Zachow R J, Weitsman S R, Magoffin D A
Department of Obstetrics and Gynecology, CSMC Burns and Allen Research Institute, Los Angeles, California 90048, USA.
Endocrinology. 1997 Feb;138(2):691-7. doi: 10.1210/endo.138.2.4950.
During ovarian follicle growth, precise regulation of the onset of androgen production by ovarian theca-interstitial cells (TIC) is necessary for maintaining follicle viability. Thus, temporary suppression of TIC androgen production in preantral follicles is the key to promoting follicle development. Evidence indicates that this process is coordinated via intraovarian growth factors. Hepatocyte growth factor (HGF) can induce granulosa cell (GC) proliferation and suppress follicular atresia, indicating a role for HGF in promoting follicle growth and viability. To determine whether HGF could reversibly suppress androgen production, this study investigated the effect of HGF on TIC differentiation and steroid production. Twenty-six-day-old rats were used in all studies. HGF messenger RNA (mRNA) expression in TIC and GC was determined by reverse transcription-PCR. Agarose gel electrophoresis of the PCR products yielded a single band corresponding to the 290-bp HGF product for both TIC and GC. HGF expression in cultured TIC and GC was not blocked by gonadotropins or HGF. To investigate the effects of HGF on TIC steroidogenesis, TIC were isolated from the ovaries of hypophysectomized rats. TIC (3.0 x 10(4) cells/well) were cultured with LH (0-3 ng/ml) and/or HGF (0-100 ng/ml) for 48 h, and androsterone levels were measured by RIA. HGF did not alter androsterone levels in the absence of LH; however, HGF reversibly impaired LH-dependent androsterone production by as much as 57% (IC50 = 1.5 +/- 0.01 ng/ml). LH (0.3 ng/ml) stimulated progesterone (P4) synthesis by TIC (1201 +/- 190 pg/ml) compared to that by control cells (210 +/- 30 pg/ml). HGF stimulated basal P4 production, and LH-dependent P4 synthesis was augmented 2.6-fold by HGF (ED50 = 0.3 +/- 0.01 ng/ml). The DNA content and cell viability in TIC cultures were not affected by HGF. The effect of HGF on steroidogenic enzyme gene expression in TIC was also investigated via PCR. HGF did not alter the level of basal or LH-induced P450 side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase mRNAs; however, LH-dependent P45017 alpha hydroxylase/C17,20 lyase mRNA content was reduced 4.5 fold in the presence of HGF. Thus, HGF is expressed in both TIC and GC obtained from the immature rat ovary, suggesting its presence in growing follicles. In TIC, HGF stimulated P4 synthesis, but impaired androgen production, concurrent with a down-regulatory effect on P45017 alpha hydroxylase/C17,20 lyase gene expression. Collectively, these results indicate that HGF reversibly impairs LH-stimulated androgen production in TIC. Such effects may help promote folliculogenesis.
在卵泡生长过程中，卵巢膜间质细胞（TIC）雄激素生成起始的精确调控对于维持卵泡活力至关重要。因此，暂时抑制窦前卵泡中TIC的雄激素生成是促进卵泡发育的关键。有证据表明，这一过程是通过卵巢内生长因子来协调的。肝细胞生长因子（HGF）可诱导颗粒细胞（GC）增殖并抑制卵泡闭锁，表明HGF在促进卵泡生长和活力方面发挥作用。为了确定HGF是否能可逆性抑制雄激素生成，本研究调查了HGF对TIC分化和类固醇生成的影响。所有研究均使用26日龄大鼠。通过逆转录PCR测定TIC和GC中HGF信使核糖核酸（mRNA）的表达。PCR产物的琼脂糖凝胶电泳显示，TIC和GC均产生一条对应于290bp HGF产物的单带。促性腺激素或HGF不会阻断培养的TIC和GC中的HGF表达。为了研究HGF对TIC类固醇生成的影响，从垂体切除大鼠的卵巢中分离出TIC。将TIC（3.0×10⁴个细胞/孔）与促黄体生成素（LH，0 - 3 ng/ml）和/或HGF（0 - 100 ng/ml）一起培养48小时，并用放射免疫分析法测定雄酮水平。在无LH的情况下，HGF不会改变雄酮水平；然而，HGF可使LH依赖性雄酮生成可逆性受损达57%（半数抑制浓度 = 1.5 ± 0.01 ng/ml）。与对照细胞（210 ± 30 pg/ml）相比，LH（0.3 ng/ml）刺激TIC合成孕酮（P4，1201 ± 190 pg/ml）。HGF刺激基础P4生成，且HGF使LH依赖性P4合成增加2.6倍（半数有效剂量 = 0.3 ± 0.01 ng/ml）。TIC培养物中的DNA含量和细胞活力不受HGF影响。还通过PCR研究了HGF对TIC中类固醇生成酶基因表达的影响。HGF不会改变基础或LH诱导的P450侧链裂解酶和3β - 羟基类固醇脱氢酶mRNA水平；然而, 在存在HGF的情况下，LH依赖性P45017α羟化酶/C17,20裂解酶mRNA含量降低4.5倍。因此，HGF在从未成熟大鼠卵巢获得的TIC和GC中均有表达，表明其存在于生长中的卵泡中。在TIC中，HGF刺激P4合成，但损害雄激素生成，同时对P45017α羟化酶/C17,20裂解酶基因表达有下调作用。总体而言，这些结果表明HGF可可逆性损害LH刺激的TIC雄激素生成。此类作用可能有助于促进卵泡发生。
