Primary culture of ovarian follicular cells of Sterlet, Acipenser ruthenus to develop an in vitro system

Abstract

The aim of the present study was to develop an in vitro system for functional investigation of ovarian follicular cells in Sterlet, Acipenser ruthenus. Oocytes for the primary culture were obtained from the ovaries of a 6 years old Sterlet 729 g in weight and 47 cm in total length. The oocytes were in advanced vitellogenesis stage (PI >10). A part of the ovary (containing about 300 follicles) was removed, ovarian follicles isolated by manually removing those from the interstitial tissue and washed with sterile phosphate buffered saline (PBS) containing antibiotics and Amphotericin B. Follicular cells were separated by treating oocytes with 0.25% trypsin-EDTA in Ca2+ and Mg2+ free PBS and cultured in medium L-15 supplemented with 20% FBS, streptomycin sulphate (Gibco, 100 mg.ml-1), penicillin G potassium (Gibco, 100 IU.ml-1) and Amphotericin B (Gibco, 2.5 mg.ml-1) at 22 °C. The concentrations of Testosterone (T), Estradiol-17β (E2), Progesterone (P4) and 17α-hydroxyprogestron (17αOHP) in the medium were measured at days 3, 5 & 7 by the Enzyme-Linked Immunosorbent Assay. According to the results, the ovarian follicular cells of Sterlet proliferated in L-15 medium were steroidogenically active as expressed by the secretion of T, E2, P4 & 17αOHP. Testosterone was the dominant hormone secreted by cultivated follicular cells, which was correlated closely with the end of vitellogenesis in the isolated oocytes. Decrease in production of these hormones was greater at days 3 & 4 in comparison with those at days 5 & 6. By successfully culturing ovarian follicular cells of Sterlet in L-15 culture medium, an in vitro system was developed which enables functional studies to be carried out similar to the in vivo situation in the ovarian follicles.