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

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 ۶ years old Sterlet ۷۲۹ g in weight and ۴۷ cm in total length. The oocytes were in advanced vitellogenesis stage (PI >۱۰). A part of the ovary (containing about ۳۰۰ 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 ۰.۲۵% trypsin-EDTA  in Ca۲+ and Mg۲+ free PBS and cultured in medium L-۱۵ supplemented with ۲۰% FBS, streptomycin sulphate (Gibco, ۱۰۰ mg.ml-۱), penicillin G potassium (Gibco, ۱۰۰ IU.ml-۱) and Amphotericin B (Gibco, ۲.۵ mg.ml-۱) at ۲۲ °C. The concentrations of Testosterone (T), Estradiol-۱۷β (E۲), Progesterone (P۴) and ۱۷α-hydroxyprogestron (۱۷αOHP) in the medium were measured at days ۳, ۵ & ۷ by the Enzyme-Linked Immunosorbent Assay. According to the results, the ovarian follicular cells of Sterlet proliferated in L-۱۵ medium were steroidogenically active as expressed by the secretion of T, E۲, P۴ & ۱۷αOHP. 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 ۳ & ۴ in comparison with those at days ۵ & ۶. By successfully culturing ovarian follicular cells of Sterlet in L-۱۵ 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.

Keywords:

Sterlet , Acipenser ruthenus , Primary culture , Follicular cells , In vitro