脊椎動物促濾泡激素生物活性之比較研究—離體大白鼠生精細管環單磷酸腺?之生成

Abstract

本研究之目的為(一)建立環單磷酸腺?之競爭性蛋白質結合分析法(二)利用大白鼠生精細管環單磷酸腺?之生成以建立促濾泡激素之離體生物活性測定系統(三)將此系統應用於多種脊椎動物純化之促濾泡激素或腦下垂體磨碎液生物活性之測定與比較研究。 環單磷酸腺?之結合蛋白質是從新鮮之牛腎上腺製備。所用標識之環單磷酸腺?為重氫標識（3H-Cyclic AMP; 40Ci/mmol）。以活性碳分離未與結合蛋白質結合的3H-Cyclic AMP。此法之測定範圍為0.5－9.0pmol（164.5－2961pg/tube）；與Cyclic IMP，Cyclic GMP，Cyclic UMP之交互反應，分別為8.4％，0.9％，0.52％而與ATP, ADP, AMP, 2′, 3′-Cyclic AMP之交互反應，均小於0.01％。其靈敏度約為0.35pmol/試管。再現性高。 促濾泡激素之離體生物活性測定方法之建立是以Collagenase處理12－16日齡大白鼠睪丸，分離出生精細管，在lmM Methyl-isobutyl-xanthine之Mediam 199 (pH 7.40)之培養液中，在34℃，連續通氣(95％ O2－5％ CO2)每分鐘搖動100次之狀況下，培養30分鐘後，定量所生成之環單磷酸腺?。羊促濾泡激素(NIH－FSH－S13，10-500ng) 能促進良好劑量反應相關之環單磷酸腺?之生成；因此，作為本系統之基準激素(Reference Hormone)。 本論文研究首次廣泛地同時比較多種脊椎動物之促濾泡激素、促性腺激素或腦下垂體磨碎液對大白鼠生精細管促進環單磷酸之生成效應。結果顯示純化之羊、豬、鼠、人之促濾泡激素，人類停經後促性腺激素和孕馬血清促性腺激素可產生良好劑量相關之環單磷酸腺?生成；白鰱與鴨之促性激素則無反應；雞、鴨、鵝及魚之腦下垂體磨碎液反應差，但大白鼠和小白鼠之腦下垂體磨碎則有良好之反應；而蜥蜴之腦下垂體磨碎液則在較高劑量下似乎有反應產生。 本研究之結果提示大白鼠生精細管環單磷酸腺?生成僅適用於哺乳類動物促濾泡激素生物活性之測定與其種別差異之比較。而大多數非哺乳類脊椎動物之促濾泡激素或促性腺激素不能引起大白鼠生精細管之反應；可能僅限於少數種別有反應，但仍需要更多實驗證明。因此本哺乳類系統（大白鼠生精細管系統）不適於非哺乳類之促濾泡激素生物活性之測定與比較。

The purposes of this study were: (1) to establish the competitive protein binding assay of Cyclic AMP; (2)to establish an in vitro follicle-stimulating hormone (FSH) bioassay system using the cyclic AMP formation of rat seminiferous tubules; and (3)to determine and compare the biological activity of FSH's and pituitary extracts from a variety of vertebrate species in such rat seminiferous tubule system. The cyclic AMP binding protein was prepared from the fresh bovine adrenal cortex. 3H-Cyclic AMP was used as a label tracer and charcoal was used to separate free and bound 3H-cyclic AMP. The sensitivity and determination range of the competitive protein binding assay were 0.35 pmol / tube and 0.5 - 9.0 pmol/tube, respectively. The cross reaction of binding protein to cyclic IMP, cyclic GMP, cyclic UMP were 8.4% , 0.9% , and 0.52%, respectively; however, the cross reaction for ATP, ADP, AMP, 2', 3'-cyclic AMP were all less than 0.01%. The seminiferous tubules from 12-to-16-day-old rat testes were isolated by collagenase treatment and incubated with various doses of FSH's, gonadotropins, or pituitary extracts in Medium 199 (PH 7.40) containing 1 mM Xanthine at 34℃ for 30 minutes under continuous aeration of 95% 02-5% CO2, shaked at 100 cycles per min. The cyclic AMP formed was quantified by the competitive protein binding assay. The biological activities of FSH's, gonadotropins or pituitary extracts from a variety of vertebrate species were compared extensively for the first time. The results indicate that the rat seminiferous tubule system is highly responsive to the mammalian hypophysial FSH's (ovine-, rat-, porcine-, and human-FSH), plancental gonadotrooins (pregnant mare serum gonadotropin, human chorionic gonadotropin) and human menopausal gonadotropin.; and good dose-related resposes were obtained between cyclic AMP formation and FSH's or gonadotropins. By contrast, duck gonadotropin and silver carp gonadotropin were inactive in this system. The pituitary extracts from mammalian species (mouse and rat) were also highly active in the cyclic AMP formation; but those from nonmammalian vertebrate (chicken, duck, goose, and grass carp) were unable to stimulate the formation of cyclic AMP by the rat seminiferous tubules, although lizard pituitary extract, at extremely high dose, seemed to evoke some-formation of cyclic AMP. The findings from the present thesis indicate that the cyclic AMP formation system by rat seminiferous tubules in vitro is suitable only for the assessment of the biological activities of FSH's from mammalian species and for comparing the species difference or phylogenetic patterns of mammalian FSH's per se. Such system is thus not suitable for biological assays of FSH activity from nonmammalian vertebrate species.