Growth hormone and prolactin affect oxytocin, vasopressin, progesterone and cyclic nucleotide secretion by bovine granulosa cells in vitro.

The effects of growth hormone (GH) and prolactin (PRL) (1, 10, 100, 1000 or 10,000 ng/ml medium) on oxytocin, vasopressin, progesterone, cAMP and cGMP release by cultured bovine granulosa cells were studied. It was found that GH significantly stimulated oxytocin, vasopressin and cAMP but suppressed progesterone secretion. PRL tended to have the same pattern of action on nonapeptide, cAMP and steroid release, but its effect was not as great, with only a high supraphysiological dose (10,000 ng/ml) producing a statistically significant effect. No significant influence of GH on cGMP output was observed. Physiological doses of PRL (1, 10, 100 or 1000 ng/ml) significantly inhibited cGMP production whilst a high dose (10,000 ng/ml) resulted in stimulation. These observations suggested that GH may regulate ovarian oxytocin, vasopressin, progesterone and cAMP secretion. The effects of PRL on the release of these substances appeared to be non-specific, possibly resulting from its structural similarity to GH.

Direct action of LH RH and its antagonist on isolated bovine granulosa cells steroidogenesis.

Progesterone, 4-androstene-3,17-dione, testosterone and estradiol-17 beta secretion by bovine granulosa cells culture without or in the presence of 10, 100 or 10.000 ng/ml LH RH or of its antagonist (D Phe2, D Phe6) LH RH were analyzed. It was observed that both LH RH and its antagonist significantly activated progesterone and estradiol output. LH RH also stimulated, but (D Phe2, D Phe6) LH RH inhibited granulosa 4-androstene-3,17-dione secretion. Both LH RH and its analogue decreased testosterone release by the cell culture. This is the first demonstration of a direct influence of LH RH on bovine ovarian steroidogenesis. The lack of correlation between LH RH and its agonist action on hypophysis and on different gonadal steroids secretion may suggest the differences in the features of receptors to LH RH-related peptides in the various target cells.

The role of cAMP- and Ca(2+)-dependent intracellular mechanisms in the control of oxytocin and vasopressin secretion by bovine ovarian granulosa cells in vitro.

The secretion of oxytocin and arginine-vasopressin was demonstrated in bovine granulosa cell culture. It was found that dbcAMP or 3-isobutyl-1-methyl-xanthine (an inhibitor of intracellular cAMP metabolization) additions increased both oxytocin and vasopressin release. The Ca2+ ionophore A23187 also stimulated, while the Ca2+ channel blocker verapamil inhibited the secretion of both nonapeptide hormones. These results suggest the involvement of cAMP- and Ca(2+)-dependent intracellular mechanisms in the stimulation of both oxytocin and vasopressin secretion by bovine granulosa cells.

Cooperation between LH-RH and LH in the direct action on the ovary: LH-RH stimulation of LH/hCG receptors, basal and LH-induced cAMP and cGMP release by porcine granulosa cells in vitro.

LH-RH effects on LH/hCG receptors content, on basal and LH-stimulated cAMP and cGMP release by cultured porcine granulosa cells, were investigated by radioreceptor analysis and radioimmunoassay. It was found that LH-RH additions (10, 100, 1000 or 10,000 ng/ml medium) increased the number of LH/hCG binding sites in granulosa cells. LH or hCG (0.1, 1, 10 or 100 ng/ml) increased both cAMP and cGMP secretion by the cell culture. LH-RH alone (0.1, 1, 10, 100 or 1000 ng/ml) had the same stimulating effect on both cyclic nucleotides. The addition of LH-RH (100 ng/ml) to the medium supplemented with LH (10, 100, 1000, 10,000 or 100,000 ng/ml) enhanced LH-stimulated cAMP and cGMP output from the culture. The present observations suggest that LH-RH can act as synergist of LH and of hCG increasing a number of LH/hCG receptors and stimulating basal and LH-induced cyclic nucleotide release by ovarian cells.

Release of a prolactin-like substance by bovine granulosa cells in vitro: effects of ovarian steroids.

The time-dependent accumulation of prolactin (PRL) immunoreactivity in serum-free and in serum-supplemented cultures of bovine granulosa cells was observed. Blockade of endogenous progesterone by specific antiserum had no significant influence on PRL immunoreactivity, but progesterone addition (10-10,000 ng/ml) lead to a dose-dependent decrease. Antisera against testosterone and against estradiol-17 beta were inhibitors, and additions of these hormones (100-10,000 ng/ml) stimulated the release of PRL immunoreactivity. The results obtained suggest the secretion of a PRL-like peptide by bovine ovarian cells. Growth hormone, testosterone and estradiol may be stimulators, and progesterone a possible inhibitor of this process.

Effects of prolactin on estrogen, cAMP and oxytocin secretion by porcine granulosa cells in vitro.

Granulosa cells were isolated from ovaries (without pre-ovulatory follicles or corpora lutea) of cycling gilts slaughtered at 8 months of age. They were cultured in the presence or absence of exogenous porcine prolactin (1, 10, 1,000, 10,000, or 100,000 pmol/ml medium). The secretion of estrogen, cAMP and oxytocin into the incubation medium was analysed by radioimmunoassay. It was found that prolactin (10, 1,000, 10,000 and 100,000 pmol/ml) inhibited estrogen secretion (p < 0.001) and stimulated cAMP output (p < 0.001, p < 0.001, not significant, and p < 0.01, respectively). Small doses of prolactin (1 and 10 pmol/ml) decreased (p < 0.001, p < 0.05), whilst high doses (1,000, 10,000 and 100,000 pmol/ml) increased (p < 0.001) oxytocin secretion by granulosa cell culture. The data suggest a direct action of prolactin on porcine ovarian cyclic nucleotides, steroid and nonapeptide hormone release.

Steroid hormones regulate cAMP and cGMP production by porcine granulosa cells in vitro.

The influence of steroid hormones on the output of the cyclic nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) by porcine granulosa cells was investigated. Both progesterone (100, 1000, 10,000 and 100,000 pg/ml medium) and estradiol (100, 1000, 10,000 and 100,000 pg/ml medium) activated cAMP and cGMP production. Testosterone (100 or 1000 pg/ml medium) also stimulated cAMP output. The stimulating effect of steroid hormones on cyclic nucleotide production may suggest the involvement of cAMP- and cGMP-dependent intracellular mechanisms in the action of steroid hormones on porcine ovarian cells.

The action of LH-releasing hormone and five analogues on oestradiol, oxytocin and vasopressin secretion by bovine granulosa cells in culture.

The release of oxytocin, arginine-8-vasopressin and oestradiol-17 beta by bovine granulosa cells in culture was analysed either with or without LH-releasing hormone (LHRH), its agonists (cyclo [Pro1DPhe6] LHRH and des 1-3, 10[DAla6] LHRH) or antagonists ([DPhe2DPhe6] LHRH, [DPhe2DPhe(NH2)6] LHRH or cyclo [Pro1DPhe2DPhe6] LHRH). All preparations used stimulated granulosa oxytocin and oestradiol secretion. Vasopressin release was significantly increased after all treatments with LHRH antagonists, but not after LHRH or its agonists. Our observations demonstrated a direct influence of LHRH and its analogues on the secretion of oestrogen and nonapeptide hormones by bovine granulosa cells. A comparison of the effects of LHRH, its agonists and antagonists suggests that the action of these peptides at the hypophysial and ovarian level is relatively independent.

[The effect of recombinant somatotropin hormone on the secretion of ovarian hormones in sexually mature swine in vivo and in vitro]. / Vplyv rekombinantného somatotropného hormónu na sekréciu ovariálnych hormónov pohlavne dospelých prasniciek in vivo a in vitro.

The development of recombinant somatotropic hormone (STH) creates a prerequisite for a wide application of exogenous stimulation of meat and milk performance of cows and pigs. Reproduction effect and its mechanism is till now restricted by a lack of complex information though there are several studies of principal character and basic effects of STH on reproduction process are also known. The result of some experimental studies, quoted in scientific literature, was a hypothesis that STH effect is accompanied by increased production of so-called insulin-like growth factors. Moreover, it is known that these factors have a steroidogenic effect. A hypothesis of STH action, just by means of insulin-like growth factors, was expressed on the basis of the above mentioned fact. Our study was aimed at the analysis of the effect of recombinant porcine somatotropic hormone (rpSTH, Research and Development, Pittman-Moore, Inc.; Terre Haute, Indiana, the U.S.A., LG-COMP-1776/93) on the dynamics of progesterone, cAMP, cGMP concentrations in the blood plasma of sexually mature gilts with surgically introduced permanent canula in v. jugularis. Gilts of early and medium follicular stages of the oestrous cycle (found laparoscopically) without any developmental or any other anomalies in reproductive organs. Blood samples were collected each 15 minutes in the period of 30 minutes before and 150 minutes after rpSTH application (5 mg per gilt). The control of gilts was administered 0.9% NaCl solution under the same conditions. Blood plasma samples collected and processed were stored not earlier than after radioimmunoassay of substances. A part of the study was to analyze different rpSTH doses (1-100,000 ng/ml) on granulosis cells on gilts' ovaries obtained after slaughtering of sexually mature gilts without any visible changes in the reproductive system. Granulosis cells were separated after aspiration of follicles of the size 2 to 5 mm without marked paleness, after thrice resuspending in essential cultivation medium TCM 199 and centrifugigng (g = 200). Followed the last straining of supernatant the cells were resuspended in the cultivation enriched medium TCM 199. 2 ml of cell suspension in the cultivation medium with known counts and viability of cells (10(6) in 1 ml and 75-95% vitality) were cultivated in defined conditions as a basic unit of the experiment. Resulting concentrations of analyzed hormones were determined on the basis of production activity of cells and statistically assessed by ANOVA test.(ABSTRACT TRUNCATED AT 400 WORDS)

The interrelationships between nonapeptide and steroid hormones secretion by bovine granulosa cells in vitro.

The effects of adding oxytocin (OT) and arginine-vasopressin (AVP) on progesterone and estradiol-17 beta secretion by bovine granulosa cells in culture were studied. The influence of these steroids on OT and AVP release was also evaluated. OT (1, 10, 100 or 1000 mIU/ml) stimulates both progesterone and estradiol output. Small doses (10 pM/ml) of exogenous progesterone or estradiol stimulated a surge in OT, while the intermediate doses (100 or 1000 pM/ml) had no influence, and large doses (10,000 pM/ml) inhibited OT secretion by granulosa cells. Thus, a potential regulatory loop between OT and steroid hormone release by granulosa cells was demonstrated. Stimulation of a surge in steroids by OT, activation of OT release by small doses of steroids and inhibition of OT secretion by excess steroids may suggest the existence of a feedback mechanism regulating these hormones production. Addition of AVP (1, 10, 100 or 1000 pM/ml) inhibited progesterone and stimulated a surge in estradiol, while steroid hormones did not induce AVP release. These data suggest the regulation of ovarian steroidogenesis by AVP, feedback influences are less likely.

[Production of progesterone and testosterone in ovarian granulosa cells in sows after administration of nonapeptide hormones in vitro]. / Produkcia progesterónu a testosterónu bunkami granulózy vajecníkov osípaných po aplikácii nonapeptidových hormónov in vitro.

In the present paper the effects of nonapeptide hormones and of some of their chemical analogues were investigated on progesterone and testosterone production in granulosa cells of sow ovaries; the experiments were made in vitro. This objective was given by data on potential regulatory roles of nonapeptides at the level of hypothalamus, pituitary and reproductive organs. The goal of this experiment was to analyze the effects of various doses of oxytocin (OT), arginine-8-vasopressin (AVP), arginine-8-vasotocin and of some of their analogues on progesterone and testosterone production in vitro in granulosa cells of sow ovaries. The production activity of granulosa cells was investigated which were obtained from slaughtered sows without any changes in their reproductive process and abnormalities in their reproductive organs. Follicles of the size 2-5 mm without marked paleness in the early follicular phase were selected for aspiration. Granulosa cells with determined viability (more than 75%) and concentration (2 million/ml) were cultivated in defined culture conditions (37.5 degrees C, 5% CO2) after threefold resuspension and centrifugation of follicle fluid. These hormonal preparations were used in the experiments: pFSH, synthetic OT, synthetic AVP, synthetic AVP with antidiuretic effects and synthetic AVT. Progesterone and testosterone concentrations were analyzed radioimmunoanalytically using commercial kits of the Institute of Radio ecology and Nuclear Technology at Kosice. Statistically significant differences between the groups were evaluated by Student's t-test. The administered preparations were found to influence progesterone and testosterone production in dependence on the doses applied (Figs. 1-6). OT stimulation of progesterone production in granulosa cells indicated its regulatory role in relation to secretion of this hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of FSH, LH, LH-RH and arginine-vasotocin on the production of steroids, nonapeptide hormones and cGMP by rabbits granulosa cells isolated at different stages of reproductive cycle.

Granulosa cells isolated from ovaries of non-cycling, cycling and pregnant rabbits of the same age were cultured in vitro either without or with pFSH (1 micrograms/ml), bLH (1 IU/ml), LH-RH (25 ng/ml) or arginine-8-vasotocin (100 ng/ml). The production of immunoreactive progesterone, estradiol-17 beta, oxytocin, arginine-8-vasopressin and cGMP was analyzed. The gonadotropins did not show any significant effects on the cells isolated from non-cycling and cycling rabbits, but not from these of pregnant ones. LH-RH inhibited and vasotocin stimulated progesterone production. All hormones used stimulated estradiol release from cells of non-cycling rabbits, while in a case of cycling animals no change was found. In the cell from pregnant females the release of estradiol was enhanced after LH treatment only. The treatment with FSH and LH (but not with LH-RH or vasotocin) resulted in a remarkable rise of granulosa vasopressin surge irrespectively to the reproductive stage. Oxytocin production by granulosa cells incubated either without or with LH, LH-RH or vasotocin was undetectable. However, FSH strongly stimulated oxytocin release. FSH and in lesser extent, LH or LH-RH (but not vasotocin) activated granulosa cGMP production in the cells from cycling and pregnant (but not from non-cycling) animals. It was also found that, in contrast to other reproductive stages, basal progesterone release from the cells of pregnant rabbits was increased, while in a case of non-cycling animals the basal estradiol release was decreased and that of cGMP was increased.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of oxytocin, vasopressin and their analogues on progesterone and testosterone production by porcine granulosa cells in vitro.

Effects of nonapeptide hormones and some of their chemical analogues on progesterone and testosterone production by culture of porcine granulosa cells have been investigated. Oxytocin (0.01-10 IU/ml), arginine-8-vasopressin (0.01-10 micrograms/ml), arginine-8-vasotocin (0.01-10 micrograms/ml) and, in a lesser degree, 2-0-methyl-tyrosine (deamino-1-karba)-oxytocin (0.01-10 micrograms/ml, but no 1-deamino-8-vasopressin (0.01-10 micrograms/ml) stimulated a progesterone surge. Testosterone production was significantly stimulated by oxytocin and inhibited by vasopressin or vasotocin additions. 2-0-methyl-tyrosine (deamino-1-karba)-oxytocin or 1-deamino-8-vasopressin had little or no effect on testosterone secretion. The present results suggest the existence of a direct influence of nonapeptide hormones on porcine ovarian progestagen and androgen production.

Reciprocal control of oxytocin and cAMP production by porcine granulosa cells in vitro.

Oxytocin and cAMP accumulation in the medium after 4 days incubation of porcine granulosa cells was measured and confirmed the production of these substances by porcine ovaries. Both cAMP analogue dbcAMP (10, 100, 1,000 or 10,000 microM/ml) and 3-isobutyl-1-methyl-xanthine (intracellular cAMP metabolization inhibitor, 0.1, 1, 10 or 100 microM/ml) additions increased granulosa cells oxytocin production in a dose-dependent manner. Low doses of oxytocin or posterior pituitary extract (10 or 100 mIU/ml) had no significant effect on granulosa cAMP output, but higher dose (1,000 mIU/ml) inhibited it. Thus, porcine ovarian oxytocin production can be stimulated via cAMP-dependent intracellular mechanisms. On the other hand, excess of oxytocin can inhibit ovarian cAMP production through a ultra-short loop negative feedback regulation.

Prolactin-like substance secretion by granulosa cells isolated from bovine ovaries.

By the 5-day culture of bovine granulosa cells both in serum-free and in serum-supplemented medium the time-dependent accumulation of PRL immunoreactivity was observed. FSH additions (10-10,000 ng/ml medium) led to a dramatic rise of immunoreactive PRL in a dose-dependent manner. LH stimulated the increase of PRL-like substance only in a great dose (10 IU/ml). Lower LH doses (0.01-1 IU/ml) had no significant influence on this process. Low doses of oxytocin (1 or 10 mIU/ml) blocked, and higher ones (100 or 1,000 mIU/ml) stimulated the granulosa PRL-like substance accumulation. Arginine-8-vasopressin (1-1,000 ng/ml), arginine-8-vasotocin (10-10,000 ng/ml), or LH-RH (10-10,000 ng/ml) failed to influence the PRL immunoreactivity accumulation in the culture medium. Present data may suggest the production of PRL-like substance by bovine ovarian cells, as well as the involvement of gonadotropins and oxytocin in the regulation of this process.

[Secretory activity of ovarian granulosa cells in cows in relation to production of prolactin or prolactin-like substances in vitro]. / Sekrecná aktivita buniek granulózy vajecníkov kráv vo vztahu k produkcií prolaktínu alebo prolaktínu podobnej substancie in vitro.

Ovarian functions are regulated by a wide variety of substances of hypothalamic, pituitary and intraovarian origin. In particular, prolactin (PRL) plays an important role in the control of ovaries. The aim of our in-vitro experiments was to prove a possibility of PRL production by bovine granulosa cells and to search into the endocrine regulators of this process. In the course of experiment 1 it was observed that the marked time-dependent accumulation of immunoreactive PRL took place during long-time cultivation of granulosa cells both in serum-free and in serum-dependent medium. After 12-24 hours of cultivation this level was reduced, but after 120 hours of cell culture the medium PRL-immunoreactivity gradually rose to exceed the starting level 2.1-2.4 times. FSH additions (10-10,000 ng/ml) led to a dramatical rise of PRL-immunoreactivity in a dose-dependent manner. A greater increase in FSH doses (1000 or 10,000 ng/ml) activated this process 14.0-18.0 times. In the other experiments the effects of LH, LH-RH and various nonapeptide hormones on the PRL-like substance production were investigated. LH stimulated PRL-like substance production at a great dose only (10 IU/ml). The lower doses did not have any significant influence on the process. Low doses of oxytocin (1 or 10 IU/ml) blocked, and higher doses (100-1000 IU/ml) stimulated the granulosa PRL-like production. Arginine-8-vasopressin (AVP) (1-1000 ng/ml), arginine-8-vasotocin (AVT) (10-1000 ng/ml), or LH-RH (10-10,000 ng/ml) failed to influence the immunoreative PRL accumulation in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS)

[Fluctuations in the level of progesterone in the peripheral blood of nutria during pregnancy]. / Kolísanie hladiny progesterónu v periférnej krvi nutrií pocas gravidity.

The fluctuations in the concentrations of progesterone in peripheral blood of three intact coypus and three coypus after natural mating were investigated for the period of four months. Immediately after mating the concentrations of progesterone are low (1.5-2.4 ng.ml-1), the maximum values reach the peak between the 14th and 15th week of gravidity (12.50-20.72 ng.ml-1), whilst there is almost no change in the intact females. On the last days of gravidity the values of progesterone decrease rapidly and they are very low on the day of parturition (1.0-2.5 ng.ml-1). The concentration of progesterone in the blood plasma of intact females in the test period is low (0.09-3.18 ng.ml-1).