Localização de proteínas, quantificação de RNAm e papel das citocinas TNF-α e IL-1β no desenvolvimento folicular in vivo e in vitro em bovinos

Abstract

The aims of this study were: 1) to investigate the expression and distribution of messengers RNAs and proteins of TNF-α and interleukin 1 systems members in the ovaries of cyclic cows; 2) to evaluate the effects of TNF-α, Dexamethasone and IL-1β, on the survival and activation of bovine primordial follicles in vitro; 3) to characterize the expression profile of TNF-α and IL-1β systems in bovine granulosa cells of preovulatory follicles during ovulation induced by GnRH/LH in vivo; 4) to evaluate the influence of gonadotropins (FSH/LH) in the expression of mRNA for TNF-α system and IL-1β system in cumulus cells cultured in vitro; and 5) to analyze the effect of TNF-α and IL-1β on cumulus cells expansion, ultrastructure integrity of cultured COCs; mRNA expression of HAS-2, CASP3, CASP6 and iNOS of cumulus cells after in vitro culture. Bovine ovaries were processed for immunohistochemistry and Western Blot analyzes for the location of proteins for TNF-α system and interleukin system 1 in preantral and antral follicles. To evaluate the in vitro follicular survival and activation, ovarian tissues were cultured for 6 days in the presence of IL-1β, TNF-α or Dexamethasone at different concentrations. To evaluate the profile of expression of TNF-α and IL-β systems after GnRH treatments in vivo, granulosa cells from bovine preovulatory follicles were collected at different time points. COCs were cultured for 12 and 24h in the presence of gonadotropins, TNF-α and IL-1β. TNF-α system (TNF-α, TNFR1 and TNFR2) and interleukin I system (IL-1β, IL-1RA, IL-1RI and IL-1RII) were detected in preantral and antral follicles by immunohistochemistry. Variable levels of mRNA for the interleukin 1 system members were observed at different stages of development. Concerning to in vitro culture of bovine ovarian tissue, the presence of TNF-α (10 ng/mL) reduced the follicular survival and increased the number of apoptotic cells, whereas the addition of dexamethasone (10 ng/mL) maintained the follicular ultrastructure. On the other hand, the presence of IL-1β (10 or 50 ng/mL) was able to maintain the percentage of normal follicles and promote the primordial follicles activation. In vivo, an increased expression of mRNA for TNF-α and TNFR1 was observed 12 h after GnRH treatment. Higher levels of mRNA for TNFR2 were observed at 3, 6 and 12 h after GnRH administration. However, it was seen an increase in mRNA levels for IL-1RA 24h after GnRH induction. In vitro, TNF-α and IL-1β did not shown additive effect of cumulus cells expansion. The levels of mRNA for TNF-α, TNFR1 and TNFR2 were increased in COCs cultured for 12 h in the presence of gonadotropins. The presence of gonadotropins increased the mRNA levels for IL-1R1 and IL-1RA in cumulus cells after 24h of culture. The presence of TNF-α reduced levels of HAS-2, CASP3 and CASP6, whereas IL-1β increased levels of mRNA for IL-β, IL-1RA and HAS-2 in cultured cumulus cells. Furthermore, IL-1β increased iNOS expression after 24h of culture. Ultrastructural analysis confirmed the integrity of COCs cultured in presence of TNF-α or IL-1β. In conclusion, TNF-α system members and interleukin-1 system members are differentially expressed in ovarian cells. TNF-α reduces the follicular survival in vitro, while dexamethasone enhances the ultrastructure of cultured follicles. On the other hand, IL-1β promotes the development of primordial follicles in vitro. The expression of TNF-α system and the IL-1 system is regulated by gonadotropins in vivo and in vitro. The presence of TNF-α and IL-1β maintain the normal ultrastructure of cultured COCs. These results indicate an important role of TNF-α and interleukin 1 systems in the regulation of bovine folliculogenesis and ovulation.