Kit ligand promotes the transition from primordial to primary follicles after in vitro culture of ovine ovarian tissue

In vitro studies have showed that Kit Ligand (KL) maintains survival, promotes growth (Jin et al, Mol. Reprod. Dev. 70, 82–90 2005; Reynaud et al, Mol. Reprod. Dev. 56, 483–494, 2000) and activation (Celestino et al, Mol. Reprod. Dev. 77, 231 - 240, 2010; Lima et al, Cells Tissues Org. 195, 260–271, 2012; Parrott e Skinner, Endocrinology 140, 4267-4271, 1999) of ovarian preantral follicles in different species. However, the effect of different concentrations of KL on in vitro development of preantral follicles enclosed in ovine ovarian tissue has not been evaluated. The aim of this study was to evaluate the effect of KL on the morphology and development of ovine preantral follicles in vitro. After collection of ovaries (n=8) in a slaughterhouse, one fragment of ovarian tissue was fixed for histological analysis, corresponding to fresh control. This fragment was dehydrated, diafanized and stained with HematoxylinEosin. The remaining fragments were cultured for 7 days in α-Minimal Essential Medium (α-MEM – GIBCO, Invitrogen, St Louis, EUA) supplemented with hypoxantine, glutamine, ascorbic acid, bovine serum albumin (BSA) and ITS (insulin, transferrin and sodium selenite) (Sigma Chemical Co., St. Louis, MO, USA) in the absence (control medium) or presence of KL (1, 10, 50, 100 or 200 ng/mL; Sigma Chemical Co., St. Louis, MO, USA). After culturing, the morphological analysis of preantral follicles was performed by histology, and follicles were classified as normal or atretic according to the absence or presence of cytoplasmic retraction, nuclear pycnosis and/or disorganization of granulosa cells, as well as primordial, primary and secondary follicles. The percentages of normal follicles and follicles at each stage of development were compared by ANOVA and Tukey’s test (P<0.05). After 7 days of culture, all the treatments reduced (P<0.05) the percentage of morphologically normal follicles compared to fresh control. In addition, a decrease (P<0.05) in the percentage of primordial follicles and an increase in the percentage of primary follicles was observed at the concentration of 100 ng/mL KL, compared with the fresh control, control medium (α-MEM) and the other concentrations of KL. In conclusion, 100 ng/mL KL promotes the transition from primordial to primary follicles (follicular activation) after in vitro culture of ovine ovarian tissue.