Porcine theca cells produce immunoreactive beta-endorphin and change steroidogenesis in response to opioid agonist.

In earlier in vitro experiments opioids affected steroidogenesis in porcine luteal and granulosa cells. The present studies were undertaken to examine the effects of FK 33-824 (opioid agonist) alone or in combination with LH, PRL or naloxone (NAL, opioid antagonist) on steroidogenesis in cultured porcine theca cells. Moreover, we have tested beta-endorphin-like immunoreactivity (beta-END-LI) concentrations in culture media under control conditions and following treatments of theca cells with LH, PRL, progesterone (P4), oestradiol (E2) or testosterone (T). FK 33-824 and NAL significantly increased P4 release by theca cells and inhibited stimulatory effect of LH on this steroid output. PRL-induced P4 secretion from the cells was blunted only by FK 33-824. Secretion of androstenedione (A4) and T was essentially elevated in the presence of FK 33-824 and this potentiation of both androgen release was completely abolished by PRL. NAL blocked stimulatory effect of the opioid agonist only in case of T. Secretion of oestradiol and oestrone was completely free from the influence of both the opioid agonist and antagonist. Pig theca cells were able to produce beta-END-LI but none of tested hormones (LH, PRL, P4, E2 and T alone or in combination) significantly affected this production. In conclusion, these data indicate that porcine theca cells may produce beta-END-LI and change their steroidogenesis in response to opioid peptides.     

Effects of exogenous estradiol-17 beta and progesterone on naloxone-reversible inhibition of the release of luteinizing hormone in ewes

The role of endogenous opioids in controlling luteinizing hormone (LH) secretion was studied by injecting the opioid antagonist naloxone into intact and ovariectomized ewes that were treated with estradiol-17 beta (E2) and progesterone (P4). The existence of a naloxone-reversible inhibition of LH release was examined in five experiments using a total of 52 mature ewes. Naloxone at a dosage of 1 mg/kg disinhibited release of LH and abruptly increased serum concentrations of LH in a variety of experimental models. This naloxone-reversible inhibition of LH secretion was apparent in all experimental models that involved P4-induced inhibition of basal LH secretion but not in one model in which P4 inhibited the LH surge. Specific effects of E2 on naloxone-reversible inhibition of LH varied among experimental models. When prolonged administration of P4 alone appeared to lose its LH-inhibitory potency, E2 restored inhibition of LH as well as the naloxone-reversible state. Whenever E2 acted synergistically to suppress basal LH secretion in models involving brief (5 d) exposure to P4, E2 appeared to antagonize the naloxone-reversible state. In summary, P4-induced suppression of LH secretion appeared to be mediated by endogenous opioids, but the apparent interaction of E2 and opioids in LH suppression varied among experiments.     

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.     

Ingestive responses to mu and delta opioid receptor agonists in the domestic fowl

1. Four experiments were conducted using the highly specific mu and delta opioid receptor agonists morphiceptin (B-casomorphin 1-4, amide) or [Met5]-enkephalin, respectively, to evaluate the effect of mu and delta opioid receptor agonists on ingestive behaviour in the domestic fowl. 2. Intracerebroventricular (ICV) administration of 0.625, 1.25, 2.5 and 5.0 micrograms of morphiceptin significantly stimulated drinking, while having no effect on feeding. Intramuscular injection of 0.5, 1.5 and 3.0 mg morphiceptin/kg body weight induced a significant increase in feeding, whereas drinking was not altered. 3. ICV administration of 0.625, 1.25, 2.5 and 5.0 micrograms [Met5]-enkephalin, as well as intramuscular injection of 0.5, 1.5 and 3.0 mg [Met5]-enkephalin significantly stimulated feeding while having no effect on drinking. 4. These results suggest that, in the central nervous system, mu opioid receptor agonists stimulate drinking and delta receptor agonists stimulate feeding. At sites outside the blood-brain barrier, both mu and delta opioid receptor agonists stimulate feeding.     

Effect of centrally administered opioid receptor agonists on CSF and plasma oxytocin concentrations in dogs

OBJECTIVE: To measure oxytocin concentrations in blood and CSF following central administration of opioid agonists in dogs. ANIMALS: 5 male dogs. PROCEDURE: In a crossover design, CSF and blood were collected immediately before and 15 and 30 minutes after cisternal administration of D-Ala2, MePhe4, Gly-ol-enkephalin (DAMGO, a mu-receptor agonist); D-Pen, pCl-Phe4, D-Pen5-enkephalin (a delta-receptor agonist); U50488H (a kappa-receptor agonist); morphine; and saline (0.9% NaCl) solution. RESULTS: Plasma oxytocin concentration was significantly increased 15 minutes after administration of DAMGO and 30 minutes after administration of U50488H, compared with concentrations obtained after administration of saline solution. Concentration of oxytocin in CSF was significantly decreased 30 minutes after administration of U50488H, compared with concentration after administration of saline solution. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that in male dogs, activation of centrally located mu and kappa receptors elicits an overall excitatory effect on neurons that regulate peripheral release of oxytocin, whereas activation of centrally located kappa receptors elicits an overall inhibitory effect on neurons that regulate central release. These results are in contrast to those reported for other species, in which opioids have a pronounced inhibitory effect on release of oxytocin from the neurohypophysis.     

Naloxone-reversible inhibition of luteinizing hormone in postpartum ewes: effects of suckling and season

Involvement of endogenous opioids in inhibition of luteinizing hormone (LH) release and stimulation of prolactin (PRL) release was investigated by injecting the opioid antagonist naloxone into 18 ewes on d 7 and 8, d 12 and 13, and d 18 and 19 postpartum. Compared with control injections of saline, iv naloxone (1 mg/kg) increased serum concentrations of LH and decreased serum PRL in samples collected 15, 30 and 45 min after each injection. Ewes lambing in the spring (March) or autumn (September and October) that nursed one or two lambs did not differ in their LH and PRL responses to naloxone. Autumn-lambing ewes from which lambs were weaned within 1 d after parturition did not differ from ewes of the autumn-nursed group in any of the following characteristics: 1) serum LH increases following naloxone, 2) basal secretion of LH, 3) postpartum interval to first increase in serum progesterone and 4) relative decrease in serum PRL after naloxone despite large differences in basal PRL secretion. In summary, postpartum expression of a naloxone-reversible inhibition of LH release and stimulation of PRL secretion did not depend on suckling stimuli or differ between autumn and spring parturitions.     

Gonadotropin-releasing hormone inhibition of LH stimulated progesterone secretion by porcine granulosa cells in vitro

GnRH has several direct actions on rat granulosa cells. Specific receptors for GnRH have been demonstrated on rat and human ovaries. Whether the porcine ovary has specific receptors for GnRH is still debated and the physiological actions of GnRH on porcine granulosa cells have not yet been clarified. Consequently, we have examined the actions of a GnRH agonist (GnRHa) on basal and LH stimulated progesterone secretion by porcine granulosa cells. GnRHa inhibited both basal and LH stimulated progesterone secretion by granulosa cells from medium (3-5 mm) and large (6-10 mm) antral follicles during 3 day incubations. LH stimulated progesterone secretion was more sensitive to inhibition than basal progesterone secretion. Studies on the time course for GnRHa inhibition of progesterone secretion indicated that the decrease in progesterone secretion occurred 48 to 72 hr after first exposure to GnRHa. Earlier inhibition occurred in only a fraction of the experiments. GnRHa did not have to be present during the time when inhibition occurred. Incubations of 2 days with GnRHa were just as effective as 3 day incubations at inhibiting progesterone secretion on day 3. Furthermore, a 30 min exposure to GnRHa on day 1 was just as inhibitory as a full 2 day incubation with GnRHa in inhibiting LH stimulated progesterone secretion on day 3. Incubation of the cells for 3 days prior to exposure of the cells to GnRHa did not alter the time course for GnRHa action. GnRHa did not alter the DNA content of the cultures in up to 6 day incubations or the number of viable cells attached to the wells in up to 3 day incubations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of CD14/TLR4 antagonist on GnRH/LH secretion in ewe during central inflammation induced by intracerebroventricular administration of LPS

Background: Immune stress induced by lipopolysaccharide(LPS) influences the gonadotropin-releasing hormone(GnRH)/luteinizing hormone(LH) secretion. Presence of LPS interacting Toll-like receptor(TLR) 4 in the hypothalamus may enable the direct action of LPS on the GnRH/LH secretion. So, the aim of the study was to investigate the influence of intracerebroventricular(icv) injection of TLR4 antagonist on GnRH/LH secretion in anestrous ewes during LPS-induced central inflammation. Animals were divided into three groups icv-treated with: Ringer-Locke solution, LPS and TLR4 antagonist followed by LPS.Results: It was demonstrated that TLR4 antagonist reduced LPS-dependent suppression of GnRH gene expression in the preoptic area and in the medial basal hypothalamus, and suppression of receptor for GnRH gene expression in the anterior pituitary gland. It was also shown that TLR4 antagonist reduced suppression of LH release caused by icv injection of LPS. Central administration of LPS stimulated TLR4 gene expression in the medial basal hypothalamus.Conclusions: It was indicated that blockade of TLR4 prevents the inhibitory effect of centrally acting LPS on the GnRH/LH secretion. This suggests that some negative effects of bacterial infection on the hypothalamic-pituitary-gonadal axis activity at the hypothalamic level may be caused by central action of LPS acting through TLR4.     

Effects of the phytoestrogen,genistein, and protein tyrosine kinase inhibitor–dependent mechanisms on steroidogenesis and estrogen receptor expression in porcine granulosa cells of medium follicles

The use of soy-based products in pig diets had raised concerns regarding the reproductive toxicity of genistein, the predominant isoflavone in soybeans. Genistein was reported to exhibit weak estrogenic activity but its mechanism of action is not fully recognized. The aim of the study was to examine the in vitro effects of genistein on (1) progesterone (P₄) and estradiol (E₂) secretion by porcine granulosa cells harvested from medium follicles, (2) the viability of cultured granulosa cells, and (3) the mRNA and protein expression of estrogen receptors α and β (ERα and ERβ) in these cells. In addition, to verify the role of protein tyrosine kinase (PTK)–dependent mechanisms possibly involved in genistein biological action, we tested the effects of lavendustin C, the nonsteroidal PTK inhibitor, on granulosa cell steroidogenesis. We found that genistein inhibited (P < 0.05) basal P₄ secretion by granulosa cells harvested from medium follicles of pigs. In contrast, lavendustin C did not affect basal P₄ secretion by the cells. Moreover, genistein increased (P < 0.05) basal granulosal secretion of E₂. In contrast, lavendustin C did not alter basal E₂ secretion by porcine granulosa cells. In addition, we demonstrated that genistein increased mRNA and protein expression of ERβ (P < 0.05) in the examined cells. The expression of ERα mRNA was not affected by genistein and ERα protein was not detected in the cultured granulosa cells of pigs. In summary, the genistein action on follicular steroidogenesis in pigs involved changes in the granulosal expression of ERβ. However, the genistein action on P₄ and E₂ production by granulosa cells harvested from medium follicles did not seem to be associated with PTK.     

The anti-gonadotropic effects of cytokines: the role of neuropeptides

The inhibitory effect of inflammation and endotoxins on the secretion of reproductive hormones from the hypothalamo-pituitary axis is well documented. A comparison of the luteinizing hormone (LH) suppressing effects of several pro-inflammatory cytokines revealed that centrally administered IL-1β was the most potent inhibitor of pituitary LH secretion; interleukin (IL)-1α and tumor necrosis factor (TNF)α were relatively less effective, whereas IL-6 was ineffective. This order of potency suggested that the anti-gonadotropic effects of an immune challenge are most likely attributable to the action of centrally released IL-1β, and this was supported by the demonstration that IL-1β suppressed hypothalamic luteinizing hormone releasing hormone (LHRH) release. We used a multifaceted approach to identify the afferent signals in the brain that convey immune messages to hypothalamic LHRH neurons. Pharmacological studies with specific antagonists of opioid receptor subtypes demonstrated that activation of the μ1 receptor subtype was required to transmit the cytokine signal. Furthermore, icv IL-1β upregulated hypothalamic POMC mRNA and increased the concentration and release of β-endorphin, the primary ligand of μ1 receptors. We have obtained evidence that IL-1β also enhanced the gene expression and concentration of tachykinins, a family of nociceptive neuropeptides in the hypothalamus. Blockade of tachykinergic NK2 receptors attenuated IL-1β induced inhibition of LH secretion. Collectively, these results demonstrate that IL-1β, generated centrally in response to inflammation, upregulates the opioid and tachykinin peptides in the hypothalamus. These two groups of neuropeptides are critically involved in relaying the cytokine signal to neuroendocrine neurons and causing the suppression of hypothalamic LHRH and pituitary LH release.     

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.     

Progesterone production in granulosa cells of the domestic fowl: effects of incubation media, pH, cell density and some other factors

The objective of this study was to determine the optimal conditions for the short term incubation of chicken granulosa cells. Compared to mechanically-dispersed cells, collagenase-treatment yielded granulosa cells of greater viability and responsiveness to ovine LH (oLH) stimulation. The rate of progesterone secretion by enzyme-dispersed chicken granulosa cells was subsequently compared in five different culture media during incubation periods of up to 24 hr. Under room air as the gas phase, Medium 199 (M199) containing Hank's salts and Ham's F-12 medium (F-12) were the most effective, whereas Krebs-Ringer bicarbonate-buffered glucose solution (KRBG) and Dulbecco's medium were the poorest. When pH and the ionic strength of KRBG was maintained by continuous gassing with O₂:CO₂ (95:5), progesterone production was similar to that obtained with Hepes-buffered M199. The pH optimum was found to be 7.4, although within the range of 6.6–8.5 granulosa cells remained responsive to LH stimulation. The optimal cell density was observed to be 1 × 10⁴ to 5 × 10⁵/ml. Although time course studies showed that both basal and stimulated progesterone production peaked by about 12 hr of incubation regardless of the media composition, the amounts released were significantly greater in M199 and F-12 during more prolonged (up to 24 hr) incubations. Glucose, a key medium ingredient for hormone-stimulated steroidogenesis, could be replace by pyruvate. On the other hand, lactate was inhibitory. It is concluded that the mature ovarian follicles of the domestic fowl are an excellent source of pure granulosa cells that can be obtained in high yield after a brief treatment with collagenase. These cells remain viable up to 24 hr and continue to produce large amounts of progesterone in response to LH when incubated in an appropriate medium and at optimal cell density.     

Development of a culture system for bovine granulosa cells: effects of growth hormone, estradiol, and gonadotropins on cell proliferation, steroidogenesis, and protein synthesis

The objectives of the present studies were 1) to develop a culture system that has the positive effect of serum on granulosa cell attachment and allows subsequent expression of hormonal effects in serum-free medium and 2) to determine the effect of insulin, epidermal growth factor (EGF), estradiol (E2), and growth hormone (GH) on growth, steroidogenesis, and(or) protein synthesis of bovine granulosa cells. Cells from small (1 to 5 mm) and large (greater than 8 mm) follicles were collected from cattle and cultured for either 4 or 6 d. When cells from small follicles were cultured, insulin (5 micrograms/ml) increased (P less than .05) cell numbers (cells x 10(5)/well) severalfold compared with controls. Alone, EGF (10 ng/ml), FSH (200 ng/ml), LH (200 ng/ml), E2 (2 micrograms/ml), or GH (0 to 1,000 ng/ml) had no effect on cell numbers. However, when included with insulin, 30, 100, and 300 ng/ml of GH increased (P less than .05) granulosa cell numbers on d 4 of culture. Insulin alone increased (P less than .05) progesterone production (ng.10(5) cells-1.24 h-1) by severalfold on d 4, but EGF, FSH, LH, or GH alone had no effect and E2 inhibited progesterone production. In the presence of insulin, FSH and GH (100 ng/ml) increased (P less than .05) progesterone production on d 4 of culture, whereas EGF (10 ng/ml) elicited a decrease (P less than .05) in production. In cells from both sizes of follicles, GH (300 ng/ml) increased synthesis of cellular proteins (greater than 10 kDa). In cells from only large follicles, LH (200 ng/ml) decreased synthesis and secretion of proteins (greater than or equal to 3.5 kDa). These results support the hypothesis that GH may have direct effects on bovine ovarian function.     

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.     

Endogenous opioid peptides and the control of gonadotrophin secretion

The endogenous opioid peptides are a group of recently discovered compounds which occur in the brain of a wide variety of species. Originally named because of their opiate-like activity, they have since been demonstrated to have multifaceted actions, one of which appears to be the modulation of luteinising hormone (LH) secretion. Because of the prime position of LH in the ovulatory process, this role for the opioids has attracted considerable interest. Their mode of action is essentially one of suppression and they work by inhibiting the release of hypothalamic gonadotrophin releasing hormone. Through this mechanism they have been implicated in the suppression of LH secretion during the prepubertal period and the modulation of LH during the oestrous cycle. It is well established that gonadal steroids suppress LH secretion by negative feedback upon the hypothalamic-pituitary axis, and this action may be brought about, in part, through intermediary opioidergic neurones. Much of the research to date has been carried out upon laboratory rodents and primates, but there is evidence now accruing that the opioids have similar actions in domestic animals. Knowledge of the role of these compounds may therefore aid in the understanding of an area of commercial importance, namely the control of ovulation in farm livestock.     

Evaluation of the interaction of mu and kappa opioid agonists on locomotor behavior in the horse.

This study was designed to determine the interactive effects of mu and kappa opioid agonists on locomotor behavior in the horse. Three doses of a mu agonist, fentanyl (5, 10, 20 micrograms/kg) and a kappa agonist U50,488H (30, 60, 120 micrograms/kg) were administered in a random order to six horses. Locomotor activity was measured using a two minute footstep count. Each dose of U50,488H was then combined with 20 micrograms/kg of fentanyl to determine the interactive effects of the drugs on locomotor activity. A significant increase in locomotor activity was seen with 20 micrograms/kg of fentanyl and all the drug combinations. The combination of U50,488H with fentanyl resulted in an earlier onset of locomotor activity. At the highest doses of the combination (U50,488H 120 micrograms/kg, fentanyl 20 micrograms/kg), the duration of locomotor activity was significantly increased when compared to the other doses. We conclude that locomotor activity is maintained or enhanced in horses when a receptor specific kappa agonist is combined with a mu receptor agonist.     

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.     

Follicle-stimulating hormone and luteinizing hormone regulate the synthesis mechanism of dihydrotestosterone in sheep granulosa cells

Steroid hormones and receptors play important roles in female reproduction, and their expression patterns affect follicular growth and development. To examine the expression of dihydrotestosterone (DHT) synthases (5α-reductases (5α-red1 and 5α-red2)) and androgen receptor (AR) during follicular development, and the regulation of DHT signalling by follicle-stimulating hormone (FSH) and luteinizing hormone (LH), we have used enzyme-linked immunosorbent assays, quantitative real-time polymerase chain reaction, immunohistochemical staining and Western blotting to examine DHT synthesis in small (≤2 mm), medium (2–5 mm) and large (≥5 mm) sheep follicles. Expression of 5α-red1, 5α-red2 and AR was observed in ovine ovaries, and with the development of follicles, the expressions of 5α-red1 and 5α-red2 mRNA and protein increased, but the levels of AR mRNA, protein and DHT level decreased. In addition, granulosa cells were treated with FSH (0.01, 0.1 and 1 international unit (IU)/ml), LH (0.01, 0.1 and 1 IU/ml) and testosterone (T, 10^–7 M) to evaluate the effects of FSH and LH on DHT and oestradiol (E2) synthesis and 5α-red1, 5α-red2 and AR expression. We found that FSH and LH upregulated 5α-red1 and 5α-red2 in sheep granulosa cells, but downregulated the concentration of DHT and expression of AR. Meanwhile, FSH and LH significantly upregulated the expression of aromatase (P450arom) and secretion of E2. This result indicates that although FSH and LH promote the expression of 5α-red1 and 5α-red2, T is not transformed into DHT, but E2. This study reveals the reason why DHT concentration is downregulated in large follicles and lays a foundation for further exploring the synthesis mechanism of DHT during follicular development.     

Opioid control of growth hormone in the suckled sow is primarily mediated through growth hormone releasing factor

Endogenous opioid peptides mediate the effect of suckling on LH and PRL in the domestic pig. However, the role of opioids in modulating GH during lactation in swine is not known. Primiparous sows that had been immunized against GRF(1-29) conjugated to human serum albumin (GRF-HSA, n = 5) or HSA (n = 4) were used to determine changes in GH after naloxone. Treatments were imposed in all sows on day 21 of lactation when antibody titers were 9100 +/- 1629. All sows received (i.v.) naloxone (0.25 mg/kg) or saline (0.0125 ml/kg) at 15 min intervals for 165 min. Active immunization against GRF-HSA during lactation decreased (P less than 0.05) mean concentration (4.8 +/- 0.2 vs 2.6 +/- 0.1 ng/ml) and frequency (1.5 +/- 0.3 vs 0.4 +/- 0.2 peaks/4 hr). Concentrations of LH and PRL were similar in GRF-HSA and HSA immunized sows. Naloxone suppressed (P less than 0.05) GH in all sows. In HSA sows, naloxone abolished episodic release of GH and decreased average, but not basal, concentrations of GH. In sows immunized against GRF-HSA, naloxone decreased (P less than 0.05) average and basal GH but failed to decrease frequency of GH release. Naloxone failed to alter frequency of LH release. Concentrations of PRL decreased (P less than 0.05) after naloxone in all sows. In conclusion, immunization against GRF-HSA blocked most of the effect of lactation on GH. Blocking opioid receptors with naloxone decreased GH and PRL in all sows. In contrast to previous findings naloxone had no effect on LH. Opioids alter concentrations of GH through a GRF dependent and GRF independent pathway.     

Endogenous opiates and the hypothalamic-pituitary-gonadal axis in male sheep

The effects of morphine and the opiate receptor antagonist, naloxone, on the secretory pattern of luteinizing hormone (LH) were assessed in male sheep. Morphine infusion (250 mg/hr) abruptly stopped LH pulsatile secretion in castrates (wethers) and decreased mean serum LH concentrations by nearly 70 percent. Response of the pituitary to exogenous LH releasing hormone was not affected by morphine suggesting that the effects of morphine on LH secretion were mediated through the hypothalamus. Estradiol-implanted wethers, characterized by a nonpulsatile LH secretory pattern, responded to intravenous injection of naloxone (20, 50 and 200 mg Lv.) with an immediate release (pulse) of L.H. Similarly, LH release was significantly increased following naloxone infusion (200 mg/hr for four hours) in intact rams and wethers implanted with testosterone or estradiol. In contrast, naloxone infusion altered the pattern of LH secretion in wethers but without affecting mean serum LH concentrations. These results support the notion that LH secretion in male-sheep is tonically regulated by endogenous opiates and further suggests that opioid modulation of the hypothalamic-pituitary-LH axis in sheep involves an interaction with the steroid negative feedback system.