Epidermal growth factor (EGF) induces cell proliferation during in vitro culture of ovine preantral follicles

Efficient in vitro culture systems of ovarian preantral follicles can promote oocyte growth and proliferation of somatic cells. After in vitro culture of ovine ovarian tissue, it was observed that control medium (α-MEM+ ) alone or added by 1 ng/mL Epidermal Growth Factor (EGF) can promote the initiation of primordial follicle development, process also called follicular activation (SANTOS et al., 2013, Anais da XXVII Reuniao Anual da SBTE). However, there is not information about oocyte growth and proliferation of granulosa cells. Thus, the aim of the present study was to determine the effect of EGF (1 ng/mL) on oocyte diameter and granulosa cell proliferation after in vitro culture of ovine ovarian tissue. After collection of ovine ovaries (n=10) in the slaughterhouse, ovarian cortex was fragmented and one fragment was immediately fixed and destined to histology (fresh control). The remaining fragments were cultured in vitro for 7 days in α-Minimal Essential Medium (α-MEM – GIBCO, Invitrogen, St Louis, EUA) supplemented with ITS (10 µg/mL insulin, 5.5 µg/mL transferrin, 5,0 ng/mL selenium), 2 mM glutamine, 2 mM hypoxanthine, 1.25 mg/mL bovine serum albumine (BSA) and 50 µg/mL ascorbic acid (α-MEM+ - control medium) or in α-MEM+ added by 1 ng/mL EGF. After culture, the fragments were destined to histology, the oocyte diameter was analyzed and immunohistochemistry was performed to evaluate Proliferating Cell Nuclear Antigen (PCNA; 1:500; Santa Cruz Biotechnology; Santa Cruz, CA, EUA). Proliferating or PCNA-positive cells (brown) were counted in the sections and expressed as percentages. The diameter was analysed by ANOVA and the percentages of PCNA-positive cells were compared by Qui-square test (P<0.05). There was no influence of α- MEM+ or EGF on oocyte diameter after in vitro culture. Concerning cell proliferation, medium supplementation with EGF significantly increased PCNA-positive cells (55.0%) in relation to the fresh control (13.0%) and control medium (5.0%). In conclusion, 1 ng/mL EGF promotes proliferation of granulosa cells in preantral follicles during in vitro culture of ovine ovarian tissue.