Isolation of common carp ovarian follicular cells and evaluation of their endocrine activity in primary cell culture

To study viability and activity of isolated common carp, Cyprinus carpio, ovarian follicular cells (granulosa and theca cells), 17-alpha-Hydroxy progesterone (17 alpha-OHP) and 17 beta-Estradiol (E-2) levels were estimated in the culture media of cultivated carp ovarian follicular cells, using radioimmunoassay (RIA). Oocytes were isolated from the ovaries of female carp. Interstitial tissue was manually removed in order to obtain single oocytes surrounded only by the follicular envelope. Such a preparation was trypsinized at room temperature. Follicular cell suspension contained both cells and cell clumps. It was the mixture of theca (T) and granulosa (G) cells. The cells were grown as monolayer in 24-well microplates in M199 medium supplemented with FBS. Culture media were analyzed for estrogen and progesterone content by appropriate radioimmunoassay. Trypsinization of ovarian follicles resulted in the formation of the cell suspension which contained a mixture of G and T cells. The follicular cells attached to glass and grew well during culture period. E-2 was the main steroid hormone secreted by cultivated cells. Estrogen secretion increased by 415.52 +/- 25 pg/mL at the first 3 days up to 530.25 +/- 55.8 pg/ml on day 5 and it didn't change significantly until the end of the experiment. 17 alpha-OHP secretion, however, gradually increased from 23.84 +/- 8.2 pg/ml at the beginning of culture up to 35.76 +/- 5.4 pg/ml at the end of cultivating. As the result of the present study the fish follicular cells grown in tissue culture were steroidogenically active cells as expressed by the secretion of E-2 and P-4 and The E-2 was a dominant hormone secreted by isolated follicular cells, which it correlated closely with vitellogenes stage