Elikplimi K. Asem

Progesterone production in granulosa cells of the domestic fowl: effects of incubation media, pH, cell density and some other factors

Abstract The objective of this study was to determine the optimal conditions for the short term incubation of chicken granulosa cells. Compared to mechanically-dispersed cells, collagenase-treatment yielded granulosa cells of greater viability and responsiveness to ovine LH (oLH) stimulation. The rate of progesterone secretion by enzyme-dispersed chicken granulosa cells was subsequently compared in five different culture media during incubation periods of up to 24 hr. Under room air as the gas phase, Medium 199 (M199) containing Hanks salts and Hams F-12 medium (F-12) were the most effective, whereas Krebs-Ringer bicarbonate-buffered glucose solution (KRBG) and Dulbeccos medium were the poorest. When pH and the ionic strength of KRBG was maintained by continuous gassing with O 2 :CO 2 (95:5), progesterone production was similar to that obtained with Hepes-buffered M199. The pH optimum was found to be 7.4, although within the range of 6.6–8.5 granulosa cells remained responsive to LH stimulation. The optimal cell density was observed to be 1 × 10 4 to 5 × 10 5 /ml. Although time course studies showed that both basal and stimulated progesterone production peaked by about 12 hr of incubation regardless of the media composition, the amounts released were significantly greater in M199 and F-12 during more prolonged (up to 24 hr) incubations. Glucose, a key medium ingredient for hormone-stimulated steroidogenesis, could be replace by pyruvate. On the other hand, lactate was inhibitory. It is concluded that the mature ovarian follicles of the domestic fowl are an excellent source of pure granulosa cells that can be obtained in high yield after a brief treatment with collagenase. These cells remain viable up to 24 hr and continue to produce large amounts of progesterone in response to LH when incubated in an appropriate medium and at optimal cell density.

Synergistic effect of gonadotropin releasing hormone on LH-stimulated progesterone production in granulosa cells of the domestic fowl (Gallus domesticus) ☆

Abstract A direct gonadal action of gonadotropin releasing hormone (GnRH) has been demonstrated in several mammalian species. The effect of mammalian GnRH on steroidogenesis in avian granulosa cells (GC) was investigated. When such cells from mature follicles were preincubated with GnRH, the subsequent production of progesterone in response to oLH was significantly enhanced. In GC from medium or small follicles (F 3 and F 5 ), no effect of GnRH was observed. Similarly, when steroidogenesis was stimulated with dibutyryl cyclic AMP preincubation with GnRH failed to alter progesterone responses. On the other hand, oFSH-stimulated steroidogenesis in GC from small follicles (F 4 and F 5 ) was moderately but consistently and significantly attenuated by GnRH. Further studies with homologous GnRH may be necessary for the elucidation of the possible ovarian action of this hormone.

Comparison of turkey luteinizing hormone (LH)- and ovine LH-induced progesterone production in granulosa cells of the turkey (Meleagris gallopavo) and of the domestic fowl (Gallus domesticus)

Progesterone production was compared in short-term cultures of turkey and fowl granulosa cells isolated from preovulatory follicles of different maturities. In both species cells from the most mature follicle produced the greatest amount of progesterone under stimulated conditions and in response to ovine or turkey LH, the latter being significantly more potent. Whereas basal production of progesterone was higher in turkey cells, the response to LH or cAMP stimulation was significantly greater in chicken granulosa cells both in absolute terms and particularly in relation to unstimulated levels. These species differences may reflect difference in the intensity of reproductive activity of the turkey and the domestic fowl selected for egg production.

Steroidogenesis in ovarian cells of the Japanese quail (Coturnix coturnix japonica)

The influence of follicular maturation on steroidogenesis and steroid metabolism by isolated Japanese quail granulosa and theca cells was examined. When stimulated with LH, granulosa cells of the largest follicle (F1) responded with a sixfold increase over unstimulated progesterone levels, whereas progesterone production in cells of F3 less than doubled even when maximally stimulated. Forskolin stimulated progesterone synthesis in both F1 and F3 granulosa cells, but its effect was less pronounced than that of LH. Furthermore, F1 cells metabolized 25-hydroxycholesterol to a greater extent than did F3 cells. There was no appreciable metabolism of [3H]progesterone by granulosa cells. Theca cells from the smaller follicles (F3-F5) responded to LH stimulation with greater estrogen and androstenedione production than theca cells from F1. [3H]Progesterone was metabolized mainly to androstenedione in theca cells. Thus, the overall pattern of in vitro steroidogenesis in quail granulosa cells is similar to that described for the chicken and turkey even though the quantitative differences in the steroidogenic capacity between developing and mature follicles are more striking in the quail. Furthermore, although the LH-stimulated androstenedione and estrogen production appears similar in developing quail and chicken theca cells, the profile of [3H]progesterone metabolism is different in quail theca cells from that found previously in chicken theca cells.

Trifluoperazine inhibits progesterone and cyclic AMP production in granulosa cells of the hen (Gallus domesticus)

Unstimulated (basal) as well as luteinizing hormone (LH)-promoted progesterone production in collagenase-dispersed hen granulosa cells was inhibited in a dose-related manner by two phenothiazines, trifluoperazine (TFP) and chlorpromazine (CP), both of which are known calmodulin antagonists. Using TFP, the more potent antagonist of the two, it was found that LH-stimulated cyclic AMP production was also suppressed. Moreover, TFP attenuated the steroidogenic effects of both 8-bromo-cyclic AMP and isobutylmethylxanthine but had no effect on the conversion of pregnenolone to progesterone. The inhibitory effects of TFP on steroidogenesis were reversible. It is concluded that phenothiazines inhibit steroidogenesis in ovarian granulosa cells by acting at multiple sites both proximal and distal to cyclic AMP generation without influencing the enzyme complex responsible for the conversion of pregnenolone to progesterone. The results are discussed in relation to calmodulin- and non-calmodulin-mediated actions of phenothiazines.

In vitro effect of prostaglandins on the accumulation of cyclic AMP in the avian oviduct.

The effects of prostaglandins E1, E2, and F2 alpha (PGE1, PGE2, and PGF2 alpha, respectively) on cyclic AMP production in tissue samples from the uterus (U), uterovaginal sphincter (UVS), and vagina (V) of regularly laying hens as well as the effects of PGE2 and forskolin on U and V of Japanese quail were studied. Both PGE1 and PGE2 enhanced cyclic AMP production in the hen oviductal segments, particularly in the V in a dose-related manner, PGE1 being the more effective agonist. PGF2 alpha had no significant effect. Quail U appeared to be less responsive to PGE2 which caused a 40% increase in cyclic AMP levels at the highest concentration tested (28 microM), whereas forskolin at 10 microM increased cyclic AMP production in quail U and V 7- and 20-fold, respectively. It is suggested that the primary function of PGE2 and PGE1 is to relax the terminal portion of the oviduct, allowing the expulsion of the egg under the contractile influence of PGF2 alpha and other oxytocic factors.

Trifluoperazine inhibits ovarian mitochondrial side-chain cleavage enzyme activity in the absence of calcium.

In a previous report we described the inhibitory effect of trifluoperazine (TFP) on steroidogenesis in avian granulosa cells. To clarify the possible site of action of TFP we measured the cholesterol side-chain cleavage enzyme (CSCC) activity in a mitochondrial preparation of granulosa cells isolated from mature and developing ovarian follicles. Using a calcium free medium, TFP inhibited CSCC in a dose related manner with an IC50 of 50 microM. Kinetic parameters (apparent Km and Vmax) obtained in the presence of TFP are indicative of uncompetitive inhibition of CSCC. Moreover, enzyme activity increased during follicular maturation while the efficacy of TFP was similar in both young and mature follicles. Because the inhibitory effects of TFP were manifest in medium from which calcium was omitted, it is suggested that the drug acts independently of the calcium-calmodulin system to suppress CSCC activity.

Influence of kaurenol on basal, LH- and forskolin-stimulated progesterone and cAMP production in avian granulosa cells

The effects of kaurenol, a diterpene alcohol, were evaluated on progesterone and cyclic AMP (cAMP) production in freshly dispersed avian granulosa cells. Kaurenol (50 microM) alone caused a fourfold increase in progesterone synthesis without a measurable influence on cAMP levels. When granulosa cells were challenged with near-maximally stimulating concentrations of LH (50 ng/ml) or forskolin (10 microM), kaurenol (10-100 microM) dose-dependently suppressed steroidogenesis. Similarly, cAMP production in response to LH and forskolin stimulation was also inhibited. When progesterone synthesis was stimulated by the addition of pregnenolone or 25-hydroxycholesterol substrates to the culture medium, the typical dose response to the latter precursor, but not to pregnenolone, was abolished by kaurenol. Whereas the mechanism of kaurenols stimulatory effect on basal steroidogenesis remains unknown, it is suggested that its inhibitory action on LH- and forskolin-promoted progesterone production may be due to the inhibition of the adenylate cyclase cAMP effector system as well as to the impairment of the action of the mitochondrial cholesterol side chain cleavage enzyme system.