建立雌二醇酵素免疫分析法應用於臺灣水牛生殖狀態之監測

Abstract

雌二醇（estradiol, E2）由雌性動物之卵巢粒性細胞（granulosa cell）轉換雄性素而來，為膽固醇之衍生物。動情周期中，雌二醇為維持卵母細胞發育所必需，經由正迴饋於下視丘及腦垂腺之方式刺激LH之潮湧及排卵的發生。由監測雌二醇之變化，可以直接反映出卵巢活動的狀況，因此必須發展一種靈敏度高且簡便快速的雌二醇酵素免疫分析系統。酵素免疫分析法（enzyme immunoassay, EIA）原理為偵測特異性抗原抗體的結合狀態，為一種相當普遍的技術。本分析法之免疫原為17β-E2-6-CMO: BSA之兔抗雌二醇多株抗體，分析內變異為8.53%，分析間則為9.19%；靈敏度可達7.13 pg/mL，標準曲線之相關係數為0.958 （picogram level） ~ 0.984 （nanogram level）；抗血清之交叉反應中除了雌酮（estrone; 2.04%）、雌三醇 （estriol; 1.51%）及雄固烷醇酮（androstanolone; 0.08%）外，其他類固醇、膽固醇及牛血清蛋白等皆小於0.05%；分析時間只需1-1.5小時。此雌二醇酵素免疫分析法可用於牛濾泡液及牛濾泡細胞培養液之檢測。 自1990年以來台灣水牛（Bubalus bubalis carabanesis）之頭數逐年減少，從近21,876頭降至2008年為2,950頭而亟需保育。過去文獻曾指出水牛血液中孕酮（progesterone, P4）與雌二醇之含量極低，而使動情周期（estrous cycle）中濾泡期（follicular phase）及黃體期（luteal phase）不易區分，因此本研究使用台灣水牛作為驗證雌二醇酵素免疫分析系統之動物模式，利用酵素免疫法分析三隻母水牛之血液及糞便試樣中之雌二醇及孕酮，以評估動情周期中之變化。結果顯示在濾泡期血清中孕酮濃度為1.1 +/- 0.5 ng/mL，雌二醇為39.9 +/- 10.0 pg/mL，糞孕酮與糞雌二醇各為161.0 +/- 30.2 及49.9 +/- 14.1 ng/g；黃體期之血清中孕酮為4.4 +/- 1.0 ng/mL，雌二醇為30.4 +/- 8.2 pg/mL，糞孕酮與糞雌二醇各為230.9 +/- 52.1 ng/g 及39.9 +/- 10.0 ng/g。依據上述內泌素推算台灣水牛之動情周期帄均為19.8 +/- 0.7天，濾泡期5.0 +/- 0.6 天，黃體期為14.8 +/- 1.2 天。 本研究所建立之雌二醇酵素免疫分析法具有高靈敏度及分析便捷等優點，已成功應用於牛卵巢濾泡液、濾泡細胞培養液之雌二醇分析，以及監測台灣水牛之生殖狀態，證明此方法可應用於動物生殖內分泌學的研究。

Estradiol （E2） is derived from cholesterol through androgens by granulosa cells in female ovaries. E2 is necessary to maintain the development of oocytes in the estrous cycle via a positive feedback system, which induces luteinizing hormone （LH） surge and ovulation through the hypothalamic-pituitary events. The monitoring of E2 primarily reflects the activities of the ovaries. The Enzyme Immunoassay （EIA） is a common technique, by detecting the specific binding of antigen and antibody. Thus, it is necessary to develop a highly sensitive and rapidly detective E2 EIA system. The rabbit anti-E2 polyclonal antiserum was made from rabbits which against E2-17β-carboxymethyloxime : BSA. The sensitivity of this E2 EIA revealed 7.13 pg/mL, and the r-square value of the standard curve was 0.958~ 0.984. Except estrone （2.04%）, estriol （1.51%） and androstanolone （0.08%）, the cross-reactivity of other steroids and BSA were lower than 0.05%. The coefficient of variation of intra- （n=5） and inter-assay （n=5） in this system were 8.53% and 9.19%, respectively. The total experimental duration from incubation to assay was 1- 1.5 hours. To validate this E2 EIA system, different kinds of samples were tested, including bovine follicular fluid and follicle cell culture medium. The population of Taiwan Buffalo has decreased from 21,876 to 2,950 since 1990 to 2008. Therefore, it is urgent to conserve Taiwan Buffalos. In the previous experiment, the blood levels of progesterone （P4） and E2 were quite low and the difference between follicular and luteal phase was too small to distinguish. Other than the result above, we also used Taiwan Buffalo as our animal model to prove the E2 EIA validation. In our experiments, blood and fecal samples of three buffalo cows were collected to measure the concentrations of E2 and P4 by EIA for assessing the pattern of estrous cycle. In the follicular phase, the serum P4 and E2 level was 1.1 +/- 0.5 ng/mL and 39.9 +/- 10.0 pg/mL, respectively. The fecal P4 was 161.0 +/- 30.2 ng/g and fecal E2 was 49.9 +/- 14.1 ng/g. During the luteal phase, the serum P4 concentration was 4.4 +/- 1.0 ng/mL, serum E2 was 30.4 +/- 8.2 pg/mL, fecal P4 was 230.0 +/- 52.1 ng/g and fecal E2 was39.9 +/- 10.0 ng/g. Based on these data of hormones, the length of estrous cycle was estimated to be 19.8 +/- 0.7days, within the follicular and luteal phase were 5.0 +/- 0.6 and 14.8 +/- 1.2 days, respectively in buffalo cows. In this study, the E2 EIA system has many advantages such as highly sensitive and rapid reaction. We successfully used this system for measurement the level of E2 in the bovine follicular fluid and follicle cell culture medium, and monitoring of the Taiwan buffalo reproductive status. It is expected that this E2 EIA system would be a valid technique to further understand animal reproduction.