The role of Ca2+ and calmodulin in the regulation of atrial natriuretic peptide-stimulated guanosine 3',5'-cyclic monophosphate accumulation by isolated mouse Leydig cells.

We have investigated the role of Ca2+ and calmodulin in the stimulation of cGMP formation by mouse Leydig cells in response to rat atriopeptin-II (rAP-II). Removal of extracellular Ca2+ had no influence on the levels of cGMP accumulated by the cells stimulated with rAP-II. The amounts of testosterone produced by unstimulated and rAP-II-stimulated cells were, however, reduced by 50% in the absence of Ca2+ from the incubation medium. Addition of ionomycin to the Leydig cells led to a dose-related inhibition of rAP-II-stimulated cGMP formation, but the basal cGMP level was not affected. These experiments were carried out in the presence of a phosphodiesterase inhibitor. The inhibitory effect of ionomycin was absolutely dependent upon the presence of Ca2+ in the medium. The guanylate cyclase activity required the presence of a cation, and Mn2+, Mg2+, or Ca2+ could function as the required cation. There was no direct inhibition of the cyclase activity by Ca2+ up to as high a concentration as 8 mM. Furthermore, three structurally unrelated calmodulin antagonists, W7, trifluoperazine, and calmidazolium, but not W5, caused a dose-related inhibition of rAP-II-stimulated cGMP accumulation by the cells. The inhibitory effect of calmodulin antagonists was not exerted directly at the level of guanylate cyclase activity, since the particulate enzyme was not inhibited by any of these drugs. We conclude, therefore, that extracellular Ca2+ is not essential for rAP-II-mediated stimulation of cGMP formation by mouse Leydig cells, at least under the short term incubation conditions used. An excessive ionophoretic influx of Ca2+ into the cells impairs the ability of rAP-II to stimulate cGMP formation. Therefore, it appears that a finely regulated level of intracellular Ca2+ is required for optimal activation of atrial natriuretic peptide-responsive guanylate cyclase in mouse Leydig cells, and calmodulin plays an important role in this process.

Phorbol ester stimulates progesterone production by isolated bovine luteal cells.

The tumor promoter, phorbol 12-myristate-13-acetate (PMA), is known to modulate the response of several steroidogenic tissues presumably by activating a Ca++- and phospholipid-dependent protein kinase (protein kinase C). The presence of this kinase has been demonstrated in bovine corpus luteum, although its role in steroidogenesis by these cells is unknown. We report here the effects of PMA on progesterone production by the enzymically dispersed bovine luteal cells in vitro. PMA (1-50 nM) produced a dose- and time-related increase in progesterone production by the luteal cells. The maximum stimulation was achieved with 10 nM PMA. Higher concentrations of PMA led to a decline of steroidogenesis close to the basal level. A nonpromoting derivative, 4 alpha-phorbol 12,13-didecanoate had no effect. The PMA-induced stimulation of progesterone production was not associated with a change in the cAMP level. PMA added together with suboptimal doses of human CG, 8Br-cAMP, cholera toxin, or forskolin significantly increased the amount of progesterone produced. PMA as well as human CG-induced steroidogenesis was sensitive to cycloheximide inhibition. The conversion of exogenous pregnenolone or 25-hydroxycholesterol to progesterone was not altered by PMA. We conclude that PMA at nanomolar concentrations is able to stimulate progesterone production by bovine luteal cells and that the site of action of PMA is distal to the formation of cAMP but before the formation of pregnenolone. The observed effects of PMA in luteal cells are probably linked to its ability to activate protein kinase C, since a diacylglycerol could mimic the steroidogenic action of PMA.

The relationship between prorenin levels in follicular fluid and follicular atresia in bovine ovaries.

Bovine follicles having a higher concentration of progesterone than estradiol in the follicular fluid can be considered as atretic. Since we observed previously that there was an inverse relationship between the follicular fluid estradiol to progesterone (E/P) ratio and the prorenin level, we have proposed that a high prorenin level may be associated with follicular atresia. The aim of the present study was to corroborate this hypothesis by including additional indices to distinguish unambiguously between atretic and nonatretic follicles and to compare the prorenin levels in these two groups of follicles. The present study included examination of more than 200 follicles in the follicular fluid of which we have measured steroid and prorenin levels. The results obtained show a highly significant negative correlation between the prorenin level on the one hand and the E/P ratio, estrogen to total androgen ratio, or estradiol concentration on the other hand. As a further criterion for atresia, we have examined the histological characteristics of the follicles by light and electron microscopy and have found that 90% of histologically characterized atretic follicles had an E/P ratio less than 1 and an average prorenin level four to five times higher than nonatretic follicles. Finally, when we determined the FSH-stimulated cAMP response and the aromatase activity, in terms of the ability to convert exogenous androgen to estrogen in granulosa cells isolated from individual follicles, we observed a markedly higher prorenin level in the fluid of follicles whose granulosa cells responded poorly to FSH and showed a low aromatase activity, compared to follicles whose granulosa cells responded strongly to FSH and contained high aromatase activity. In summary, follicles that were classified as atretic on the basis of a number of biochemical and histological parameters contained significantly higher prorenin levels in their follicular fluid than nonatretic ones. Thus, a high follicular fluid prorenin level is a valid indicator for follicular atresia in bovine ovaries. However, the reason for this increase in follicular fluid prorenin level and whether this increase is a cause or a consequence of atresia remains to be determined.

A low molecular weight substance obtained from serum which has luteinizing hormone like activity ("mini LH").

A low molecular weight substance of 1000-2000 daltons has been isolated in dialysates of normal human serum which, on testing in a radio-immunoassay, a radioligand receptorassay and an in-vitro bioassay exhibited LH activity comparable to the range of LH activity found in normal serum. The substance was heat labile. Its reactivity in a RIA suggests peptide nature. No "mini LH" activity could be derived from LH-free serum samples of hypophysectomized patients.

A direct immunoradiometric assay for human plasma prorenin: concentrations in cycling women and in women taking oral contraceptives.

Two synthetic penta deca peptides corresponding to the N-terminal portion (amino acid sequence 1-15) and the C-terminal portion (sequence 32-46) of the pro moiety of the human prorenin (PR) molecule were coupled to BSA and used as antigens to generate antibodies against PR. In a RIA system using the 125I-labeled peptide as tracer, it could be shown that antibodies against peptide 32-46 bound the peptide and the native PR in the follicular fluid (FF) to a similar degree, whereas antibodies generated against peptide 1-15 did not specifically recognize native PR. The specificity of the PR-(32-46) antibodies for PR was demonstrated by comparative measurements of PR by RIA and by an indirect procedure (involving trypsin treatment of PR) in different individual FF samples, plasma samples, and fractions of FF obtained by gel filtration or immunoaffinity chromatography. Normal plasma PR levels could not be measured by RIA, since they were below the detection limit of the assay (0.5 micrograms PR/L approximately 10 pmol/L). For measurement of the low PR levels in plasma, a sensitive direct immunoradiometric assay with a detection limit of 0.1 fmol PR/tube was developed. It was based on the combined action of a commercially available solid phase renin antibody and the affinity-purified and 125I-labeled PR-(32-46) antibody. The measurement of 35 individual plasma samples with different PR concentrations showed an excellent correlation (r = 0.99) between the new direct and the conventional indirect assays. The direct assay of PR concentrations in plasma of healthy women during the length of a menstrual cycle resulted in a biphasic pattern of PR concentrations, with peak levels (approximately 3-fold increase) at the time of the LH surge. The intake of monophasic and triphasic contraceptives caused a suppression of normal PR concentrations (96.9 +/- 34 ng/L; early follicular phase; n = 12) by 39% and 25%, respectively, which was also observed in the pill-free phase of the artificial cycle.

PIP: This paper studies the effects of different oral contraceptives on plasma prorenin (PR) levels in women as determined by a direct immunoradiometric assay (IRMA). 3 healthy and normally cycling women, aged 22, 30, and 37, volunteered for venipuncture every second day of one complete menstrual cycle. 18 healthy women taking oral contraceptives were studied for the effects of oral contraception on plasma PR concentrations. Results of this study showed that in a renin-angiotensin (RIA) system, using the I-labeled peptide, antibodies against peptide 32-46 bound the peptide and the native PR in the follicular fluid (FF) to a similar degree, while antibodies generated against peptide 1-15 did not recognize native PR. The absolute PR levels measured by the direct assay were approximately 20% lower than in the indirect assay, although both assay procedures indicate the same molecular measurement. The measurement of the different PR concentrations of the 35 individual plasma samples indicates an excellent correlation (r = 0.99) between the new direct and the conventional indirect assays. Sequential changes in the plasma PR levels of healthy women demonstrate that PR in the circulation was mainly due to ovarian origin. Lastly, PR concentration was significantly lower at any time during the artificial cycle than in the early follicular phase of spontaneous cycles. In conclusion, this PR IRMA is believed to be superior to the conventional indirect procedures.

Testosterone production by mouse Leydig cells is stimulated in vitro by atrial natriuretic factor.

The synthetic atrial peptides, rat atrial natriuretic peptide, atriopeptin I and atriopeptin II, stimulated testosterone production by mouse Leydig cells in a time- and concentration-dependent manner. The maximum stimulation of the steroidogenesis in response to the peptides was 6-10-fold over the basal level, as compared with 20-24-fold stimulation obtained with saturating concentrations of hCG. The stimulation of steroidogenesis by the most potent peptide, atriopeptin II, was markedly enhanced in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, suggesting an involvement of cyclic nucleotides. However, neither basal nor hCG-stimulated levels of cAMP were altered by the peptide, though testosterone production in response to submaximal concentrations of hCG was increased in the presence of atriopeptin II. The nature of the second messenger involved and the mechanism of action of the atrial peptides may be elucidated by further research in progress.

Effect of a tumour-promoting phorbol ester on atrial peptide-induced testosterone production and cyclic GMP accumulation by isolated mouse Leydig cells.

The objective of this study was to investigate the effects of 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA)--a potent activator of protein kinase C--on the responsiveness of mouse Leydig cells to stimulation with rat atriopeptin II (rAP-II). We report that, in these cells, the stimulation of testosterone production by rAP-II could be inhibited in a dose-dependent manner by 4 beta-PMA (1-200 nM). In contrast, the basal steroidogenesis was stimulated 2-fold by 4 beta-PMA. There was no inhibition of testosterone production when the cells were stimulated with 8-bromo cyclic GMP (8Br-cGMP) in the presence of 4 beta-PMA. Furthermore, addition of 4 beta-PMA resulted in a marked reduction in the amount of cGMP accumulated in response to rAP-II stimulation. 4 alpha-Phorbol 12-myristate 13-acetate (4 alpha-PMA) was found to have no effect at all. The inhibitory effect of 4 beta-PMA on steroidogenesis could be completely reversed by the addition of 0.25 mM 3-isobutyl 1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Also, the 4 beta-PMA-induced lowering of cGMP content could be partially reversed by IBMX. Membrane fractions from cells treated with 4 beta-PMA or 4 alpha-PMA did not differ in their contents of either basal or rAP-II-stimulated guanylate cyclase activities. We conclude that the 4 beta-PMA-mediated inhibition of testosterone production by Leydig cells stimulated with rAP-II results from an activation of a phosphodiesterase enzyme, hypothetically through an activated protein kinase C. This leads to a reduction in the cellular cGMP content through an increased metabolic removal of cGMP formed in response to rAP-II stimulation.

Human seminal fluid contains significant quantities of prorenin: its correlation with the sperm density.

The relevance of the tissue prorenin-renin-angiotensin system (PRAS) to male reproduction has been suggested by several investigators in the past. Although the presence of angiotensin converting enzyme in semen has been demonstrated, unequivocal evidence for the presence of prorenin and renin in the semen is not yet available. We have used a specific immunoradiometric assay based on an antibody directed against the pro-segment of the prorenin molecule to demonstrate that significant quantities of prorenin are present in human semen samples. Although semen is a rich source of proteases and protease inhibitors, the assay used by us, unlike the usual enzymatic renin assay, is not affected by such proteases, and their inhibitors. Furthermore, Western blotting data clearly demonstrated that prorenin is present in semen as a 48 kDa protein. In a majority of semen samples, the prorenin content was found to be several fold greater than that measured in EDTA-plasma samples. Interestingly, the level of prorenin was found to be directly proportional to the sperm density in semen samples. Our results suggest that seminal prorenin is produced locally within the male reproductive system, although its exact origin is yet to be defined, that a complete prorenin-renin-angiotensin system exists in human semen and that this system may be relevant to sperm function.

Histones inhibit human chorionic gonadotrophin-stimulated but not atrial peptide-stimulated testosterone production and cyclic nucleotide formation by isolated mouse Leydig cells.

Recently it has been reported that histone type H2A can inhibit gonadotrophin-stimulated cAMP formation and steroidogenesis by ovarian cells. In the present study we have investigated if similar antigonadotrophic effects of commercially available histones can also be demonstrated on testicular steroidogenic cells. Using percoll-purified mouse Leydig cells, we have demonstrated that several types of histones could almost completely inhibit hCG-stimulated testosterone production and cAMP formation. The inhibition was dose-dependent and could be reversed by the addition of excess of hCG. The most potent histone types were H2AS and H8S, both of which could inhibit hCG-stimulated cAMP formation half-maximally at concentrations of 4-5 micrograms/ml. Forskolin-stimulated cAMP formation was not affected by histones. When the cells were stimulated with either db-cAMP or rAP-II, histone H2AS and H8S failed to inhibit the testosterone production. In fact there was a marked increase in the amount of testosterone produced, the reason for which is not yet understood. The amount of cGMP accumulated in response to rAP-II was not affected by the presence of H2AS or H8S. In unstimulated cells, neither the cyclic nucleotide level nor the amount of steroid produced was affected by the histones. Based on the [125I]hCG binding data it is possible to conclude that histone H2AS inhibits the binding of hCG to its receptors on Leydig cells and thereby causes the inhibition of hCG-stimulated cAMP formation and steroidogenesis.

Preliminary report about a modified gonadotropin (human menopausal gonadotropin/human chorionic gonadotropin) treatment in infertile patients with premature luteinization.

Premature luteinization is a frequent phenomenon observed in infertile women undergoing human menopausal gonadotropin/human chorionic gonadotropin (hMG/hCG) therapy for corpus luteum insufficiency or anovulatory cycles. By inducing hyperprolactinemia in these women with sulpiride, we intended to create a dysfunctional state in these women, which supposedly induces a better reaction to hMG/hCG therapy. The rationale behind this combined treatment was the observation that hyperprolactinemic amenorrheic patients have a much higher pregnancy rate under hMG/hCG treatment than the above group. Three cases are reported in detail, illustrating the altered ovarian response to combined sulpiride-hMG/hCG treatment. Of 60 infertile women with repeated premature luteinization, 12 conceived after sulpiride-hMG/hCG therapy. Their expected pregnancy rate would have been very low (5% or less) if conventional hMG/hCG treatment had been continued.

Different hormonal patterns in human menopausal gonadotropin-treated, clomiphene citrate-treated, and untreated conception cycles.

Serum concentrations of seven different hormones were analyzed during 189 singleton conception cycles. One hundred nine women were treated with human menopausal gonadotropin (hMG), 52 women received clomiphene citrate (CC), and 28 became pregnant spontaneously. Serum progesterone (P) levels in hMG-treated women started to increase on day 18 of the cycle and reached peak concentrations between days 28 and 32 of the cycle. Serum estradiol (E2) concentrations paralleled the P patterns. In hMG-treated women, there were significant correlations between serum E2 and P concentrations and the number of the mature follicles observed before ovulation (both P less than 0.01). CC-treated and spontaneous conception cycles revealed significantly lower serum levels of P, E2, testosterone, 17 alpha-hydroxyprogesterone, and prolactin, compared with hMG-treated cycles (P less than 0.05). Elevations of the gonadal steroids in hMG-treated women possibly reflect the multiple corpora lutea formed after multiple ovulations.

24-hour profiles of salivary progesterone.

OBJECTIVES: To assess whether the known pulsatility of P secretion by the corpus luteum, which is detected in blood by P measurements, translates into fluctuations of saliva P concentrations, and to determine how well saliva P measurements reflect plasma P concentration. A second objective was to see whether there is a window in the luteal phase, where P secretion has reached its maximum capacity, but the amplitude is not very accentuated, which would be an ideal time to measure P. DESIGN: Twenty-one ovulatory women were randomly assigned to be studied on day 5, 7, or 8 after the luteinizing hormone surge. Blood samples were drawn every 20 minutes, and saliva samples were obtained hourly over a 24-hour period. Comparison between saliva plasma P was performed, and pulse analysis of plasma P was done. RESULTS: The percent variation of saliva P concentration over a 24-hour period was much higher when compared with the percent variation of plasma P concentration over the same time period (saliva P: 149%; plasma P: 107%). Also, the ratio of saliva to plasma P varied significantly between individuals (range: 0.0050 to 0.0148). A single plasma P concentration (8:00 A.M.) correlated better with the 24-hour mean plasma concentration than the respective single saliva value or the mean of two or three saliva samples (8:00 A.M. and 12:00 P.M.; 8:00 A.M., 12:00 P.M., and 8:00 P.M.). Plasma pulse frequency, mean pulse interval, pulse width, pulse amplitude, and 24-hour mean P level did not differ between the 3 study days. CONCLUSIONS: A single plasma P determination reflects more accurately 24-hour P secretion than repeated saliva P samples measured in the same individual. We could not identify a window in the luteal phase when P measurements are more representative of corpus luteum function.

Steroidogenic effect of atrial natriuretic factor in isolated mouse Leydig cells is mediated by cyclic GMP.

The effects of different atrial natriuretic peptides on cyclic GMP formation and steroidogenesis have been studied in Percoll-purified mouse Leydig cells. Rat atrial peptides rANP (rat atrial natriuretic peptide), rAP-I (rat atriopeptin I) and rAP-II (rat atriopeptin II), in the presence of a phosphodiesterase inhibitor, stimulated cyclic GMP formation in a concentration-dependent manner. In the presence of saturating concentrations of the peptides, a 400-600 fold stimulation of cyclic GMP accumulation was observed. Among the peptides, rAP-II appeared to be the most potent. ED50 values (concentration causing half-maximal effect) for rAP-II, rANP and rAP-I were 1 X 10(-9) M, 2 X 10(-9) M and 2 X 10(-8) M respectively. A parallel stimulation of cyclic GMP formation and testosterone production by the cells was observed after incubation of the cells with various concentrations of rAP-II. In the presence of a saturating concentration of rAP-II (2 X 10(-8) M), maximum stimulation of intracellular cyclic GMP content was obtained within 5 min of incubation. Testosterone production by mouse Leydig cells could be stimulated by 8-bromo cyclic GMP in a concentration-related manner. At a 10 mM concentration of the cyclic nucleotide, steroidogenesis was stimulated to a similar extent as that obtained with a saturating concentration of human chorionic gonadotrophin (5 ng/ml). On the basis of these results we conclude that cyclic GMP acts as a second messenger in atrial-peptide-stimulated steroidogenesis in mouse Leydig cells. The steroidogenic effect of atrial peptides appears to be species-specific, since none of these peptides stimulated testosterone production by purified Leydig cells of rats, though in these cells a 40-60-fold stimulation of cyclic GMP formation in response to each of the three peptides was observed. However, 8-bromo cyclic GMP could stimulate testosterone production in rat Leydig cells. Therefore we conclude that the lack of steroidogenic response in rat Leydig cells to atrial-natriuretic-factor-stimulation results from an insufficient formation of cyclic GMP in these cells. This species difference would appear to result from a lower guanylate cyclase activity in rat Leydig cells.

Phorbol esters inhibit LH stimulated steroidogenesis by mouse Leydig cells in vitro.

The effect of phorbol esters on the stimulation of testosterone production in response to LH was studied in mouse Leydig cells incubated in vitro. The tumor promoting phorbol esters, Phorbol-12-myristate-13-acetate and Phorbol-12-13-didecanoate at nanomolar concentrations effectively inhibited testosterone production by Leydig cells in response to stimulation by LH, whereas non-tumor promoting phorbol esters were ineffective. When the cells were stimulated by 8Br-cAMP, instead of LH, the testosterone production was stimulated similarly as in the presence of LH, but phorbol esters were without any effect. This suggests that the tumor promoting phorbol esters may act in the Leydig cells by suppressing the stimulation of cAMP production in response to hormonal activation and/or by interfering with the hormone-receptor interaction.

Positive relationship between the nocturnal concentrations of melatonin and prolactin, and a stimulation of prolactin after melatonin administration in young men.

The relationship between the concentrations of melatonin and prolactin over the 24-h cycle has been investigated in a group of young men at three times in the year. Melatonin and prolactin showed a significant positive correlation (P less than 0.001) for all times during the 24-h period but with a greater contribution from concentrations during the nocturnal period, when both hormones were elevated. The positive correlation for nocturnal concentrations was evident in February and March (P less than 0.01) but was of greatest significance in June (P less than 0.001). In blood samples taken at 15-min intervals during the morning (0800-1200) and evening (2000-2400), melatonin and prolactin concentrations were not significantly correlated. Melatonin concentrations increased before prolactin during the evening and decreased before prolactin in the morning. Oral administration of 6 mg melatonin significantly stimulated prolactin release above concentrations measured after placebo administration, in both the morning (P less than 0.05) and evening (P less than 0.01) time periods; the prolactin response being greater in the evening. These results provide evidence for melatonin controlling the nocturnal increase of prolactin via its ability to stimulate prolactin release.

Correlation between hcg (lh)-binding capacity, leydig cell number and secretory activity of rat testis throughout pubescence.

Male rats varying in age from 10 to 110 days were examined for testosterone concentration of serum and testicular tissue and 125-I-HCG-binding capacity of testicular tissue. In addition, the Leydig cell number of testes was determined using two different staining methods. It could be demonstrated that specific 125-I-HCG-binding capacity of rat testis increased with age up to 60 days of life. This rise was accompanied by an increase in serum and testicular testosterone concentrations and can be explained by a concomitant increase in Leydig cell number up to this age. The calculated specific 125-HCG-binding capacity per Leydig cell does not change from day 30 to 110 of life. This finding is in contrast to the 125-I-HCG-binding capacity per Leydig cell at day 10 and 20, the former being 5-6 times higher than in the age groups of 30 to 110 days. The observation of high 125-I-HCG-binding capacity per Leydig cell at day 10 and 20 is discussed in the light of simultaneous elevated serum and testicular testosterone concentrations.

Oxytocin and vasopressin have no effect on progesterone production and cyclic AMP accumulation by rat luteal cells in vitro.

Enzymically dispersed luteal cells obtained from PMSG-hCG-treated immature pseudopregnant rats were incubated with oxytocin and vasopressin. In response to increasing doses of hCG the rat luteal cells produced progesterone and accumulated intracellular cAMP in a dose-dependent manner. A neuropeptide GnRH agonist (4 X 10(-6) M) produced a significant inhibition of hCG-stimulated progesterone production and of accumulation of intracellular cAMP. However, neither the basal nor the hCG-stimulated rate of progesterone production and level of intracellular cAMP was affected by the neurohypophysial peptides tested. Therefore, it is concluded that oxytocin and vasopressin do not have a direct action on steroidogenesis by rat luteal cells.