Effects of dbcAMP on progesterone synthesis by cultured goat luteal cell subpopulations isolated from early and late luteal stage corpora lutea

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 3β-hydroxysteroid dehydrogenase (3β-HSD). Cells stained positively for 3β-HSD activity (5 x 104 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.(AU)

Effects of dbcAMP on progesterone synthesis by cultured goat luteal cell subpopulations isolated from early and late luteal stage corpora lutea

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 3β-hydroxysteroid dehydrogenase (3β-HSD). Cells stained positively for 3β-HSD activity (5 x 104 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.

Effects of cholesterol, FSH and LH on steroidogenic activity of cat granulosa cells cultured in vitro

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 Dulbeccos Modified Eagles 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 μg/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.(AU)

Effects of cholesterol, FSH and LH on steroidogenic activity of cat granulosa cells cultured in vitro

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 μg/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.