菜鴨排卵前濾泡內類固醇生成關鍵酵素表現量之變化

Abstract

產蛋家禽排卵前濾泡內，卵巢孕酮（progesterone, P4）主要是由粒性細胞所生成；而膜性細胞主要為生成睪固酮（testosterone, T）與雌二醇（estradiol, E2），但調控粒性與膜性細胞中類固醇生成機制，尚未完全明瞭。本研究的目的探討菜鴨（Tsaiya duck；Anas platyrhynchos var. domestica）排卵前濾泡內類固醇內泌素生成及其關鍵酵素表現量的關聯性。 試驗一：製備芳香化酶（aromatase, P450arom）多株抗體，所使用之P450arom抗原，是經人工合成牛P450arom蛋白片段（第371到396個胺基酸），經免疫兔子後，所收集之抗血清，以西方墨染法（Western blot）之序列稀釋（serial dilution）檢測最佳稀釋倍數，以及檢測抗體對不同種別動物之辨識能力，結果顯示此多株抗體的力價可達20,000倍，且具有辨識牛、羊、豬、大鼠及鴨等動物的類固醇生成組織之芳香化酶。 試驗二：收集菜鴨排卵前濾泡F1-F5之粒性及膜性細胞層，應用西方墨染法以偵測類固醇生成急性調節蛋白(steroidogenic acute regulatory protein, StAR)、P450膽固醇側鏈截切酵素(P450 cholesterol side-chain cleavage enzyme, P450scc)、3β-羥類固醇去氫酶(3β-hydroxysteroid dehydrogenase, 3β-HSD) 與芳香化酶的表現量。結果顯示，在菜鴨的排卵前濾泡粒性細胞層的StAR、P450scc與3β-HSD表現量都是以F1的表現量最高，且此三種蛋白質的表現量隨濾泡的逐漸成熟，而增加（P < 0.05）；而P450arom在不同時期濾泡中的表現量則沒有差異。在F1-F5濾泡膜性細胞層中，發現StAR、P450scc和3β-HSD的表現量並沒有差異；而P450arom則是在F4/5的表現量有明顯的高於其他時期的濾泡（P < 0.05），且F3的表現量亦高於F2與F1（P < 0.05）。在P4及T含量方面，濾泡細胞層內是以粒性細胞高於膜性細胞層內的P4含量，而在粒性細胞層內以F1含量最高，且排卵前濾泡的粒性細胞層的P4含量，是隨濾泡的逐漸成熟，而增加（P < 0.05）。相反地，濾泡細胞層內T及E2的含量是以膜性細胞高於粒性細胞，且於不同濾泡時期其含量具顯著差異，而以F4/5的膜性細胞層含量最高（P < 0.05）。然而，在粒性細胞層內的T含量在F1-F5濾泡內含量，則無顯著差異。 結論：（1）菜鴨排卵前濾泡內生成之P4與其相關之關鍵生成酵素（StAR、P450scc和3β-HSD）的表現量是呈正相關；（2）F1濾泡藉由增加與P4生成相關酵素（P450scc與3β-HSD）的酵素活性及其表現量，進而誘發排卵的發生，故推測排卵前F1濾泡在排卵機制上必定扮演一重要角色；（3）F3-F5膜性細胞層生成高量的T，但其StAR的表現量卻沒增加，推測菜鴨排卵前濾泡膜性細胞層，其生成T之原料並不僅由膽固醇提供，可能有部分是源自粒性細胞層所提供之P4，此仍有待進一步證實。

In preovulatory follicles of egg laying avians, progesterone is synthesized primarily by the granulosa cells while testosterone and estradiol are produced mainly by the theca cells. The mechanism of steroidogenesis in granulosa and theca cells is not yet clear. The purpose of this study was to investigate the correlation of steroidogenesis with the expression of key steroidogenic enzymes in preovulatory follicles of Tsaiya duck (Anas platyrhynchos var. domestica). Experiment one: The immunogen of aromtase protein was obtained from synthesized peptide based on bovine amino acid sequence (371-396). After 4th immunization, serum from rabbit was collected for its antibody titer and further specificity examination conducted by using serial dilution and Western blotting techniques. We found that this antiserum specifically recognizes protein extracted from cattle, goat, pig, rat and duck steroidogenic tissues with titer dilution up to 20,000-fold. Experiment two: Preovulatory follicles (F1-F5) were collected from ducks and the granulosa and theca layers of the five follicles isolated, and proteins extracted. The obtained protein was then used for Western blot to detect the expression of StAR (steroidogenic acute regulatory) protein, P450scc (P450 cholesterol side-chain cleavage), 3β-HSD (3β-hydroxysteroid dehydrogenase) and P450arom (aromatase) enzymes. The results showed that expression of StAR, P450scc and 3β-HSD proteins level was highest in F1 granulosa layer and the expression levels gradually increased with the follicle maturity (P < 0.05). However, the expression level of P450arom showed no significant difference in different stages of granulosa layers development. In theca layers of preovulatory follicles, expression of StAR, P450scc and 3β-HSD protein levels showed no difference, but the P450arom expression levels of F4/5 layer was significantly higher than in other stages, with F3 being higher than F1 and F2 (P < 0.05). Progesterone and testosterone contents in F1-F5 tissues showed interesting result. Progesterone contents in granulosa layers was higher than theca layers, especially in F1 and it increased gradually with the follicle maturity. In contrast, the testosterone (T) and estradiol (E2) contents in theca layers were higher than granulosa layers. The T and E2 content of F4/5 layer were the highest than in other stages (P < 0.05), but there was no difference in granulosa layers obtained from different stages of preovulatory follicle. In brief, it could be concluded that; (1) The steroidogenesis of P4 have positive correlation with the expression of key steroidogenic enzymes (StAR, P450scc and 3β-HSD) in preovulatory follicles of Tsaiya duck. (2) The F1 granulosa layer mainly secrete P4 by increased enzymatic activity and expression levels of StAR, P450scc and 3β-HSD that induces ovulation. Therefore, it may be tempting to speculate that the F1 follicle plays an important role in ovulatory mechanism. (3) The F3 and F4/5 theca layer produce a lot of T, but the expression level of StAR protein was not different in theca layers. This finding makes us speculate that the substrate source for testosterone production in theca layers is not solely cholesterol. However, some source of testosterone production in theca layers may be from progesterone secreted by the granulosa cells that is required further study.