Effect of beta-endorphin on cAMP and progesterone accumulation in rat luteal cells.

The aim of this study is to investigate the effect of beta-endorphin on cAMP and progesterone accumulation in rat luteal cells. Luteal cells of 4-day-old corpora lutea were cultured for 3 h in the absence or presence of 0.001 or 0.01 IU/ml hCG, and cAMP, progesterone and beta-endorphin levels in the medium were measured by RIA. hCG stimulated the production of cAMP, progesterone and beta-endorphin. In the presence of hCG, treatment with islet-activating protein (IAP) led to overall augmentation of cAMP and progesterone accumulation in comparison with untreated controls. In the absence or presence of low doses of hCG (0.001 IU/ml), beta-endorphin did not affect progesterone production, but inhibited cAMP accumulation. This inhibitory effect was abolished by pre-treatment with IAP. In the presence of high doses of hCG (0.01 IU/ml), however, beta-endorphin stimulated progesterone production without a corresponding increase in cAMP. This stimulatory effect was also abolished by IAP-treatment. These results suggest that luteal cells produce and release beta-endorphin that affects cAMP and progesterone production via IAP-sensitive mechanisms.

Antigonadotropic activity of hop extract.

Biological activity of water soluble fractions F-1 and F-2, which were extracted from hop, was studied and its action mechanism was speculated using the immature female SD rats. Administration of the substance significantly inhibited the effects of pregnant mare serum gonadotropin (PMSG) on 22-day-old female rats. Thus, PMSG-induced increases in ovarian weight, estrogen secretion, number of ovulated egg, progesterone production, uterine thymidine kinase activity, and plasma LH level were suppressed significantly. Furthermore, addition of the substance to incubated ovarian cells of the second day after PMSG injection resulted in suppression of FSH-induced estradiol secretion in vitro, probably via cAMP-dependent mechanism. But addition of the substance to incubated pituitary cells from ovariectomized rats did not change in LH secretion.

Effect of beta-endorphin on cAMP accumulation in rat luteal cells.

The effect of beta-endorphin on cAMP levels in 4-day-old rat luteal cells was investigated. In both the presence and absence of low doses of human chorionic gonadotropin (hCG, 0.001 IU/ml), beta-endorphin inhibited cAMP accumulation, whereas in the presence of high doses of hCG (0.01 IU/ml) it did not. This inhibitory effect was abolished by pre-treatment with islet-activating protein (IAP). Moreover, treatment with IAP resulted in an overall enhancement of hCG-stimulated cAMP accumulation when compared with untreated controls. These results suggest that beta-endorphin suppresses adenylate cyclase activity via Gi, which may be coupled to the LH receptor.

Adenosine receptor-mediated effects on adenylate cyclase activity in rat luteal tissue: a putative local regulatory role of adenosine in the corpus luteum.

The influence of adenosine analogs on adenylate cyclase activity was investigated in membrane preparations of luteinized ovaries and in cell homogenates of isolated luteal cells. The adenosine receptor agonist 5'-(N-ethyl)-carboxamido adenosine (NECA) dose-dependently stimulated adenylate cyclase activity in membrane preparations of 5-day-old luteinized ovaries with an apparent EC50 of 0.58 microM. The other adenosine analogs tested were less potent in stimulating the adenylate cyclase activity with the following rank order of potency: NECA less than 2-chloro-adenosine greater than N6-(R-phenyl-isopropyl)- adenosine less than N6 -(S-phenyl-isopropyl)-adenosine. In homogenates of isolated cells from 5-day-old corpora lutea, NECA stimulated adenylate cyclase with the same EC50 as in the membranes from luteinized ovaries. The effect of NECA was antagonized by the adenosine receptor antagonist 8-phenyltheophylline. In incubated luteal cells of both 2- and 5- to 6-day-old luteinized ovaries, NEC stimulated cyclic adenosine 3', 5'-monophosphate (cAMP) accumulation and markedly potentiated luteinizing hormone-stimulated cAMP accumulation. Progesterone synthesis was also stimulated by NECA in incubated cells. The study demonstrates effects of adenosine analogs on adenylate cyclase and cAMP accumulation that fulfill the criteria for adenosine A2 receptor-mediated effects in luteal cells and membranes. These data suggest that adenosine may have a local regulatory action in luteal tissue through adenosine receptor activation.

[Effects of ACTH or hCG on corticosterone and progesterone secretion from adrenal glands of the rats at various ages].

Effects of ACTH or hCG on the secretion of corticosterone (CB) and progesterone (PRG) from adrenal glands of the female rats at various ages were studied. In in vitro experiment, adrenal gland cells were isolated from fetal, neonatal, immature and adult rats by the method described by us before and were incubated in MEM. We chose to designate the hormones in the medium as hormone secretion. In in vivo experiment, hormones in the serum were determined. In vitro experiment, in which ACTH was added to the medium, indicated that CB was low in the media containing the cells from fetal and neonatal glands compared to those from immature and adult. PRG was elevated by ACTH addition at all of the ages. hCG addition had no effects on both CB and PRG in the media of immature and adult rats. In vivo experiment, in which ACTH was injected s.c., indicated that the response of serum PRG was slightly earlier than the CB response and peaked with a subsequent decline in immature rats. The results suggest that adrenal gland responds to ACTH from fetal age and secrets PRG, but little of CB in fetal age.

Priming procedure and cell isolation for study on luteal cell response to peptide hormone in the rat.

The objective of this study was to develop a method of isolating luteal cells from the ovaries of immature rats pretreated with pregnant mare serum gonadotrophin (PMSG). After the ovaries were digested by collagenase and trypsin, the corpora lutea were obtained from the tissues, gently pressed in a test tube, and then placed on a sucrose density gradient. The two bands that appeared in the tube after centrifugation were designated S1 (top band) and S2 (bottom band). Progesterone and 20 alpha-dihydroprogesterone (20 alpha-DHP) secreted by the isolated cells during short-term incubation were measured by radioimmunoassay (RIA). A larger amount of progesterone, i.e., 60 to 260 ng/10(5) cells, was secreted by S1 cells than by S2 cells during the 18-h incubation. These results suggest that this simple procedure for isolation of luteal cells may provide a suitable model for in vitro studies of the luteal function.

[The extraction of biologically active substances from hop].

Substances which suppressed the effect of gonadotropin in the rat were obtained from hop. These biologically active substances were fractionated from the hop cone, from which lipophilic components (total resins) were removed with acetone. They were water soluble, 70,000-80,000 in molecular weight and were composed mainly of neutral sugars and uronic acid. Administration of the substances to immature rats primed with pregnant mare's serum gonadotropin (PMS) resulted in the following: a significant decrease of ovarian weight gain, whereas there was no change in uterine weight gain, and significant reductions of serum LH and progesterone from cultured luteal rat cells.

Extraction of the hormonal substance from hop.

Substances that suppressed a gain in the weight of pregnant mare's serum gonadotropin (PMSG)-primed immature rat ovaries, were obtained from dried powder of the hop cone. The substances were designated F1a-I and F1a-II. In immature rats at 4 days after PMSG priming, ovarian weights decreased by 58.0 +/- 3.7 and 66.9 +/- 6.6% the control by injections with 4 mg each of F1a-I and F1a-II, respectively. Apparent M1s of the partially purified fractions were nearly 80000 (F1a-I) and 66000-80000 (F1a-II). They were water-soluble. Acidic and neutral sugars were detected in the hydrolysate of the substance.

[Preparation of progesterone secreting cells from rat ovaries by a density gradient].

Isolation of progesterone secreting cells from the ovaries of immature rats pretreated with pregnant mare serum gonadotropin (PMS) was conducted by a simple procedure which combined the collagenase digestion with a density gradient method. After digestion of the ovarian tissue slices by the enzyme, the residue was gently pressed and placed on a sucrose density gradient. Three bands appearing in the tube after centrifugation were designated as S-1, S-2 and S-3 from the top to the bottom, respectively. The S-1 cells from the ovaries at 6 days after PMS secreted the greatest amount progesterone, i.e. approximately 430 ng per 10(5) cells during the 18th incubation. Progesterone secretion from the S-2 cells was less than 48% of that from the S-1 cells. A physiological interrelation between the S-1 and S-2 cells remains unexplained by the present experiment. The luteal cells were yellow, spheroidal and 15 to 40 mus in diameter. Many vesicle-like particles were found on the cell surfaces. Progesterone secretion from the prepared cells was stimulated significantly by hCG in vitro. This result indicates that progesterone secreting cells isolated by the collagenase-sucrose density gradient preserve their native function as luteal cells. The procedure for preparation of luteal cells in the present report may provide a suitable model for in vitro studies on the corpus luteum.