The aim of the study was to determine mRNA expression of genes involved in steroidogenic pathways in chicken ovarian follicles by real-time PCR method.
Hy-Line Brown hens (n=20), 28 weeks of age, laying regular sequences of about 20 or more eggs per clutch were used in the experiment. Birds were decapitated ~2 h after ovulation. From the ovary, white prehierarchical follicles (18 mm) and the three largest yellow preovulatory follicles F3F1 (F3<F2<F1; 2235 mm) were dissected. White follicles were divided into two classes: small white follicles (SWF, 14 mm) and large white follicles (LWF, 68 mm). Granulosa and theca layers of F3F1 follicles were separated according to the Gilberts method. Total RNA extracted from the specified tissues was reverse transcribed, and real-time PCR analysis was performed using TaqMan Gene Expression Assay (Applied Biosystems) designed for StAR, P450scc, 3β-HSD, P450c17 and P450arom genes. Results showed that mRNA of all examined genes was expressed in ovarian follicles. The expression profile of StAR protein mRNA and P450scc enzyme were similar during follicular growth. Their levels were 4.6- and 7-fold higher in LWF than in SWF while a gradual decline in mRNA levels was observed in the theca layer of F3F1 (opposite effect was in granulosa). A very low 3β-HSD mRNA levels were found in SWF and LWF while in preovulatory ones the mRNA levels were ~160- and 10-fold higher in granulosa and theca layers, respectively. The expression of P450c17 and P450arom mRNAs in the theca layers was higher in white follicles in comparison with theca layers of F3-F1, where a gradual decline in mRNA levels was observed.
Obtained results show quantified differences in mRNA expression of steroidogenic genes during maturation and growth of the follicles in the chicken ovary. This results can be useful for advanced studies of steroidogenic genes in avian species.
Study supported by grant N N311 006436.
Prague, Czech Republic
24 - 28 Apr 2010
European Society of Endocrinology