵母细胞共培养导致[³H]胸苷掺入量呈剂量依赖性增加（在0.5个DO/微升时比对照水平高20倍）。然而，当与11至15日龄小鼠减数分裂无能的DO（3%生发泡破裂；GVB）共培养时，[³H]计数与对照水平相比保持不变，无论DO的剂量或GC（MGC或窦前GC）的发育状态如何。在一些处理中，用5微摩尔米力农（一种卵母细胞特异性环核苷酸磷酸二酯酶的选择性抑制剂）培养可阻止有能力的卵母细胞自发减数分裂恢复。发现卵母细胞的促有丝分裂能力在卵母细胞成熟期间和成熟后下降。当与减数分裂抑制（米力农处理）的GV期DO共培养时，MGC中[³H]胸苷掺入量最高（比对照高11倍），与成熟卵母细胞共培养刺激其掺入量增加5.5倍，与排卵的卵母细胞共培养刺激其增加3倍，而与合子共培养则无刺激作用。无论是否存在DO，米力农的剂量均未改变MGC中[³H]胸苷掺入量。中期I标志着卵母细胞促进MGC DNA合成能力下降的开始。这些结果表明，卵母细胞促进颗粒细胞增殖的能力遵循一个发育程序，与卵母细胞减数分裂状态密切相关，随着减数分裂能力的获得而增加，并在卵母细胞成熟期间和成熟后下降。
