Changes in Key Steroidogenic Enzymes Expression Pattern in Pre-ovulatory Follicles of Tsaiya Duck
| Autor: | Wen -Wang Kuo, 郭文旺 |
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| Rok vydání: | 2006 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 94 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. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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