Effects of luteinizing hormone-releasing hormone agonist and calcium upon adenylyl cyclase activity of human corpus luteum membranes

We have investigated the ability of the agonist analog of luteinizing hormone-releasing hormone (LH-RH), D-Trp6-LH-RH (LH-RHa), and of CaCl2 to inhibit directly gonadotropin stimulation of adenylyl cyclase in a cell-free system prepared from human corpus luteum. In the presence of a submaximally effective concentration of hCG, addition of 10(-5)M final concentration of LH-RHa did not alter the gonadotropin-stimulated enzyme activity, nor did LH-RHa alone show any effect upon basal levels of the enzyme. The failure to inhibit adenylyl cyclase would indicate that the LH-RHa does not affect gonadotropin receptor binding or cAMP synthesis and/or degradation in this membrane system, suggesting that the luteolytic effects of LH-RH are unlikely to involve a direct antigonadotropic activity at the level of the human corpus luteum. In great contrast to LH-RHa, addition of CaCl2 resulted in a dose-dependent inhibition of hCG-stimulable adenylyl cyclase. Thus, in the presence of either a maximally or submaximally effective concentration of hCG, inhibition was significant at 0.5 mM CaCl2 added in excess of ATP (2 mM) and EDTA (1 mM), being about 90% upon addition of 2.5 mM CaCl2. We also found that calcium reduced enzyme stimulation by forskolin and the GTP analog, guanyl 5'-yl imidodiphosphate [GMP-P(NH)P] in a dose-related manner and that activation by NaF was less sensitive to inhibition by calcium. Accordingly, at 2.5 mM CaCl2, guanyl nucleotide and forskolin stimulations were inhibited 96% and 86%, respectively, while NaF stimulation was reduced by 40%. Because previous studies have shown that calcium does not impair gonadotropin binding activity, the calcium-dependent inhibition of gonadotropin responsiveness reported here would imply an alteration in the functional coupling of the components of the luteal adenylyl cyclase system. These data suggest that calcium may play a role in the regulation of gonadotropin action in the human corpus luteum.     

The effect of porcine luteinizing hormone in the synchronization of ovulation and corpus luteum development in nonlactating cows

The objective of this study was to determine the effects of different doses of porcine luteinizing hormone (pLH) versus 100 μg gonadotropin-releasing hormone (GnRH) on ovulatory response (during diestrus and proestrus) and corpus luteum (CL) development in nonlactating cows. In Experiment 1, 75 cows received an intravaginal insert containing 1.9 g progesterone (P4) for 10 d to synchronize estrus (Day 0), with prostaglandin F_2α (PGF) at insert removal. On Day 5, all follicles ≥8 mm were ablated, and on Day 12, cows received 8, 12.5, or 25 mg pLH or 100 μg GnRH. Mean (±SEM) plasma P4 concentrations on Day 12 did not differ among treatments (5.6 ± 0.2 ng/mL). Mean plasma LH concentration was greatest (P < 0.01) in cows given 25 mg pLH (4.3 ± 0.4 ng/mL). The ovulatory response to 25 mg pLH (84%) or 100 μg GnRH (72%) was greater (P < 0.05) than that to 8 mg pLH (32%), but not different from that of 12.5 mg pLH (58%). In Experiment 2, 68 cows were given two injections of PGF 10 d apart to synchronize estrus (Day 0). On Day 7, cows received PGF, and, 36 h later, pLH or GnRH (as in Experiment 1). The interval from treatment to ovulation was most variable in cows given 8 mg pLH; only 65% of these cows ovulated during the initial 27 h versus 88% of cows given 25 mg pLH (P < 0.05). Cows given 25 mg pLH or 100 μg GnRH had larger CL area and greater plasma P4 concentrations (P < 0.05) than that of those given 8 mg pLH. In summary, diestrous cows given 25 mg pLH had the greatest plasma luteinizing hormone concentrations, but ovulatory response did not differ from that of those given 100 μg GnRH. Proestrous cows given 25 mg pLH or 100 μg GnRH had greater CL area and P4 concentrations than that of those given 8 mg pLH.     

Gel-filtration analysis of soluble adenylate cyclase from bovine corpus luteum.

1. Sepharose 6B gel-filtration analysis of soluble adenylate cyclase from bovine corpus luteum is described. Both zonal and frontal techniques of analysis were used. 2. Under conditions of zonal analysis recoveries of activity were low. It was concluded that dissociation of two or more components of the adenylate cyclase complex was occurring on the column and that the maintenance of the complex was essential for the high-activity state of the catalytic unit. Two peaks of adenylate cyclase activity, of approximate mol. wts. 45,000 and 160,000 were detected. 3. The theory of frontal analysis (or steady-state gel filtration), applied to the study of the interacting components of the adenylate cyclase complex is discussed, and activity profiles are predicted. Activity profiles obtained experimentally be frontal analysis compared well with the theoretically predicted profile and provide evidence that dissociation of a high-activity complex, with concomitant loss of activity, does occur. Recoveries of activity under conditions of frontal analysis were higher than with zonal analysis. 4. The effects of concentration and removal of detergent on the activity of the soluble enzyme are discussed.     

Subcellular localization and partial characterization of bovine corpus luteum adenylate cyclase.

The subcellular localization of adenylate cyclase (ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1) in bovine corpus luteum was studied using isotonic and hypotonic homogenization and fractionation conditions. All fractions prepared were assayed for adenylate cyclase, marker enzymes and DNA. Only plasma membrane marker enzyme, 5'-nucleotidase paralleled the distribution of adenylate cyclase under both isotonic and hypotonic conditions (conditionsoth isotonic and hypotonic conditions (coefficient of correlation = 0.95). Two main fractions prepared under hypotonic conditions were subfractionated by discontinuous sucrose gradient centrifugation. The highest amount of adenylate cyclase was found in a fraction having a density approximately equal to 1.13 g/cm3. The specific activity of this fraction was 4--6 times higher than that of the homogenate. The electron microscopic study of this fraction revealed the presence of a single type of particulate material consisting of small vesicles exhibiting a typical unit membrane structure. It is concluded that this adenylate cyclase is primarily localized in the plasma membranes. Basal adenylate cyclase activity of plasma membranes was stimulated 2--3 times by luteinizing hormone (10 mug/ml), 3--4 times by prostaglandin E2 (10 mug/ml), 4--6 times by NaF (0.01 M) and two times by methanol (0.2%).     

Effect of luteinizing hormone and human chorionic gonadotropin on cell populations in the ovine corpus luteum

Two experiments were conducted to examine the effect of treatment with human chorionic gonadotropin (hCG) or ovine luteinizing hormone (LH) on the number and size distribution of steroidogenic luteal cells. In Experiment I, 27 ewes were assigned to one of three groups: 1) hCG (300 IU, i.v.) administered on Days 5 and 7.5 of the estrous cycle (Day 0 = Estrus); 2) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the cycle; 3) saline (i.v.) administered as in the LH treatment group. Blood samples were drawn daily from the jugular vein for quantification of progesterone. On Day 10, corpora lutea were collected, decapsulated, weighed, and dissociated into single cell suspensions. Cells were fixed, stained for 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activity, and the size distribution of 3 beta HSD-positive cells was determined. Treatment with hCG, but not LH, increased (p less than 0.05) concentrations of progesterone in serum and the weight of corpora lutea. Treatment with either hCG of LH increased the proportion of cells greater than 22 micron in diameter and decreased the proportion of cells less than or equal to 22 micron (p less than 0.01). The ratio of small to large luteal cells decreased after treatment with either hCG or LH (p less than 0.05). In Experiment II, 9 ewes were assigned to one of two groups: 1) LH (120 micrograms, i.v.) administered at 6-h intervals from Days 5 to 10 of the estrous cycle, and 2) saline (i.v.) administered as in the LH treatment group.(ABSTRACT TRUNCATED AT 250 WORDS)     

FSH receptors in the bovine corpus luteum

Binding of follicle stimulating hormone (FSH) to a crude membrane fraction of bovine corpus luteum (CL) has been detected. This binding meets the usual criteria for a receptor based on specificity, time course of reaction and association constant (Ka = 8.5 x 10(10)M(-1)). Physiological studies with CL removed from heifers at specific times after estrus indicate that day-6 CL had the highest FSH binding. However, a correlation with physiological function was not obvious since some functional mid-cycle CL were high in progesterone and luteinizing hormone (LH) receptor but had nondetectable FSH receptor. Conversely, some late-cycle CL had low progesterone and LH receptor but significant quantities of FSH receptor.     

Intercellular communication in the bovine corpus luteum

There is a growing body of evidence that intercellular communication is important in the regulation of luteal function. Although the nature of the interactions between small and large luteal cells are not yet clear, it seems likely that they do exist. Many of the substances to which luteal cells respond, such as prostaglandins, growth factors, oxytocin and progesterone, are produced locally. These substances may act as paracrine factors to modulate the response of luteal cells to hormonal signals. Endothelial cells also produce factors that can modify steroidogenesis, and luteal cell-stimulation of endothelial cell proliferation is necessary for the extensive angiogenesis that occurs during luteinization Finally, bidirectional intercellular communication likely occurs between luteal cells and resident immune cells. Immune cells produce cytokines that can modify progesterone and prostaglandin synthesis by luteal cells. Cytokines may also have direct cytotoxic effects on luteal cells, and dead cells are then phagocytized by resident macrophages. Also, factors secreted by luteal cells can serve as chemoattractants for immune cells, and can enhance or suppress immune cell functions. There is little doubt that intercellular communication within the corpus luteum is very complex. One must remember, however, that nearly all evidence collected thus far is based on in vitro studies. Eventually, technology will allow for study of these interactions in vivo, and may lead to new methods for control of luteal function.