幼齡大白鼠生精細管雌二醇生成系統與調節因數之探討

Abstract

本論文之研究目的為（一）利用幼齡大白鼠生精細管足細胞中雌二醇之生成，以建立一套較快速簡便且特異性高的促瀘泡激素離體生物活性測定法；（二）探討內泌激素及其它因數對該系統雌二醇生成之影響。 促濾泡激素離體生物活性測定法之建立是以colla-genase (Type Ⅰ）處理10-12日齡大白鼠睪丸，分離出生精細管，將細胞混懸於培養液中，分裝至多孔培養盤，在培養液C（培養液A+0.125mM MIX+0.17μM胰島素）中與lμM 190H-A在35℃，連續通氣（95% O2-5% CO2)，每分鐘搖動50次的狀況下，前培養（preincubation) 2小時後，加入不同劑量的促瀘泡激素，在搖動培養槽中共同培養20小時。羊促瀘泡激素（NIADDK-oFSH-17）能促進特異性高且劑量相關（dose-related）之雌二醇生成。雖然本系統較簡便，但靈敏度卻因初培養的省略而稍差，因此在前培養 2小時之前增加了初培養2 小時的步驟，提昇靈敏度約50％。 探究內泌激素及其它因數對雌二醇生成之影響 ，是將有關因數加入培養系統，與羊促濾泡激素共同培養。結果顯示，大鼠鐵傳遞蛋白，三碘甲素，皮質醇，性釋素，生長素釋因，神經勝?Y，P 物質及松果腺激素在生理劑量時對雌二醇生成有顯著促進；松果腺激素在生理劑量時呈現抑制作用，在藥理劑量時呈現促進作用。腎上腺素，乙醯膽鹼和甲狀腺素略有促進作用。小鼠鐵傳遞蛋白，enka-phalinamide，擬胰島素生長因數Ⅱ，正腎上腺素，泌乳激素和生長激素無效應。上皮細胞生長因數，酸性纖維母細胞生長因數和新生小牛血清呈抑制作用；17％濃度時的新生小牛血清顯著抑制雌二醇生成。 本研究建立之促瀘泡激素生物活性測定法，不但較為簡便，對促瀘泡激素也有高度特異性；在內泌激素及其它因數的探討上，有顯著效應者對大白鼠足細胞雌二醇生成系統的機制及生理意義，有待進一步探討。

The purposes of this study were:(1) To establish a simplified in vitro FSH bioassay system, by directly employing the dispersed seminiferous tubule cells; (2) To study the influence of hormones and other factors on the simplified FSH bioassay and its estradiol formation system. Decapsulated testes, obtained from 10-12 days old rats were dispersed with collagenase in Medium A twice. The dispersed seminiferous tubule cells were initially cultured for 2 hr. After centrifu-gation, cell pellets dispersed in Medium A were preincubated with 190H-A for 2 hr in 16-mm 24-well multiwell culture plate and then incubated with varying doses of FSH in Medium C (Medium A + 0.125mM MIX + 0.17μM insulin) at 35℃ for 20 hr, under continuous aeration in Dubnoff incubator shaken at 50 cycles/min. A highly sensitive, specific and dose-related estradiol-17β were produced by Ovine FSH (NIADDK-oFSH-17), such modified procedures proved to be a simplified and rapid bioassay for FSH. Hormones and factors dissolved in Medium C were incubated together with oFSH to investigate their effects on estrogen formation. Rat transferrin, triiodothyronine, hydrocortisone, GnRH, GRF, neuro-peptide Y and substance P enhanced FSH-evoked estradio1-17β formation ; melatonin had biphase effects-inhibitory at physiological doses while stimulatory at pharmacological doses. Mouse transferrin, Leu-enkaphalinamide, IGFⅡ,.epinephrine, prolactin and growth hormone showed no influence on estradiol formation. EGF, aFGF and new-born calf serum decreased ESH-stimulated estrogen formation; new-born calf serum at a concerntration of 17% greatly inhibited estradiol -17β formation.