THE MECHANISM OF LUTEINIZATION IN VITRO AND IN VIVO

对颗粒细胞黄素化机制的研究将继续进行 强调本地控制过程。 毛囊的作用 从大型（6 - 12 mm）猪分离的液体黄体化刺激剂（LS） 将检查卵泡。 LS是一种蛋白质， 道尔顿，将从大猪卵泡液中分离、纯化， 具有化学和生理特性。 该LS将在 其增强FSH诱导LH/hCG受体的能力的基础， 培养的猪颗粒细胞孕酮分泌。 LS将是 通过适当的醇沉组合纯化， Sephacryl-S-300柱层析，DEAE离子交换层析 在TSK 5 PW柱上进行三丙烯酰基和HPLC离子交换。 C3反相HPLC 相柱和tiyo soda 3000 SW凝胶排阻柱 按要求出。 将通过SDS凝胶表征馏分 电泳和蛋白酶测定。 最终产品将受到 端基分析和手动Edman降解。 的细胞部位 将在培养的猪颗粒层中检查纯化的LS的作用 细胞，即其改变LH/hCG受体从头合成的能力 利用重氨基酸的掺入，改变腺苷酸环化酶的能力， 改变一般蛋白质合成的能力。 大鼠颗粒细胞将 也可以作为实验系统。 一种抗血清，单克隆和 将针对猪LS产生多克隆的抗体。 交叉反应性 将检测人卵泡液中的LS活性。 的 猪颗粒细胞和卵泡膜细胞分泌LS的能力将是 在不同的荷尔蒙条件下进行检查。 LS在体内的作用 将首先在未成熟大鼠中进行检查，如果阳性结果 将在未成熟的恒河猴中进行观察 猴子. 另外，抗LS的抗血清在体内对卵巢的影响 将在未成熟大鼠或猴中检查功能。 的角色 小型猪卵泡液黄体化抑制剂（LI） 将确定卵泡（1 - 3mm）和中等大小（3 - 5mm）的卵泡。 将使用FSH处理的猪颗粒层分离和纯化LI 细胞培养作为生物测定。 抑制FSH诱导的能力 LH/hCG受体、孕酮分泌与形态学黄体化 将被跟踪。 将制备抗LI的抗血清并进行评价 体外和体内抗LI的作用。 荷尔蒙控制 将检查颗粒细胞合成LI活性。 相互作用 将考虑LI和LS控制卵泡成熟。

Studies on the mechanism of granulosa cell luteinization will be pursued with an emphasis on local control processes. The role of a follicular fluid luteinization stimulator (LS) isolated from large (6-12mm) porcine follicles will be examined. The LS, which is a protein about 60,000 daltons, will be isolated from large porcine follicular fluid, purified and characterized chemically and physiologically. This LS will be assayed on the basis of its ability to enhance FSH induction of LH/hCG receptors and progesterone secretion in cultured porcine granulosa cells. The LS will be purified by an appropriate combination of alcohol precipitation, Sephacryl-S-300 column chromatography, ion exchange chromatography on DEAE trisacryl and HPLC ion exchange on a TSK 5 PW column. HPLC on a C3 reverse phase column and a tiyo soda 3000 SW gel exclusion column will be carried out as required. Fractions will be characterized by SDS gel electrophoresis and by protease assay. The final product will be subject to end group analysis and Manual Edman degradation. The cellular site of action of the purified LS will be examined in cultured porcine granulosa cells, i.e. its ability to alter de novo synthesis of LH/hCG receptors using incorporation of heavy amino acids, ability to alter adenyl cyclase, & ability to alter general protein synthesis. Rat granulosa cells will also be used as an experimental system. An antiserum, monoclonal and polyclonal, will be generated against porcine LS. The cross reactivity against LS activity in human follicular fluid will be examined. The ability of porcine granulosa and thecal cells to secrete LS will be examined under different hormonal conditions. The effect of LS in vivo will be examined initially in the immature rat and if positive results are obtained the observations will be carried out in the immature rhesus monkey. Alternately the effects of antiserum to LS in vivo to ovarian function will be examined in the immature rat or monkey. The role of a follicular fluid luteinization inhibitor (LI) present in small porcine follicles (1-3mm) and medium (3-5mm) sized follicles will be determined. The LI will be isolated and purified using FSH treated porcine granulosa cell cultures as a bioassay. The ability to inhibit FSH induction of LH/hCG receptors, progesterone secretion and morphological luteinization will be followed. An antiserum against LI will be prepared and evaluated for actions against LI in vitro and in vivo. The hormonal control of granulosa cell synthesis of LI activity will be examined. Interaction of LI and LS in control of follicular maturation will be considered.