The potential implication of neurokinin B in the modulation of prolactin secretion at the pituitary level in cyclic gilts.

Based on the previous studies, neurokinin B (NKB) participation in the modulation of prolactin secretion at the pituitary level can be assumed, but information concerning this topic is largely insufficient. Therefore, in the present study, we aimed: 1) to evaluate changes in the expression of NKB precursor (Tac3) and its receptor (Tacr3) genes as well as the content of NKB and TACR3 proteins in the porcine anterior pituitary throughout the estrous cycle (days 2 - 3, 9 - 10, 12 - 13, 15 - 16, 19 - 20); 2) to determine in vitro the influence of NKB on the expression of Prl, D2r and Trhr genes in the anterior pituitary cells (incubated for 4 h) as well as on prolactin secretion by these cells (incubated for 4 and 24 h) during chosen days of the estrous cycle (9 - 10, 15 - 16, 19 - 20). The experiments have shown alterations in the expression of Tacr3 mRNA and TACR3 protein content, but not in Tac3 mRNA and NKB protein. The treatment with NKB stimulated the expression of Prl (days 15 - 16), D2r (days 9 - 10) and Trhr (days 19 - 20) genes, but its potential to modulate prolactin secretion was observed only following 24-h incubation, specifically inhibition by NKB alone and stimulation by NKB with dopamine on days 19 - 10 of the cycle. These results indicate some implications of NKB in the modulation of prolactin secretion at the pituitary level in cyclic pigs, however further experiments are required to better clarify its role in this process.

The effect of selective agonists of opioid receptors on in vitro secretion of steroid hormones by porcine endometrium during the estrous cycle and early pregnancy.

Steroid hormones play an important role in the regulation of cyclic changes in the uterus and preparation of intrauterine environment for the egg fertilization, embryo implantation and maintenance of pregnancy. Their secretion by porcine uterus has been demonstrated. The present study aimed to establish the effect of opioid receptors (µ, δ and κ) activation by selective agonists (DAMGO, DPLPE and U 50.488, respectively) on in vitro secretion of steroid hormones (during 6-h and 24-h incubations) by the endometrial explants of gilts on days 2 - 3, 10 - 11, 12 - 3, 15 - 16, 18 - 20 of the estrous cycle and 10 - 11, 12 - 13, 15 - 16 of pregnancy. The agonists at certain of tested concentrations (10-9, 10-8 and 10-7 M) affected secretion of steroid hormones. Progesterone secretion was increased by µ-opioid receptor agonist on days 18 - 20 (6 h) and by δ-agonist on days 2 - 3 and 18 - 20 (24 h) of the cycle. During pregnancy (days 15 - 16), κ-agonist increased it (6 h), but µ-opioid agonist decreased (24 h). Androstenedione secretion was decreased during shorter incubation; by µ- and δ-receptor agonists on days 2 - 3, by all agonists on days 12 - 13, and by κ-receptor agonist on days 18 - 20 of the cycle. However, it was increased during longer incubation with agonists of κ- and µ-opioid receptors on days 10 - 11 and 18 - 20 of the cycle, respectively. Estradiol secretion was elevated by κ- and µ-agonists (6 h) on days 2 - 3 and 15 - 16 of the cycle, respectively, as well as following 24-h incubation with µ-agonist on days 15 - 16, and µ- and κ-agonists on days 18 - 20 of the cycle. During pregnancy, its secretion was increased (24 h) ondays 15 - 16 by µ- and κ-opioid agonists. Cortisol secretion did not significantly change (versus control) in response to applied treatments. These results indicate a potential involvement of EOPs in the modulation of endometrial steroidogenesis in the pig during the estrous cycle and pregnancy.

Periconceptional undernutrition affects in utero methyltransferase expression and steroid hormone concentrations in uterine flushings and blood plasma during the peri-implantation period in domestic pigs.

Female undernutrition during early pregnancy may affect the physiological pattern of genomic DNA methylation. We hypothesised that in utero DNA methylation may be impaired in females fed a restrictive diet in early pregnancy. In this study we evaluated whether poor maternal nutritional status, induced by applying a restricted diet during the peri-conceptional period, may influence: (1) the potential for in utero DNA methylation, expressed as changes in the mRNA expression and protein abundance of methyltransferases: DNA methyltransferase 1 (DNMT1) and DNMT3a in the endometrium and the myometrium, (2) the intrauterine microenvironment, measured as oestradiol 17ß (E2) and progesterone (P4) concentrations in uterine flushings and (3) plasma concentration of E2 and P4 during the peri-implantation period. Our results indicate that maternal peri-conceptional undernutrition affects maintenance and de novo DNA methylation in the endometrium, de novo methylation in the myometrium and a results in a decrease in intrauterine E2 concentration during the peri-implantation period. The intrauterine concentration of P4 and plasma concentrations of E2 and P4 did not change. These findings suggest that undernutrition during the earliest period of pregnancy, and perhaps the pre-pregnancy period, may create changes in epigenetic mechanisms in the uterus and intrauterine milieu of E2 during the peri-implantation period.

Modulatory effects of steroid hormones, oxytocin, arachidonic acid, forskolin and cyclic AMP on the expression of aquaporin 1 and aquaporin 5 in the porcine uterus during placentation.

Aquaporins (AQPs) are proteins forming trans-membrane channels responsible for water transport. AQP1 and AQP5 are present in structures of the female reproductive system. In the uterus, these AQPs are involved in water movement between the intraluminal, interstitial and capillary compartments and their uterine expression is essential throughout the pregnancy, including its early stages. Thus, the study aimed to assess the influence of P4 (progesterone), E2 (estradiol), OT (oxytocin), AA (arachidonic acid), cAMP and FSK (forskolin) on the AQP1 and AQP5 mRNA and protein expression in the uterine tissue of gilts on Days 30 - 32 of gestation (the placentation period), following short (3 h) and long (24 h) incubations. Steroid hormones influenced the expression of AQP1 and AQP5; E2 up-regulated, but P4 down-regulated mRNAs of these AQPs, whereas the protein level of studied AQPs was increased by both steroids. OT treatment decreased AQP1 (after 24 h), but increased AQP5 (after 3 h) mRNA expression. Treatment with AA significantly reduced the AQP1 expression at the mRNA level, but stimulated at the protein level. The expression of AQP5 mRNA and protein was stimulated by AA. FSK markedly decreased AQP1 mRNA, but increased of AQP5 after 3-h incubation. In turn, cAMP stimulated and inhibited transcription of AQP5 after 3- and 24-h incubations, respectively. Immunohistochemical analysis confirmed the uterine localization of AQP1 in the apical and basal membranes of endothelial cells and AQP5 in the apical membranes of epithelial cells under control condition. Treatments with P4, E2, AA, cAMP or FSK have caused additional appearance of AQP5 labeling in the basolateral membranes of epithelial cells. These results suggest a participation of steroid hormones (P4 and E2), AA derivatives and cAMP in controlling the expression of AQP1 and AQP5 as well as the distribution of AQP5 in the uterine tissue of pregnant gilts during placentation (Days 30 - 32 of gestation).

The effects of mu-, delta- and kappa-opioid receptor activation on luteinizing and follicle-stimulating hormone secretion from porcine pituitary cells.

Endogenous opioid peptides (EOP) are involved, among others, in the regulation of endocrine systems, including gonadotropin (LH and FSH) secretion in females during the estrous cycle. EOP preferentially act through three major types of opioid receptors: mu (MOP), delta (DOP) and kappa (KOP). Their influence on gonadotropin secretion at the hypothalamic level was extensively studied in different species, but information pertaining to their modulatory action on LH and FSH secretion at the pituitary level is scarce. Therefore, the aim of the present study was to examine the effects of opioid receptor agonists (mu--DAMGO, delta--DPDPE and kappa--U 50.488) at doses 10⁻9, 10⁻8, 10⁻7 mol/L on both basal and GnRH-stimulated gonadotropin (LH and FSH) secretion in vitro from the anterior pituitary cells of gilts on days 8-10 (luteal phase) and 19-20 (follicular phase) of the estrous cycle. The exposition of pituitary cells in vitro to kappa-receptor agonist (U 50.488; 10⁻7 mol/L) significantly reduced (p<0.05) basal secretion of LH during both phases of the estrous cycle. The GnRH-stimulated LH secretion was also decreased (p<0.05) by this agonist during the luteal phase (10⁻7 mol/L) and follicular phase (10⁻9, 10⁻8 and 10⁻7 mol/L). In turn, the FSH secretion was reduced (p<0.05) by kappa-agonist only in the presence of GnRH during the luteal phase (10⁻8 and 10⁻7 mol/L) and follicular phase (10⁻7 mol/L). The delta-opioid agonist (DPDPE) significantly reduced (p<0.05) the GnRH-affected secretion of LH during the follicular phase (10⁻8 mol/L) and FSH during the luteal phase (10⁻7 mol/L). The mu-opioid agonist (DAMGO) affected neither LH nor FSH secretion. These results indicate that opioid peptides, acting mainly through kappa- and delta-opioid receptors, may participate in the modulation of gonadotropin (LH and FSH) secretion at the pituitary level in cyclic gilts.

The expression of genes encoding opioid precursors and the influence of opioid receptor agonists on steroidogenesis in porcine adrenocortical cells in vitro.

Endogenous opioid peptides are involved in the regulation of the HPA-axis function and stress response mechanism. However, there is a scarcity of data on opioid involvement in the regulation of the adrenocortical endocrine function. This study was performed to: 1) establish the expression of proenkephalin, POMC and prodynorphin genes in the porcine adrenal cortex and test in vitro the influence of ACTH, angiotensin II, CRH and epinephrine on this expression, and 2) determine the effects of opioid receptor agonists on basal and ACTH- or angiotensin II-affected secretion of cortisol, aldosterone and progesterone by the cultured adrenocortical cells. Our experiment has demonstrated the presence of mRNAs for opioid precursors in cells isolated from the adrenal cortex and the significant effects of ACTH and angiotensin II, but not CRH or epinephrine, on adrenocortical transcription of the analyzed genes. Angiotensin II reduced the expression of the POMC gene but stimulated that of prodynorphin. In turn, ACTH decreased the transcription of prodynorphin. The study has also demonstrated the effects of selective opioid receptor agonists - DPLPE (delta), FK33-824 (mu) and U50,488 (kappa) - on adrenal steroidogenesis in pigs. Basal secretion of cortisol was enhanced after the activation of mu or kappa receptors, whereas ACTH-stimulated cortisol output was increased only by the mu receptor agonist. Angiotensin II-treated cells significantly decreased aldosterone secretion in the presence of the kappa receptor agonist. The present results suggest that opioid peptides are synthesized in the porcine adrenal cortex, indicating their involvement in the regulation of adrenal steroidogenesis through autocrine and/or paracrine interactions.

The expression of genes coding for opioid precursors, opioid receptors, beta-LH subunit and GnRH receptor in the anterior pituitary of cyclic gilts.

In previous studies the effect of endogenous opioid peptides (EOP) on LH secretion was mainly considered at the hypothalamic level, while opioid involvement in the modulation of LH secretion at the pituitary level remains insufficiently elucidated. Therefore, the present study was undertaken to determine the expression of genes encoding opioid precursors--proopiomelanocortin (POMC), proenkephalin (PENK), prodynorphin (PDYN) and opioid receptors--mu, delta, kappa in the porcine anterior pituitary throughout the estrous cycle. Additionally, the mRNA content of beta-LH subunit and GnRH receptor (GnRH-R) was estimated. Pituitaries (5xN = 7) were collected from sows on days 3-5, 8-10, 13-15, 16-17 and 19-20 of the cycle and gene expression was determined using a semi-quantitative RT-PCR assay. The expression of POMC, PDYN, delta and kappa receptor genes was variable across the cycle, whereas the expression of PENK and mu receptor genes remained relatively stable. The POMC mRNA content was the lowest on days 19-20 of the cycle and the PDYN content was reduced on days 8-10. The delta receptor mRNA content was elevated on days 3-5, while the kappa receptor mRNA content was decreasing over the luteal phase. Changes in the expression of genes encoding beta-LH and GnRH-R were also demonstrated. These results indicate variable activity of pituitary opioid systems in cyclic pigs and suggest implication of EOP in the modulation of LH secretion at the pituitary level.

Expression of proopiomelanocortin, proenkephalin and prodynorphin genes in porcine luteal cells.

The objective of the study was to examine the expression of the genes coding for proopiomelanocortin (POMC), proenkephalin (PENK) and prodynorphin (PDYN) in porcine luteal cells isolated from corpora lutea (CL) collected on days 3-6, 8-10 and 13-16 of the oestrous cycle. Total RNA was purified from non-incubated cells and from cells incubated for 48 h in the absence or presence of luteinising hormone (LH). The semi-quantitative RT-PCR technique, involving coamplification of the target and control cDNA (beta-actin or 18S rRNA), was used to examine gene expression. It was found that the genes coding for opioid precursors are expressed in both non-incubated and incubated porcine luteal cells representing the early, mid- and late luteal phase. In non-incubated cells, only POMC mRNA content changed during CL development, whereas the expression of PENK and PDYN genes remained relatively constant. Additionally, the treatment of cells with LH markedly affected the expression of POMC and PENK, but no influence on PDYN expression was observed. The present study indicates that porcine luteal cells may produce opioid peptides and that gene expression of their precursors (except for PDYN) may be modulated in these cells by LH. Moreover, the present results support the involvement of opioid peptides in local regulation within the CL of the pig.

Leptin and long form of leptin receptor genes expression in the hypothalamus and pituitary during the luteal phase and early pregnancy in pigs.

Leptin is a polypeptide that plays a key role in the regulation of energy homeostasis and is also linked, among others, to mechanisms controlling reproductive processes. Data concerning the involvement of leptin in controlling reproductive functions at the level of hypothalamus and pituitary in the pig are limited. Therefore, in the present study, an expression of genes coding for leptin and long-form leptin receptor (Ob-Rb) was determined by a semiquantitative reverse transcription polymerase chain reaction (RT-PCR) in the discrete areas of porcine hypothalamus (medial basal hypothalamus - MBH, preoptic area - POA, stalk median eminence - SME) and pituitary (anterior - AP and posterior/neural - NP parts) during the luteal phase of the cycle (days 10-12 and 14-16) and two early stages of pregnancy (days 14-16 and 30-32). Leptin gene expression in MBH was found to be higher in the mid- than in the late-luteal phase, whereas in other structures studied it remained unchanged during these periods. More pronounced differences were noted in expression of Ob-Rb gene, which was increased in MBH, AP and NP during the late-luteal phase in comparison to the mid-luteal one, whilst the relationship in the POA was reversed. In turn, during pregnancy, leptin gene expression in all tested areas of hypothalamus as well as Ob-Rb mRNA content in MBH were higher on days 30-32 than on days 14-16. In contrast, in the anterior pituitary, Ob-Rb gene expression was more pronounced on days 14-16 than during later stage of pregnancy. Comparison of leptin and Ob-Rb mRNA content in studied structures between the mid-luteal phase and days 14-16 of pregnancy revealed inhibition of leptin gene expression in almost all examined tissues (MBH, POA, SME, NP) during early pregnancy whereas Ob-Rb gene expression was inhibited in POA but stimulated in both parts of the pituitary during this stage. In summary, obtained results suggest an involvement of leptin in the regulation of hypothalamic-pituitary axis activity during both the luteal phase of the cycle and early pregnancy in pigs.

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.

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 regulation of steroidogenesis by opioid peptides in porcine theca cells.

The present study was designed to investigate basal and LH-induced steroidogenesis in porcine theca cells from large follicles in response to various concentrations (1-1000 nM) of mu opioid receptor agonists (beta-endorphin, DAMGO, FK 33-824), delta receptor agonists (met-enkephalin, leu-enkephalin, DPLPE) and kappa receptor agonists (dynorphin A, dynorphin B, U 50488). Agonists of mu opioid receptors suppressed basal androstenedione (A4), testosterone (T) and oestradiol-17beta (E2) secretion and enhanced LH-induced A4 and T release by theca cells. The inhibitory effect of the agonists on E2 secretion was abolished in the presence of LH. All delta receptor agonists depressed basal progesterone (P4) output. However, the influence of these agents on LH-treated cells was negligible. Among delta receptor agonist used only leu-enkephalin and DPLPE at the lowest concentrations inhibited basal A4 release. The presence of LH in culture media changed the influence of these opioids from inhibitory to stimulatory. Similarly, DPLPE reduced T secretion by non-stimulated theca cells and enhanced T secretion of stimulated cells. All of delta agonists inhibited basal E2 secretion and unaffected its release from LH-treated theca cells. Agonists of kappa receptors inhibited basal, non-stimulated, P4 secretion and two of them (dynorphin B, U 50488) potentiated LH-induced P4 output. Basal A4 and T release remained unaffected by kappa agonist treatment, but the cells cultured in the presence of LH generally increased both androgen production in response to these opioids. Basal secretion of E2 was also suppressed by kappa agonists. This inhibitory effect was not observed when the cells were additionally treated with LH. In view of these findings we suggest that opioid peptides derived from three major opioid precursors may directly participate in the regulation of porcine theca cell steroidogenesis.

The influences of GnRH, oxytocin and vasoactive intestinal peptide on LH and PRL secretion by porcine pituitary cells in vitro.

The aim of the present study was to evaluate the possible direct effects of GnRH, oxytocin (OT) and vasoactive intestinal peptide (VIP) on the release of LH and PRL by dispersed porcine anterior pituitary cells. Pituitary glands were obtained from mature gilts, which were ovariectomized (OVX) one month before slaughter. Gilts randomly assigned to one of the four groups were treated: in Group 1 (n = 8) with 1 ml/100 kg b.w. corn oil (placebo); in Group 2 (n = 8) and Group 3 (n = 8) with estradiol benzoate (EB) at the dose 2.5 mg/100 kg b.w., respectively, 30-36 h and 60-66 h before slaughter; and in Group 4 (n = 9) with progesterone (P4) at the dose 120 mg/ 100 kg b.w. for five consecutive days before slaughter. In gilts of Group 2 and Group 3 treatments with EB have induced the negative and positive feedback in LH secretion, respectively. Isolated anterior pituitary cells (10(6)/well) were cultured in McCoy's 5a medium with horse serum and fetal calf serum for 3 days at 37 degrees C under the atmosphere of 95% air and 5% CO2. Subsequently, the culture plates were rinsed with fresh McCoy's 5A medium and the cells were incubated for 3.5 h at 37 degrees C in the same medium containing one of the following agents: GnRH (100 ng/ml), OT (10-1000 nM) or VIP (1-100 nM). The addition of GnRH to cultured pituitary cells resulted in marked increases in LH release (p < 0.001) in all experimental groups. In the presence of OT and VIP we noted significant increases (p < 0.001) in LH secretion by pituitary cells derived from gilts representing the positive feedback phase (Group 3). In contrast, OT and VIP were without any effect on LH release in Group 1 (placebo) and Group 2 (the negative feedback). Pituitary cells obtained from OVX gilts primed with P4 produced significantly higher amounts (p < 0.001) of LH only after an addition of 100 nM OT. Neuropeptide GnRH did not affect PRL secretion by pituitary cells obtained from gilts of all experimental groups. Oxytocin also failed to alter PRL secretion in Group 1 and Group 2. However, pituitary cells from animals primed with EB 60-66 h before slaughter and P4 produced markedly increased amounts of PRL in the presence of OT. Neuropeptide VIP stimulated PRL release from pituitary cells of OVX gilts primed with EB (Groups 2 and 3) or P4. In contrast, in OVX gilts primed with placebo, VIP was without any effect on PRL secretion. In conclusion, the results of our in vitro studies confirmed the stimulatory effect of GnRH on LH secretion by porcine pituitary cells and also suggest a participation of OT and VIP in modulation of LH and PRL secretion at the pituitary level in a way dependent on hormonal status of animals.

The effect of stimulators and blockers of adrenergic receptors on LH secretion and cyclic nucleotide (cAMP and cGMP) production by porcine pituitary cells in vitro.

The direct effects of alpha- and beta-adrenergic agents on luteinizing hormone (LH) secretion in vitro by porcine pituitary cells and the participation of secondary messengers, adenosine 3'5'-monophosphate (cAMP) and guanosine 3'5'-monophospate (cGMP), in transduction of signals induced by adrenergic agents and gonadotropin-releasing hormone (GnRH) in these cells have been investigated. Pituitary glands were obtained from mature gilts, which were ovariectomized (OVX) 1 month before slaughter. OVX gilts, assigned to four groups, were primed with: (1) vehicle (OVX); (2 and 3) estradiol benzoate (EB; 2.5mg/100kg b.w.) at 30-36h (OVX+EB I) or 60-66h (OVX+EB II) before slaughter, respectively; (4) progesterone (P(4); 120mg/100kg b.w.) for 5 consecutive days before slaughter (OVX+P(4)). Anterior pituitaries were dispersed with trypsin and then pituitary cells were cultured (10(6) per well) in McCoy's 5a medium containing horse serum (10%) and fetal calf serum (2.5%) for 3 days, at 37 degrees C and under the atmosphere of 95% air and 5% CO(2). On day 4 of the culture, the cells were submitted to 3.5h incubation in the presence of GnRH (a positive control), alpha- and beta-adrenergic agonists (phenylephrine (PHEN) and isoproterenol (ISOP), respectively), and alpha- and beta-adrenergic blockers (phentolamine (PHENT) and propranolol (PROP), respectively). The culture media were assayed for LH (experiment I) and cyclic nucleotides (experiment II). In experiment I, addition of GnRH (100ng/ml) increased LH secretion by pituitary cells taken from gilts of all experimental groups. The effects of alpha- and beta-adrenergic agents on LH secretion by the cells depended on hormonal status of gilts. The LH secretion by pituitary cells of OVX gilts was potentiated in the presence of PHEN (10, 100nM, and 1microM) and PHENT (1microM), alone or in combination with PHEN (100nM) and by the cells derived from OVX+EB I and OVX+P(4) animals in response to PHEN (100nM) and ISOP (1microM). ISOP (1microM) also stimulated LH secretion by the cells taken from OVX+EB II gilts. In experiment II, GnRH (100ng/ml) increased cGMP production by pituitary cells obtained from all groups of gilts and cAMP secretion by the cells taken from OVX and OVX+P(4) animals. PHEN (100nM) decreased and PROP (1microM) enhanced cAMP production by pituitary cells derived from OVX+EB I and OVX gilts, respectively. Moreover, PHEN (100nM) reduced, while PHENT (1microM) stimulated the release of cGMP by pituitary cells taken from OVX+EB II animals. In turn, ISOP (100nM) decreased and increased cGMP production by the cells derived from OVX+EB II and OVX+P(4) gilts, respectively. PROP (1microM) potentiated cGMP accumulation by pituitary cells taken from OVX+EB I and OVX+P(4) animals. In conclusion, our results suggest that adrenergic agents can modulate LH release by porcine pituitary cells acting through guanyl and adenylyl cyclase and in a manner dependent on hormonal status of gilts.

The influence of GnRH, oxytocin and vasoactive intestinal peptide on the secretion of beta-endorphin and production of cAMP and cGMP by porcine pituitary cells in vitro.

The objective of this study was to determine whether gonadotrophin-releasing hormone (GnRH), oxytocin (OT) and vasoactive intestinal polypeptide (VIP) modulate beta-endorphin-like immunoreactivity (beta-END-LI) secretion by dispersed anterior pituitary cells of pigs and in vivo priming with steroid hormones, estradiol benzoate (EB) and progesterone (P(4)), influences the cell reactivity to peptide hormones tested. Additionally, the aim of this research was to examine the involvement of cyclic nucleotides (cAMP and cGMP) in transduction of signals induced by GnRH, OT and VIP in porcine pituitary cells. Pituitaries were collected from ovariectomized (OVX) gilts that were divided into four experimental groups. Animals of group 1 (OVX) received 1ml corn oil (placebo)/100 kg body weight (b.w.), group 2 (OVX+EB I) and group 3 (OVX+EB II) were treated with EB at the dose 2.5mg/100 kg b.w., 30-36 and 60-66 h before slaughter, respectively. Animals of group 4 (OVX+P(4)) were injected with P(4) at the dose 120 mg/100 kg b.w. for 5 subsequent days before slaughter. Anterior pituitaries were dispersed with trypsin and then pituitary cells were cultured (10(6) per well) in McCoy's 5A medium containing horse serum (10%) and fetal calf serum (2.5%) for 3 days at 37 degrees C under an atmosphere of 95% air and 5% CO(2). Subsequently, plates were rinsed with fresh McCoy's 5A medium and pituitary cells were treated with one of the following agents: GnRH (100 ng/ml), OT (10(-6)M) or VIP (10(-7)M) and incubated for 3.5h at 37 degrees C.GnRH did not affect beta-END-LI secretion by pituitary cells of OVX (group 1) and OVX+P(4) (group 4) gilts. When the pituitary cells were incubated in the presence of OT and VIP, significant increases were observed. After priming of OVX gilts with EB, 30-36 h before slaughter (group 2), we noted a significant increase in beta-END-LI release from pituitary cells only in the presence of VIP. Pituitary cells from gilts treated with EB, 60-66 h before slaughter (group 3), produced markedly elevated amounts of beta-END-LI after GnRH, OT or VIP addition.GnRH markedly stimulated cGMP release from cultured pituitary cells in all experimental groups and significantly increased cAMP production by the cells from OVX, OVX+EB II and OVX+P(4) animals. The addition of OT enhanced both cAMP and cGMP output in all experimental groups of pigs. VIP stimulated cAMP release from pituitary cells derived from OVX, OVX+EB I and OVX+EB II animals. cGMP output was markedly elevated under the influence of VIP from pituitary cells of OVX, OVX+EB II and OVX+P(4) gilts. In conclusion, our results suggest that GnRH, OT and VIP can modulate beta-endorphin release from porcine pituitary cells and imply the involvement of cAMP and cGMP in transduction of signals induced by studied peptides in the cells.

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.

The content of beta-endorphin-like immunoreactivity in porcine corpus luteum and the potential roles of progesterone, oxytocin and prolactin in the regulation of beta-endorphin release from luteal cells in vitro.

The amount of beta-endorphin-like immunoreactivity (beta-END-LI) in porcine corpora lutea from several stages of the oestrous cycle and the effects of progesterone, oxytocin, and prolactin on beta-END-LI secretion in vitro by luteal cells were studied. Porcine corpora lutea obtained on days 1-5, 6-10, 11-13, 14-18, and 19-21 of the cycle were used to prepare extracts for beta-END-LI determination. Additionally, corpora lutea from days 11-13 and 14-18 were enzymatically dissociated and isolated luteal cells were used for further study of beta-endorphin secretion in vitro. Cells were cultured in serum-free defined M 199 medium (106 cells/ml) at 37 degrees C under 5% CO2 in air, for 12 h. The influences of the following factors on beta-END-LI secretion by luteal cells were tested: progesterone (10-9, 10-7 and 10-5 M), oxytocin (0.01, 0.1, 1 and 10 ng/ml), and prolactin (0.1, 1, 10 and 100 ng/ml). The beta-END-LI contents in extracts and media were measured by radioimmunoassay. The tissue concentration of beta-END-LI was lowest on days 1-5 of the cycle (0.35 +/- 0.03 ng/g wet tissue). Subsequently, it constantly increased to the highest value on days 14-18 (16.58 +/- 0.52 ng/g wet tissue) and on days 19-21 it declined (11.10 +/- 0.52 ng/g wet tissue). Progesterone at a low dose (10-9 M) resulted in significant (p < 0.05) increases and decreases in beta-END-LI secretion by luteal cells from days 11-13 and 14-18, respectively. Higher doses of progesterone (10-7 and 10-5 M) had no effect on beta-END-LI release, compared with the control group. All dose-levels of oxytocin used decreased beta-END-LI secretion by luteal cells on days 11-13 and 14-18 of the cycle. Prolactin at doses of 0.1 and 1 ng/ml on days 11-13, and all doses tested on days 14-18 resulted in decreases in beta-END-LI release from luteal cells. These results document evident changes in beta-END-LI content in the pig corpus luteum during its development and indicate the potential roles of progesterone, oxytocin, and prolactin in luteal cell secretion of beta-END-LI.

Luteotrophic action of prolactin during the early luteal phase in pigs: the involvement of protein kinases and phosphatases.

Prolactin (PRL) involvement in the regulation of luteal steroidogenesis in pigs during the early luteal phase and pregnancy is well documented. The intracellular mechanism of PRL action in steroidogenic cells, however, is not fully recognized yet. In the current study, we have tested the hypothesis that protein kinase C (PKC) and tyrosine kinases (PTK) as well as serine-threonine (PP) and tyrosine phosphatases (PTP) are involved in PRL signaling in luteal cells originated from the early corpora lutea (CL) of cyclic sows. Luteal cells (50 000 cells/ml M199) were incubated for 8 h (37 degrees C) with PRL (200 ng) and low density lipoproteins (LDL) to stimulate P(4) production. In addition, treatments included: PKC inhibitors--staurosporine and chelerythrine chloride; tyrosine kinase inhibitors--genistein and tyrphostin; serine-threonine phosphatase inhibitors--okadaic acid, cantharidin (inhibitors of PP1/2A) and cypermethrin (inhibitor of PP2B); and tyrosine phosphatase inhibitor--sodium orthovanadate. Moreover, after incubation (37 degrees C) with PRL (200 ng) for 2, 5, 10 or 20 min, luteal cells were homogenized and cytosolic as well as membrane fractions have been obtained. This was followed by partial purification of the subcellular fractions by DEAE-cellulose chromatography and determination of PKC activity by measuring the transfer of (32)P from [gamma-(32)P]ATP to histone III-S. In unstimulated porcine luteal cells the major proportion of PKC activity was present in the cytosol. Incubation of luteal cells with PRL resulted in a rapid, time dependent increase in the amount of PKC activity in the membrane fraction and a decrease in the amount of PKC activity in the cytosol fraction. PKC activity in the membrane fraction was maximal after 5 min of exposure the cells to PRL. Inhibitors of PKC and PTK suppressed PRL and LDL-induced P(4) production by porcine luteal cells. It is of interest that stimulated P(4) production was also reduced by inhibitors of PTP and PP1/2A (okadaic acid, cantharidin). In contrast, cypermethrin did not affect P(4) production stimulated by PRL and LDL. The results of the current study support the hypothesis that PKC and tyrosine kinases are intracellular mediators of PRL action in porcine luteal cells during the first days of the estrous cycle. The involvement of protein phosphatases in transmission of the PRL signal in early luteal cells in pigs is also suggested.

Gene expression and peptide localization for LH/hCG receptor in porcine small and large luteal cells: possible regulation by opioid peptides.

The aim of the present studies was to investigate (1) the presence of LH receptor (LHR) in porcine separated small (SLCs) and large (LLCs) luteal cells taken from mid-luteal corpora lutea and (2) the influence of opioid agonist, FK 33-824 (FK) on LHR gene expression in these cells. Immunocytochemistry revealed intense staining for LHR in both SLCs and LLCs. Reverse transcription-polymerase chain reaction (RT-PCR) and Southern hybridization were used to check the effect of FK and hCG on LHR gene expression. The LHR gene expression was observed in non-stimulated LLCs and in both types of cells after treatment with FK or hCG. FK stimulated LHR gene expression in SLCs and inhibited the gene expression in LLCs. Moreover, FK inhibited and potentiated stimulatory influence of hCG on the gene expression in LLCs and SLCs, respectively. These results suggest that LHR gene expression in porcine luteal cells can be regulated by opioid peptides.

Large luteal cells are the source of immunoreactive beta-endorphin in the pig: effects of HCG and TNFA on its secretion by luteal cells in vitro.

OBJECTIVE: 1. To compare the release of beta-endorphin-like immunoreactivity (beta-END-LI) by large and small luteal cells of the pig; 2. to test the effects of human chorionic gonadotropin (hCG) either alone or combined with the cytokine TNFalpha on beta-END-LI secretion by these cells. METHODS: Isolated large and small luteal cells on days 8-10 of the cycle (n=7) were incubated in M199 supplemented with hydrocortisone (40 ng/ml), transferrin (5 microg/ml), insulin (2 microg/ml), gentamicin (50 microg/ml), nystatin (240 U/ml), porcine low-density lipoproteins (LDL; 100 microg/ml), 1 % BSA and 2 x 10(-5) M bacitracin, for 12 h at 37 C and under the atmosphere of 95 % air and 5 % CO2. The cells were treated either with hCG (100 ng/ml) or TNFalpha (0.1, 1 and 3 nM) alone or with both agents together. Beta-END-LI concentration in incubation media was measured by RIA. RESULTS: beta-END-LI secretion by large luteal cells was 15-fold greater than by small cells (666.38 +/- 24.59 pg/ml/106 cells vs. 44.60 +/- 2.53 pg/ml/106 cells). hCG enhanced beta-END-LI secretion by both large and small luteal cells. TNFalpha alone had no effect on beta-END-LI release by large and small luteal cells, but it abolished the stimulatory effect of hCG on beta-END-LI secretion by large luteal cells. CONCLUSION: The results indicate that large luteal cells are a major source of beta-endorphin in the porcine corpus luteum, where its release may be affected by gonadotropins (possibly LH) and TNFalpha.