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Öğe Effects of Cholesterol on Progesterone Production by Goat Luteal Cell Subpopulations at Two Different Stages of the Luteal Phase(Wiley-Blackwell Publishing, Inc, 2010) Arikan, S.; Kalender, H.; Simsek, O.Contents The aim of the present study was to evaluate the effects of cholesterol on progesterone production during long-term culturing of luteal cell subpopulations at early and late luteal stages of the goat corpora lutea. Corpora lutea were collected from Angora goats on days 5 and 15 of the oestrous cycle. Luteal cells were isolated by collagenase digestion. The cells were separated into two distinct subpopulations by Percoll density-gradient centrifugation. Both subpopulations of luteal cells staining positively for 3 beta-HSD activities (5 x 104 cell/well) were cultured with or without 22(R)-hydroxycholesterol (22R-HC) in serum-free culture medium for periods of up to 7 days. Cells were incubated with serum (10%) for the first 18 h of incubation followed by serum-free medium. Cell treatment (10 and 20 mu g/ml) was performed on days 1, 3 and 5. Treatment of cells with both concentrations of 22R-HC resulted in significant (p < 0.01) and dose-dependent stimulation (p > 0.05) on progesterone production in both fractions of cells throughout 7 days of incubation. Treatment of the cells with cholesterol resulted in 2.5- and 9.0-fold increases in progesterone accumulation on day 3 of incubation. Steroid production was maintained throughout the incubations when cells are incubated in serum-free media treated with cholesterol and ITS premix. Cells collected from higher density of percoll layers produced 2.82 and 2.32 times more progesterone, in comparison to the lover density percoll layer, on days 5 and 15 of the oestrous cycle in untreated cell groups, respectively. Progesterone accumulation was decreased as incubation time advanced in all groups of untreated cells. These results demonstrated that goat luteal cell subpopulations secrete substantial amounts of progesterone in response to cholesterol treatment at least for 7 days, and cholesterol is required as progesterone precursor for maintaining a high-level steroidogenesis during long-life culturing of both cell subpopulations.Öğe Effects of cholesterol, FSH and LH on steroidogenic activity in cat granulosa cell culture(Wiley-Blackwell, 2014) Simsek, O.; Arikan, S.…Öğe Effects of cholesterol, FSH and LH on steroidogenic activity of cat granulosa cells cultured in vitro(Brazilian Coll Animal Reproduction, 2015) Simsek, O.; Arikan, S.The aim of this study was to examine the effects of 22R-hydroxycholesterol (22R-HC), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on estradiol and progesterone production by cat granulosa cells. Granulosa cells from follicles were collected and cultured for up to 5 days in 24 well plates containing Dulbecco's Modified Eagle's Medium (DMEM)/HAM F-12 supplemented with 10(-7) M androstenedione, 0.1% ITS premix and 0.1% bovine serum albumin, in the presence or absence of 22R-HC (10 mu g/ml), FSH or LH (10, 100 ng/ml each) on first and third day. Additionally, 5% fetal calf serum was added into the culture medium for the first 24 h. Treatment of cells with 22R-HC resulted in an increase (P < 0.05) in progesterone and estradiol production on days 3 and 5 of the culture. Incubation of cells with FSH (10 and 100 ng/ml) resulted in significant stimulations of progesterone (P < 0.001) whilst incubation had no effect on estradiol production. None of the LH doses (10 and 100 ng/ml) had any effect on progesterone production by granulosa cells during the culture time. With the inclusion of 22R-HC into the culture system, progesterone synthesis was enhanced (P < 0.001) in the presence of all FSH doses.Öğe Effects of dbcAMP on progesterone synthesis by cultured goat luteal cell subpopulations isolated from early and late luteal stage corpora lutea(Brazilian Coll Animal Reproduction, 2016) Arikan, S.; Kalender, H.; Simsek, O.This research aimed to investigate the effects of dbcAMP on steroid accumulation by culturing two distinct luteal cell subpopulations isolated from early and late luteal stage corpora lutea. Cells were isolated from corpora lutea collected from eight Angora goats on either the 5th or 15th days of their estrous cycles. Cell isolation was performed by enzymatic digestion using collagenase and DNase. Isolated cells were separated into two distinct subpopulations enriched with small and large luteal cells by percoll density-gradient centrifugation. Isolated cells were stained in order to detect 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). Cells stained positively for 3 beta-HSD activity (5 x 10(4) cell/well) were incubated with dbcAMP in the absence or presence of 22(R)-hydroxycholesterol (22R-HC) for periods of up to 7 days. Large luteal cell enriched subpopulations produced more basal progesterone (P < 0.05) than did the small luteal cell enriched subpopulations. Treatment of cells with 22R-HC alone induced 4.00 to 11.60 times increase in steroid synthesis depending on type of cells incubated, luteal age and days of incubation. Incubation of cells with 1 mM dbcAMP in the absence or presence of 22R-HC induced in a significant increase (P < 0.01) in steroid accumulation in all treated groups. In contrast, when cells are treated with low dose dbcAMP (0.1 mM), treatment induced stimulation failed to reach significant level in most treated groups. In conclusion, although treatment of goat luteal cells with dbcAMP induces an increase in steroid accumulation, a high dose is necessary to reach significant levels. Stimulatory effect of dbcAMP on steroidogenesis maintains during long life culturing.
