Visfatin improves survival and promotes the activation of primordial follicles in cultured sheep ovaries

Pinto, Joisyleide Gonçalves da CostaBarberino, Ricássio de SousaGuimarães, Valéria da SilvaOliveira Júnior, Joãozito Liandro deMonte, Alane Pains Oliveira doAndrade, Kíscyla Oliveira deMatos, Maria Helena Tavares de

Abstract Visfatin is an adipokine involved in the regulation of female reproduction. However, there are no studies on the effects of visfatin on the in vitro culture of ovarian tissue in any species. Therefore, the aims of this study were to analyze the effects of visfatin on survival, primordial follicle activation, granulosa cell proliferation, and the immunostaining of tumour necrosis factor-α (TNF-α) in preantral follicles after the in vitro culture of sheep ovarian tissue. Ovarian fragments were fixed for histological analysis (fresh control) or cultured in α-minimum essential medium alone (α-MEM+: control medium) or in α-MEM+ supplemented with different concentrations of visfatin (1 or 10 ng/mL) for 7 days. Subsequently, ovarian tissue was destined to histology (morphology, activation and growth) and immunohistochemistry (granulosa cell proliferation and pro-inflammatory cytokine TNF-α immunostaining). The results indicated that treatments with visfatin (1 or 10 ng/mL) maintained the percentage of morphologically normal follicles at a level similar (P>0.05) to the fresh control and significantly higher than of α-MEM+. A significant increase in primordial follicle activation was also observed in tissue cultured for 7 days at both visfatin concentrations compared to the fresh control and α-MEM+. In addition, only the treatment containing 10 ng/mL of visfatin significantly increased follicular and oocyte diameters, and granulosa cell proliferation compared to α-MEM+, and attenuated inflammatory activity by reducing TNF-α immunostaining after in vitro culture. In conclusion, 10 ng/mL visfatin maintains survival, reduces immunostainig of TNF-α and promotes the activation of primordial follicles by stimulating granulosa cell proliferation after the in vitro culture of sheep ovarian tissue.