Orexin system in buffalo ovarian follicles and effect of orexin on estradiol production.

Abstract

Orexin is a ligand for orexin receptors OX1R and OX2R; it is a neuropeptide with pleiotropic functions, including regulation of reproduction. The current study was carried out to investigate the mRNA expression of the prepro-orexin gene (PPO) and orexin receptors (OX1R and OX2R) in ovarian follicles during different stages of their development in the ovary of water buffalo (Bubalus bubalis) and to determine the role of orexin on estradiol production. Ovarian follicles were classified into four groups based on size and estradiol (E2 ) level in the follicular fluid (FF) as follows: (i) small or F1, (ii) medium or F2, (iii) large or F3 and (iv) dominant/pre-ovulatory follicle or F4. In follicles, the mRNA expression of PPO and OX1R was greater in F3 and F4 follicles in granulosa cells (GC) and theca interna (TI) cells. OX2R expression did not vary amongst the different follicular stages in GC. Orexin A and orexin receptors were localized in the cytoplasm of GC and TI, and intensity was higher in F3 and F4 follicles. In addition, we cultured GC and treated them at 0.1,1.0, and 10 ng/ml orexin A alone or in the presence of FSH (30 ng/ml) or IGF-I (10ng/ml) for 48h. There was a significant (p < 0.05) increase in estradiol (E2 ) secretion and cytochrome P0450 family 19 subfamily A member 1 (CYP19A1) expression from GC at 1.0 and 10.0 ng/ml orexin A in the presence of 30 ng/ml follicle-stimulating hormone (FSH) or 10 ng/ml insulin-like growth factor-I (IGF-I). In conclusion, the present study provided evidence that the orexin system is expressed in buffalo ovarian follicles, and orexin-A in the presence of FSH and IGF-I has a stimulatory effect on estradiol secretion from the GC of water buffalo.