The response of phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A pathways in porcine theca interna cells to opioid agonist FK 33-824

Opioids were found as factors affecting porcine ovarian steroidogenesis. The mechanism of opioid action, however, on porcine theca interna cells is completely unknown. Therefore, the present study was designed to investigate the possible involvement of two intracellular pathways, phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A, in opioid signal transduction in porcine theca cells treated with mu opioid receptor agonist, FK 33-824. Incubation of the cells for 4 h with FK 33-824 at the dose 1 nM resulted in decreases in inositol phosphate accumulation as well as androstenedione (A(4)), testosterone (T), and estradiol (E(2)) secretions. Protein kinase C (PKC) inhibitors, staurosporine (1-100 nM), D-sphingosine (10-500 nM), and PKCi (100-2000 nM), both added alone and together with the opioid agonist, depressed release of the steroid hormones. PKC activator, phorbol ester (PMA, 1-100 nM), used alone was without effect on theca cell steroidogenesis, but added in combination with FK 33-824 abolished inhibitory influence of the opioid on A(4), T, and E(2) output. The steroid hormone secretion by PKC-deficient theca cells was inhibited by the opioid agonist. FK 33-824 also suppressed PKC activity reducing [(3)H]PDBu specific binding to theca cells, whereas ionomycin (a positive control) increased labeled phorbol ester binding to the cells. In the next experiment, cAMP release from theca cells during 2 and 4 h incubations with FK 33-824 (1-100 nM), naloxone (10 microM; opioid receptor antagonist), and LH (100 ng/mL; a positive control) was examined. FK 33-824 at the dose 1 nM inhibited cAMP secretion during 2 h incubation, but had no effect during longer incubation. LH in a manner independent on incubation time multiplied cAMP release. Protein kinase A inhibitor, PKAi (100-2000 nM), alone and in combination with FK 33-824 (1 nM), inhibited A(4), T, and E(2) secretions by theca cells. PKA activator, 8BrcAMP (10-1000 microM), stimulated the steroid hormone release, but this stimulatory effect was diminished in the presence of FK 33-824. The results allow to suggest that opioid peptides affect porcine theca cell steroidogenesis and their acute action on the cells is connected with the inhibition of phospholipase C/protein kinase C and adenylyl cyclase/protein kinase A signal transduction systems.     

The Influence of Opioid Peptides on Steroidogenesis in Porcine Granulosa Cells

The present studies were undertaken to examine the influence of mu (beta-endorphin, DAMGO, FK 33-824), delta (met-enkephalin, leu-enkephalin, DPLPE) and kappa opioid receptor agonists (dynorphin A, dynorphin B, U 50488) used at different doses (1-1000 nM) alone and in combination with LH (100 ng/ml) on steroidogenesis in porcine granulosa cells derived from large follicles. The effects of mu, delta and kappa receptor agonists on both basal and LH-induced progesterone (P4) secretion were negligible. Agonists of mu opioid receptors reduced basal androstenedione (A4), testosterone (T) and oestradiol (E2) release. Co-treatment with LH entirely abolished the inhibitory effect of these agonists on A4 and E2 secretion and resulted in an increase in T release. The addition of delta receptor agonists was followed by a decrease in basal A4, T and E2 secretion. The cells incubated in the presence of LH increased the androgen production and abrogated the inhibitory effect of delta agonists on E2 output. Basal A4, T and E2 release was also suppressed by kappa receptor agonists. The presence of LH in culture media extended the inhibitory effect of these opioids on E2 output and caused either abolition of the inhibitory influence of kappa agonists or even augmentation of both androgen release in response to the opioids. In conclusion, these data support the involvement of three major types of opioid receptors in the regulation of porcine granulosa cell steroidogenesis.     

Attenuation by an opioid agonist of the oestradiol-induced LH surge in anoestrous ewes and its reversal by naloxone

The effects of a potent opioid peptide agonist [D-ala2-Phe4, Met(0)ol5-enkephalin (FK 33-824) on the magnitude of the oestradiol-induced LH surge and on basal plasma LH concentrations were examined in intact and chronically-ovariectomized ewes during the late-anoestrous period. In intact ewes, treatment with FK 33-824 (0.5 mg i.v. every 3 hr) for a 24 hr period commencing at the time of oestradiol-17 beta administration (25 micrograms i.m. bolus) was associated with non-significant 65% reduction in the peak plasma LH level observed and a significant (P less than 0.05) 58% reduction in the total amount of LH released during the surge (calculated from the area under the curve). Concurrent treatment with the opioid antagonist naloxone (10 mg i.v. every 3 hr) partially reversed this suppressive effect on the magnitude of the LH-surge. In ovariectomized ewes no significant effects on the oestradiol-induced LH surge of either FK 38-824 alone or FK 33-824 in combination with naloxone were observed. Administration of FK 33-824 at a 6-fold higher dose rate (0.5 mg every 30 min) failed to modify basal plasma LH concentration in intact ewes. In ovariectomized ewes, however, a significant (P less than 0.05) 25% fall in basal plasma LH was observed, an effect which was completely reversed by combined treatment with naloxone (10 mg every 30 min). These results support the conclusion that endogenous opioid peptides may contribute to the neuroendocrine mechanism through which oestradiol promotes a preovulatory-like surge in the anoestrous ewe.     

Endogenous opioid modulation of luteinizing hormone and prolactin secretion in postpartum ewes and cows

A possible role for endogenous opioid peptides (EOP) in the control of luteinizing hormone (LH) and prolactin (PRL) secretion was studied by injecting the opioid antagonist, naloxone (NAL), into postpartum ewes and cows. Twelve ewes that lambed during the fall breeding season and nursed their lambs were injected iv with NAL (1.0 mg/kg) on d 10, 14, 18, 22 and 26 postpartum. Blood samples were collected at 15-min intervals from 2 h before to 2 h after NAL, and serum concentrations of LH and PRL were quantified. Following treatment on d 10, suckling lambs were removed from 6 of the 12 ewes, creating non-suckled (NS) and suckled (S) treatment groups for subsequent study on d 14 through 26. On d 10, NAL treatment increased LH (P less than .01) but concentrations of PRL were not affected. When averaged across d 14 to 26, post-NAL concentrations of LH were greater (P less than .001) than pre-NAL concentrations (6.5 +/- .7 vs 1.9 +/- .4 ng/ml). In contrast, concentrations of PRL in the post-NAL period were lower (P less than .001) than pre-NAL concentrations (129 +/- 15 vs 89 +/- 10 ng/ml). Compared with S ewes over d 14 to 26, those in the NS group had similar pre-NAL concentrations of LH, tendencies for higher (P less than .10) post-NAL concentrations of LH, lower (P less than .001) mean serum concentrations of PRL (pre- and post-NAL) and similar pre-NAL vs post-NAL differences in serum PRL. Six suckled beef cows on d 24 to 35 were injected iv with either saline or NAL (.5 mg/kg) in a replicated crossover design. Injections of NAL increased serum concentrations of LH (P less than .05), when averaged over all 12 injections in the six cows, but serum PRL was not changed. However, three of six cows did not respond to NAL with increases in serum LH. These non-responding cows were similar to the responding cows in their pre-injection concentrations of LH and PRL, but they tended (P = .10) to have higher serum concentrations of cortisol than responding cows.     

The effects of GnRH and adrenergic agents on PRL and beta-endorphin secretion by porcine pituitary cells in vitro

The direct effects of alpha- and beta-adrenergic agents on PRL and beta-endorphin (beta-END) secretion in vitro by porcine pituitary cells have been investigated. Pituitary glands were obtained from mature gilts, which were ovariectomised (OVX) one month before slaughter. Ovariectomised gilts, assigned to four groups, were primed with: (1) vehicle (OVX); (2) and (3) oestradiol benzoate (EB; 2.5 mg/100 kg b.w.) at 30-36 h (OVX+EB I) and 60-66 h (OVX+EB II) before slaughter, respectively; and (4) progesterone (P4; 120 mg/100 kg b.w.) for 5 consecutive days before slaughter (OVX+P4). Isolated anterior pituitary cells were submitted to 3.5 h incubation in the presence of GnRH, alpha- and beta-adrenergic agonists [phenylephrine (PHEN) and isoproterenol (ISOP), respectively], or alpha- and beta-adrenergic blockers [phentolamine (PHENT) and propranolol (PROP), respectively]. The culture media were assayed for PRL (exp. I) and beta-endorphin-like immunoreactivity (beta-END-LI) (experiment II). In experiment I, GnRH did not influence PRL release by pituitary cells in all experimental groups. Some of tested doses of adrenergic agonists, PHEN and ISOP, increased PRL release from pituitary cells of OVX gilts, but not from those of OVX+EB I animals. In the OVX+EB II group, PHEN alone, but ISOP with PROP, potentiated PRL secretion by the cells. In OVX+P4 animals, PHEN alone or in combination with PHENT and also ISOP alone or with PROP enhanced PRL output from the cells. In experiment II, addition of GnRH increased beta-END-LI release from pituitary cells only in the OVX+EB II group. PHEN and PHENT potentiated beta-END-LI secretion by pituitary cells in OVX+EB II and OVX+P4 groups, while ISOP and PROP increased beta-END-LI secretion by the cells of OVX and OVX+EB II animals. In turn, in the OVX+EB I group, effect of PHENT and PROP on PRL secretion by pituitary cells was inhibitory. In conclusion, our results suggest that adrenergic agents can modulate PRL and beta-END secretion by porcine pituitary cells in a manner dependent on the hormonal status of gilts.     

The control of adenohypophysial hormone secretion by amino acids and peptides in swine

Several different amino acids and peptides control secretion of adenohypophysial hormones and this control may be indirect, via the modulation of hypothalamic hormone secretion. Indeed, classical hypothalamic hormones (e.g., gonadotropin-releasing hormone [GnRH], growth hormone-releasing hormone [GHRH], somatostatin, etc.) may be released into the hypothalamo-hypophysial portal vasculature, travel to the adenohypophysis and there stimulate or inhibit secretion of hormones. Alternatively, some amino acids and peptides exert direct stimulatory or inhibitory effects on the adenohypophysis, thereby impacting hormone secretion. In swine, the most extensively studied modulators of adenohypophysial hormone secretion are the excitatory amino acids (ExAA), namely glutamate and aspartate, and the endogenous opioid peptides (EOP). In general, excitatory amino acids stimulate release of luteinizing hormone (LH), follicle-stimulating hormone (FSH), growth hormone (GH), and prolactin (PRL). Secretion of adenohypophysial hormones induced by ExAA is primarily, but perhaps not exclusively, a consequence of action at the central nervous system. By acting primarily at the level of the central nervous system, EOP inhibit LH secretion, stimulate GH release and depending on the animal model studied, exert either stimulatory or inhibitory influences on PRL secretion. However, the EOP also inhibited LH release by direct action on the adenohypophysis. More recently, peptides such as neuropeptide-Y (NPY), orexin-B, ghrelin, galanin, and substance P have been evaluated for possible roles in controlling adenohypophysial hormone secretion in swine. For example, NPY, orexin-B, and ghrelin increased basal GH secretion and modulated the GH response to GHRH, at least in part, by direct action on the adenohypophysis. Secretion of LH was stimulated by orexin-B, galanin, and substance P from porcine pituitary cells in vitro. Because the ExAA and various peptides modulate secretion of adenohypophysial hormones, these compounds may play an important role in regulating swine growth and reproduction.     

Opioid inhibition stimulates luteinizing hormone and prolactin secretion in the gilt

Ten gilts on day 6·11 of the estrous cycle (onset of estrus = day 0) were given 115 mg of naloxone (NAL), an opioid antagonist, in saline i.v. (n = 5) or saline Lv. (n = 5). Jugular blood was collected at 15 min intervals for 2 hr before and 4 hr after treatment. Serum LH concentrations were 0.4 ± 0.1 ng/ml before NAL treatment, increased (P<.01) to 4.3 ± 0.7 ng/ml at 15 min following NAL treatment and returned to control concentrations by 75 minutes. Serum PRL concentrations were 5.0 ± 0.1 ng/ml before NAL treatment, increased (P<.05) to 14.8 ± 2.9 ng/ml at 30 min following NAL treatment and returned to control concentrations by 120 minutes. Serum LH and PRL concentrations were 0.5 ± 0.1 ng/ml and 5.2 ± 0.4 ng/ml, respectively, at 15 min following saline treatment and remained unchanged throughout the blood sampling period. Four of the 5 NAL treated gilts responded with an increase in both serum LH and PRL concentrations. The mean of serum progesterone concentrations, quantitated in samples taken every 2 hr, were similar for controls (22.7 ± 1.8 ng/ml) and NAL (26.5 ± 1.4 ng/ml) treated gilts. The gilt which failed to respond to NAL had nondetectable concentrations of serum progesterone and was excluded from analysis. These data indicate that the opioids modulate LH and PRL secretion during the luteal phase of the estrous cycle.     

Gossypol inhibits LH‐induced steroidogenesis in bovine theca cells

Gossypol, a polyphenolic aldehyde found in cottonseed, has been shown to perturb steroidogenesis in granulosa and luteal cells of rats, pigs and cattle. However, little is known about the direct effect of gossypol on theca cell functions in any species. The present study was conducted to investigate the effect of gossypol on the steroidogenesis and the expression of genes involved in it in cultured bovine theca cells. Theca cells were isolated from healthy preovulatory follicles and were cultured in the presence of luteinizing hormone (LH) for up to 7 days. During the culture period, main steroid products of the theca cells shifted from androstenedione (A4) at day 1 to progesterone (P4) from day 2 onward. At days 1 and 7, theca cells were treated with gossypol (0‐25 μg/mL) for 24 h. Gossypol inhibited LH‐stimulated theca cell A4 and P4 production in a dose‐dependent manner at both occasions. The viability of theca cells was not affected by gossypol at any doses used. Gossypol down‐regulated expressions of steroidogenic enzymes CYP11A1, HSD3B1 and CYP17A1, but not that of LHR. These results indicate that gossypol inhibits thecal steroidogenesis through down‐regulating gene expressions of steroidogenic enzymes but without affecting cell viability in cattle.     

Endocrine changes in sows exposed to elevated ambient temperature during lactation

Seven sows were placed into one of two environmental chambers at 22 C, 5 d prior to farrowing. On day 9 of lactation, one chamber was changed to 30 C (n = 4) and the other remained at 22 C (n = 3). On days 24 and 25, blood samples were collected every 15 min for 9 hr and 7 hr, respectively. On day 24, thyrotropin releasing hormone (TRH) and gonadotropin releasing hormone (GnRH) were injected iv at hour 8. On day 25 naloxone (NAL) was administered iv at hour 4 followed 2 hr later by iv injection of TRH and GnRH. Milk yield and litter weights were similar but backfat thickness (BF) was greater in 22 C sows (P less than .05) compared to 30 C sows. Luteinizing hormone (LH) pulse frequency was greater (P less than .003) and LH pulse amplitude was less (P less than .03) in 22 C sows. LH concentrations after GnRH were similar on day 24 but on day 25, LH concentrations after GnRH were greater (P less than .05) for 30 C sows. Prolactin (PRL) concentrations were similar on days 24 and 25 for both groups. However, PRL response to TRH was greater (P less than .05) on both days 24 and 25 in 30 C sows. Growth hormone (GH) concentrations, and the GH response to TRH, were greater (P less than .0001) in 30 C sows. Cortisol concentrations, and the response to NAL, were less (P less than .03) in 30 C sows. NAL failed to alter LH secretion but decreased (P less than .05) PRL secretion in both groups of sows. However, GH response to NAL was greater (P less than .05) in 30 C sows. Therefore, sows exposed to elevated ambient temperature during lactation exhibited altered endocrine function.     

N-methyl-d,l-aspartate modulation of pituitary hormone secretion in the pig: Role of opioid peptides

Sixteen ovariectomized (OVX) mature gilts, averaging 139.6 ± 3.1 kg body weight (BW) were assigned randomly to receive either progesterone (P, 0.85 mg/kg BW, n=8) or corn oil vehicle (OIL, n=8) injections im twice daily for 10 d. On the day of experiment, all gilts received either the EAA agonist, N-methyl-d,l-aspartate (NMA; 10 mg/kg BW, iv) alone or NMA plus the EOP antagonist, naloxone (NAL, 1 mg/kg BW, iv), resulting in the following groups of 4 gilts each: OIL-NMA, OIL-NMA-NAL, P-NMA and P-NMA-NAL. Blood samples were collected via jugular cannula every 15 min for 6 hr. All pigs received NMA 5 min following pretreatment with either 0.9% saline or NAL 2 hr after blood collection began and a GnRH challenge 3 hr after NMA. Administration of NMA suppressed (P<0.03) LH secretion in OIL-NMA gilts and treatment with NAL failed to reverse the suppressive effect of NMA on LH secretion in OIL-NMA-NAL gilts. Similar to OIL-NMA gilts, NMA decreased (P<0.03) mean serum LH concentrations in P-NMA gilts. However, in P-NMA-NAL gilts, serum LH concentrations were not changed following treatment. All gilts responded to GnRH with increased (P<0.01) LH secretion. Additionally, administration of NMA increased (P<0.01) growth hormone (GH) and prolactin (PRL) secretion in both OIL-NMA and P-NMA gilts, but this increase in GH and PRL secretion was attenuated (P<0.01) by pretreatment with NAL in OIL-NMA-NAL and P-NMA-NAL gilts. Serum cortisol concentrations increased (P<0.01) in all gilts and the magnitude of the cortisol response was not different among groups. In summary, results of the present study confirmed previous findings that NMA suppresses LH secretion in both oil- and P-treated OVX gilts, but we failed to provide definitive evidence that EOP are involved in the NMA-induced suppression of LH secretion. However, NMA may, in part, activate the EOP system which in turn increased GH and PRL secretion in the gilt.     

Effect of morphine on serum gonadotropin concentrations in postpartum beef cows

Morphine (M), an opioid agonist, was administered to postpartum (PP) Angus cows to investigate opioid modulation of gonadotropin secretion. In Exp. 1, eight PP cows (36.9 +/- 2.3 d) received either M (1 mg/kg; n = 4) or saline solution (S) (n = 4) via i.v. injection 36 h after calf removal. Morphine decreased (P less than .01) the number of serum LH pulses (3.0 +/- 1.1 pre- vs .3 +/- .3 post-pulses/h) and, compared with pretreatment values (3.3 mg/ml), decreased (P less than .05) mean LH at 105 min (2.1 ng/ml) through 270 min 1.9 ng/ml +/- .4). Serum prolactin (PRL) increased (P less than .01) following M from 16.4 ng/ml to a peak of 59.3 ng/ml (+/- 3.9). Serum FSH concentrations were unaffected. In Exp. 2, M (.31 mg/kg i.v. injection followed by .15 mg/(kg.h) infusion; n = 6) or S (n = 6) treatments were given for 7 h beginning 36 h after calf removal. Serum LH was similar between groups during the pretreatment and the first 6 h of infusion, but M decreased (P less than .001) the number of serum LH pulses (.44 +/- .09 vs .06 +/- .04 pulses/h). Morphine increased (P less than .05) serum PRL. It is concluded that M differentially modulated gonadotropin secretion in the cow such that PRL increased, LH decreased and FSH was unchanged.     

Expression of adiponectin and its receptors (AdipoR1 and AdipoR2) in chicken ovary: potential role in ovarian steroidogenesis

Adiponectin and its receptors (AdipoR1 and AdipoR2) mRNAs are expressed in various chicken tissues including ovary. However, the cellular expression and the role of adiponectin system have never been investigated in chicken ovary. Here, we have shown that the level of adiponectin mRNA is about 10- to 30-fold higher (p < 0.001) in theca cells than in granulosa cells from each hierarchical yellow follicle studied (F4–F1). In contrast, the level of AdipoR1 mRNA expression was about two-fold lower in theca cells than in granulosa cells (p < 0.05) whereas those of AdipoR2 was similar in both ovarian cells. Whereas expression of adiponectin mRNA increased with follicular differentiation in theca cells, it decreased in granulosa cells. In contrast, mRNA expression of AdipoR1 and AdipoR2 in both theca and granulosa cells remained stable during yellow follicle development. To determine whether adiponectin is involved in the ovarian steroidogenesis, LH (100 ng/ml)-, FSH (100 ng/ml)- and IGF-1 (100 ng/ml)-induced progesterone production was measured in absence or presence of human recombinant adiponectin (10 μg/ml) for 36 h in cultured granulosa cells from F1, F2 and mixed F3 and F4 follicles. In absence of LH, FSH and IGF-1, adiponectin treatment had no effects on progesterone production whatever vitollegenic follicle studied. However, it increased by about two-fold IGF-1-induced progesterone secretion in F2 and F3/4 follicles whereas it halved progesterone production in response to gonadotropins (LH and FSH) in F3/4 follicles. Thus, in chicken, adiponectin, mainly expressed in theca cells, could exert paracrine or autocrine effect on the ovarian steroidogenesis.     

N-methyl-d,l-aspartate stimulates growth hormone and prolactin but inhibits luteinizing hormone secretion in the pig.

The effects of n-methyl-d,l-aspartate (NMA), a neuroexcitatory amino acid agonist, on luteinizing hormone (LH), prolactin (PRL) and growth hormone (GH) secretion in gilts treated with ovarian steroids was studied. Mature gilts which had displayed one or more estrous cycles of 18 to 22 d were ovariectomized and assigned to one of three treatments administered i.m.: corn oil vehicle (V; n = 6); 10 micrograms estradiol-17 b/kg BW given 33 hr before NMA (E; n = 6); .85 mg progesterone/kg BW given twice daily for 6 d prior to NMA (P4; n = 6). Blood was collected via jugular cannulae every 15 min for 6 hr. Pigs received 10 mg NMA/kg BW i.v. 2 hr after blood collection began and a combined synthetic [Ala15]-h GH releasing factor (1-29)-NH2 (GRF; 1 micrograms/kg BW) and gonadotropin releasing hormone (GnRH; .2 micrograms/kg BW) challenge given i.v. 3 hr after NMA. NMA did not alter LH secretion in E gilts. However, NMA decreased (P < .02) serum LH concentrations in V and P4 gilts. Serum LH concentrations increased (P < .01) after GnRH in all gilts. NMA did not alter PRL secretion in P4 pigs, but increased (P < .01) serum PRL concentrations in V and E animals. Treatment with NMA increased (P < .01) GH secretion in all animals while the GRF challenge increased (P < .01) serum GH concentrations in all animals except in V treated pigs. NMA increased (P < .05) cortisol secretion in all treatment groups. These results indicate that NMA inhibits LH secretion and is a secretagogue of PRL, GH and cortisol secretion with ovarian steroids modulating the LH and PRL response to NMA.     

Effects of dopamine, norepinephrine and serotonin on secretion of luteinizing hormone, follicle-stimulating hormone and prolactin in ovariectomized, pituitary stalk-transected ewes

Two experiments were conducted in ovariectomized, pituitary stalk-transected ewes to determine if dopamine (DA), norepinephrine (NE) or serotonin (5-HT) alter secretion of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL). In experiment 1, ewes were infused (iv) with saline (control), DA (66 micrograms/kg/min), NE (6.6 micrograms/kg/min) or 5-HT (6.6 micrograms/kg/min). Treatments did not alter pulse frequency, but 5-HT increased (P less than .05) amplitude of pulses of LH and mean concentrations of LH, DA and NE were without effect on basal secretion of LH. DA but not NE or 5-HT decreased (P less than .05) the release of LH in response to gonadotropin hormone-releasing hormone (GnRH, 25 micrograms, im). Concentrations of FSH were not affected by treatments. Secretion of PRL was reduced (P less than .05) by treatment with DA and NE but not 5-HT. Each amine reduced (P less than .05) the release of PRL in response to thyrotropin-releasing hormone (TRH; 3 micrograms, im). In experiment 2, ewes were given DA at doses of 0, 0.66, 6.6 or 66.0 micrograms/kg/min, iv. No dose altered basal LH, but each dose reduced (P less than .05) basal and TRH-induced release of PRL. Key findings from these studies include direct pituitary action for: (1) 5-HT enhanced basal secretion of LH, (2) suppression of GnRH-induced secretion of LH by DA. (3) DA and NE inhibition of PRL secretion, and (4) DA, NE and 5-HT inhibition of release of PRL in response to TRH.     

Prolactin signaling in porcine adrenocortical cells: involvement of protein kinases

Prolactin (PRL) was found to have a stimulatory effect on adrenal steroidogenesis in vivo and in vitro in several species including pigs. PRL signal transduction pathways, however, in adrenocortical cells are poorly recognized. Therefore, the goal of this paper is to ascertain the involvement of protein kinase C (PKC) and tyrosine kinases in PRL signaling in porcine adrenal cortex. Adrenals were harvested from locally slaughtered mature gilts. Cortical cells were dispersed by sequential treatment with collagenase. The cells were seeded into 24-well culture plates at a density of 3×10⁵/mL. Cells were incubated with or without PRL (500 ng/mL), ACTH (5 nM—a positive control), tyrosine kinase inhibitor—genistein (1; 2.5 or 5 μM), PKC inhibitor—sphingosine (20–1000 nM) and PKC activators—diacylglycerol (DiC8; 10–100 μM) and phorbol ester (PMA; 1–1000 nM). All incubations were performed for 8 h (95% air and 5% CO₂, 37°C). PRL and ACTH (P<0.05) increased cortisol and androstenedione (A₄) secretion. DiC8 and PMA mimicked the stimulatory effect of PRL. Sphingosine (P<0.05) suppressed basal and PRL-stimulated steroid secretion. Genistein inhibited (P<0.05) PRL-stimulated cortisol secretion and enhanced (P<0.05) basal and PRL-stimulated A₄ secretion. Moreover, PKC activation was assessed by measuring the specific association of [³H]phorbol dibutyrate ([³H]PDBu) with adrenocortical cells after treatment with PRL or ionomycin (a positive control). PRL (within 2–3 min) and ionomycin (within 2–5 min) increased (P<0.05) specific binding of [³H]PDBu to the porcine adrenocortical cells. In addition, PRL did not augment the cortisol and A₄ secretion by PKC-deficient adrenocortical cells. In conclusion, presented results support the hypothesis that PKC and tyrosine kinases are involved in PRL signaling in adrenocortical cells in pigs. Moreover, activation of PKC is associated with the increased secretion of cortisol and A₄.     

BMP-2 and -6 modulate porcine theca cell function alone and co-cultured with granulosa cells

Bone morphogenetic proteins (BMPs) are emerging as a family of proteins crucial in the regulation of fertility and ovulation rate. We have shown that porcine theca cells express BMP receptors, however, there is a paucity of information regarding the effect(s) of BMPs on theca cell function. The purpose of this study was to investigate the effects of BMP-2 and -6 on theca cells cultured under serum-free conditions in terms of steroidogenesis, cAMP release and proliferation. The study was further extended to determine whether BMP responses in theca cells are affected by the addition of granulosa cells to the culture system. Both BMPs suppressed progesterone and androstenedione synthesis by theca cells (P < 0.05) after 144 h in culture. Oestradiol synthesis was suppressed (P < 0.05) by BMP-2, but not BMP-6, and theca cell proliferation was stimulated (P < 0.05) by BMP-6, but not BMP-2, after 144 h in culture. Both BMP-2 and -6 inhibited cAMP release (P < 0.05) by theca cells. Furthermore, progesterone and androstenedione synthesis by co-cultured theca and granulosa cells were suppressed (P < 0.05) whereas cell proliferation was stimulated (P < 0.05). These results provide strong evidence for a functional BMP system in the porcine ovary and that theca cells are responsive to BMPs in terms of steroidogenesis and proliferation. BMP-2 and -6 may have a role as luteinisation inhibitors in this polyovular species.     

Long-term feed intake regulation in sheep is mediated by opioid receptors

These experiments were conducted to determine if 1) syndyphalin-33 (SD33), a mu-opioid receptor ligand, affects feed intake; 2) SD33 effects on feed intake are mediated by actions on opioid receptors; and 3) its activity can counteract the reduction in feed intake associated with administration of bacterial endotoxin. In Exp. 1, 5 mixed-breed, castrate male sheep were housed indoors in individual pens. Animals had ad libitum access to water and concentrate feed. Saline (SAL; 0.9% NaCl) or SD33 (0.05 or 0.1 micromol/kg of BW) was injected i.v., and feed intake was determined at 2, 4, 6, 8, 24, and 48 h after the i.v. injections. Both doses of SD33 increased (at least P < 0.01) feed intake at 48 h relative to saline. In Exp. 2, SAL + SAL, SAL + SD33 (0.1 micromol/kg of BW), naloxone (NAL; 1 mg/kg of BW) + SAL, and NAL + SD33 were injected i.v. Food intake was determined as in Exp. 1. The SAL + SD33 treatment increased (P = 0.022) feed intake at 48 h relative to SAL + SAL. The NAL + SAL treatment reduced (at least P < 0.01) feed intake at 4, 6, 8, 24, and 48 h, whereas the combination of NAL and SD33 did not reduce feed intake at 24 (P = 0.969) or 48 h (P = 0.076) relative to the saline-treated sheep. In Exp. 3, sheep received 1 of 4 treatments: SAL + SAL, SAL + 0.1 micromol of SD33/kg of BW, 0.1 microg of lipopolysaccharide (LPS)/kg of BW + SAL, or LPS + SD33, and feed intake was monitored as in Exp. 1. Lipopolysaccharide suppressed cumulative feed intake for 48 h (P < 0.01) relative to saline control, but SD33 failed to reverse the reduction in feed intake during this period. These data indicate that SD33 increases feed intake in sheep after i.v. injection, and its effects are mediated via opioid receptors. However, the LPS-induced suppression in feed intake cannot be overcome by the opioid receptor ligand, SD33.     

GnRH antagonist inhibition of gonadotropin and steroid secretion in boars in vivo and steroid production in vitro

The hormone GnRH has a stimulatory effect on gonadotropin synthesis and secretion. The objective of the first study was to evaluate concentrations of FSH and LH in plasma of boars after successive treatment with SB75, a GnRH antagonist. Thirteen boars greater than 1 yr of age (eight White Composite [WC] and five Meishan [MS]) were injected once daily with SB75 (10 microg/kg of body weight) for 4 d. Plasma concentrations of LH and testosterone (T) decreased after 1 h from the first dose of SB75. After 12 h of treatment, LH gradually returned to pretreatment concentrations, but T remained suppressed (< 2 ng/mL) until after the last injection of SB75. There was a modest, but significant, reduction in FSH during treatment with SB75. The prolonged inhibitory effect of SB75 on suppression of plasma T concentrations, in the presence of pretreatment concentrations of LH, implied direct effects of SB75 at the testis. In the second experiment, testicular tissue from adult boars was incubated in the presence of three doses of human chorionic gonadotropin (hCG; 0, .5, and 5 IU) with SB75 (250 ng/mL) or with Deslorelin, a GnRH agonist (500 ng/mL). Samples of media were collected every hour for 3 h, and concentrations of T and estrone (E1) were determined by RIA. Concentrations of T and E1 increased with time in response to treatment with hCG. Co-treatment with SB75 decreased media concentrations of T (P < .01) and E1 (P < .03) compared to controls (77.9 vs 85.7 +/- 2.0 and 4.7 vs 5.3 +/- .2 ng/g). In contrast, treatment with Deslorelin had no effect on the amount of T (P > .50) or E1 (P > .26) released with all dosages of hCG. These results indicate that a GnRH antagonist has a direct effect on the testis, decreasing amounts of T and E1 released from the Leydig cells; however, treatment with a GnRH agonist had no direct effect on release of these gonadal steroids. Thus, it remains unresolved whether the site of action of GnRH antagonist on testicular steroidogenesis is through a testicular GnRH receptor or through some other mechanism.     

Evidence for endogenous opioid modulation of serum luteinizing hormone and prolactin in the steer

Opioid modulation of LH and prolactin (PRL) concentrations in Angus steers was investigated. In Exp. 1, morphine sulfate (M) was administered at either 1, 2 or 3 mg/kg BW (n = 4) as an i.v. injection. Blood samples were obtained at 15-min intervals for 4 h pre- and post-treatment for serum hormone analyses. Mean serum LH concentration and number of LH secretory pulses decreased (P less than .1) for 2 h after M (4.1 to nadir of 2.4 ng/ml, and .33 vs. .21 pulses/h; pre- vs post-treatment). Luteinizing hormone pulse amplitude decreased (P less than .01; 7.3 vs 2.6 ng/ml; pre- vs post-treatment) during the 2 h following M. Prolactin concentrations increased 126.6%, 170.6% and 187.6% following 1, 2 and 3 mg M/kg BW, respectively (P less than .05, 1 vs 2; P less than .01, 1 vs 3). In Exp. 2, either saline solution (S, n = 6) or M (.31 mg/kg BW, i.v. injection followed by .15 mg/(kg.h) infusion; n = 6) was given for 7 h. Concentration of LH was unaffected. Response of LH to naloxone was determined in Exp. 3. Blood samples were obtained for 2 h pre- and post-administration of either naloxone (1 mg/kg BW, i.v. injection; n = 5) or S (n = 5). Response of LH at 15, 30 and 45 min posttreatment was greater (P less than .05) in naloxone- compared with S-treated steers. In summary, M had no significant effect on serum LH concentration or LH pulse frequency, but it decreased pulse amplitude and increased serum PRL concentrations. In contrast, naloxone increased LH secretion. These observations taken together indicate a physiological role for opioid modulation of LH and PRL secretion in the steer.