Intracellular mechanisms involved in copper-gonadotropin-releasing hormone (Cu-GnRH) complex-induced cAMP/PKA signaling in female rat anterior pituitary cells in vitro.

The copper-gonadotropin-releasing hormone molecule (Cu-GnRH) is a GnRH analog, which preserves its amino acid sequence, but which contains a Cu(2+) ion stably bound to the nitrogen atoms including that of the imidazole ring of Histidine(2). A previous report indicated that Cu-GnRH was able to activate cAMP/PKA signaling in anterior pituitary cells in vitro, but raised the question of which intracellular mechanism(s) mediated the Cu-GnRH-induced cAMP synthesis in gonadotropes. To investigate this mechanism, in the present study, female rat anterior pituitary cells in vitro were pretreated with 0.1 µM antide, a GnRH antagonist; 0.1 µM cetrorelix, a GnRH receptor antagonist; 0.1 µM PACAP6-38, a PAC-1 receptor antagonist; 2 µM GF109203X, a protein kinase C inhibitor; 50 mM PMA, a protein kinase C activator; the protein kinase A inhibitors H89 (30 µM) and KT5720 (60 nM); factors affecting intracellular calcium activity: 2.5 mM EGTA; 2 µM thapsigargin; 5 µM A23187, a Ca(2+) ionophore; or 10 µg/ml cycloheximide, a protein synthesis inhibitor. After one of the above pretreatments, cells were incubated in the presence of 0.1 µM Cu-GnRH for 0.5, 1, and 3 h. Radioimmunoassay analysis of cAMP confirmed the functional link between Cu-GnRH stimulation and cAMP/PKA signal transduction in rat anterior pituitary cells, demonstrating increased intracellular cAMP, which was reduced in the presence of specific PKA inhibitors. The stimulatory effect of Cu-GnRH on cAMP production was partly dependent on GnRH receptor activation. In addition, an indirect and Ca(2+)-dependent mechanism might be involved in intracellular adenylate cyclase stimulation. Neither activation of protein kinase C nor new protein synthesis was involved in the Cu-GnRH-induced increase of cAMP in the rat anterior pituitary primary cultures. Presented data indicate that conformational changes of GnRH molecule resulting from cooper ion coordination affect specific pharmacological properties of Cu-GnRH molecule including specific pattern of intracellular activity induced by complex in anterior pituitary cells in vitro.

Direct in vitro effect of LH and steroids on leptin gene expression and leptin secretion by porcine luteal cells during the mid-luteal phase of the estrous cycle.

Leptin, the product of the ob gene, is implicated in the control of reproductive functions in different species. Leptin transcript and protein are present in several tissues including adipose tissue and corpus luteum. The regulation of leptin mRNA expression and leptin secretion in porcine luteal cells is not clear. In this study, we determined leptin and OB-Rb mRNA/protein levels in porcine luteal cells during the mid-luteal phase of the estrous cycle (days 10-12) and examined, in vitro, the effects of LH, 17-ß estradiol (E(2)) and progesterone (P(4)) on leptin gene expression and leptin secretion in those cells. Leptin and long form of leptin receptor (OB-Rb) mRNA expressions as well as leptin and OB-Rb protein were detected in the luteal cells by real-time PCR and fluorescence immunocytochemistry (F-ICC), respectively. Isolated luteal cells, after preliminary culture (48 h) were treated with LH (1; 10; 100 ng/ml), E(2) (0.02; 0.2; 2; 20 ng/ml) and P(4) (20; 100; 200 ng/ml) for 24 h. LH did not induce significant changes in leptin mRNA expression in the luteal cells. A higher level of leptin transcript was found in the presence of E(2) (0.02 ng/ml) or P(4) (200 ng/ml). Estradiol (0.2 ng/ml) and P(4) (200 ng/ml) increased leptin secretion by luteal cells. Our results indicate that leptin and OB-Rb genes and proteins are expressed in porcine luteal cells and suggest that steroid hormones (E(2) and P(4)) affect leptin mRNA expression and leptin secretion during the mid-luteal phase of the estrous cycle.

Long form of leptin receptor gene and protein expression in the porcine trophoblast and uterine tissues during early pregnancy and the oestrous cycle.

Leptin, the product of the OB gene, is a 16-kDa polypeptide of 146 amino acid residues produced mainly by adipocytes that regulates metabolism and reproduction. The actions of leptin are mediated mainly via the long form of the leptin receptor (OB-Rb). The identification of leptin and OB-Rb mRNAs and proteins in human and mouse endometrium, and placental trophoblast suggests that leptin may be involved in the implantation process. Thus, the aim of this study was to compare the expression levels of porcine OB-Rb mRNA and protein in the endometrium and myometrium during mid- and late-luteal phases of the oestrous cycle (days 10-12 and 14-16, respectively) as well as during two stages of pregnancy respondent to the beginning of the implantation process (days 14-16) and the post-implantation period (days 30-32), and in trophoblast during both periods of pregnancy. OB-Rb gene expression in endometrium during the examined stages of pregnancy and the mid- and late-luteal phases of the cycle was at the same level. In contrast, in myometrium leptin receptor gene expression decreased on days 14-16 of pregnancy compared to both phases of the cycle, and on days 30-32 of pregnancy in relation to late-luteal phase. OB-Rb protein expression in the tissues was lower during the examined stages of pregnancy in comparison to the mid- and late-luteal phases of the cycle. In trophoblast, OB-Rb mRNA and protein expression was higher on days 30-32 than during days 14-16 of pregnancy. In conclusion, our results might suggest that leptin can participate in the control of pig reproduction by exercising its action at the uterine and trophoblast level and have a direct effect on these organ during both the luteal phase of the cycle and early pregnancy. Moreover, changes in OB-Rb gene and protein expression in tissues of pig reproductive tract strongly suggest that their sensitivity to leptin varies throughout luteal phase of the cycle and early pregnancy.

Long form of leptin receptor gene and protein expression in the porcine ovary during the estrous cycle and early pregnancy.

Leptin, a hormone secreted by adipocytes, plays an important role in the regulation of metabolism and reproduction. The effect of leptin is mediated mainly via the long isoform of the leptin receptor (OB-Rb). Expression of leptin and its receptor has been identified in the central nervous system (hypothalamus, pituitary) and reproductive tract (uterus, ovary and placenta) in prepubertal gilts. Therefore, in the present study, the porcine OB-Rb expression was examined by a semiquantitative reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization and Western blotting in the corpus luteum (CL) and ovarian stroma (OS) during the luteal phase of the estrous cycle (days 10-12 and 14-16) and two stages of early pregnancy (days 14-16 and 30-32). The OB-Rb gene expression in both ovarian structures was higher during early pregnancy in comparison to the mid- and late-luteal phase of the estrous cycle. Significant differences in OB-Rb gene expression between the two periods of the pregnancy were observed only in OS. The results of in situ hybridization are generally supported by data of semiquantitative RT-PCR. The CL expression of leptin receptor protein was significantly lower during early pregnancy compared to the cycle. In OS, the OB-Rb protein content was higher during pregnancy compared to the late-luteal phase of the estrous cycle. In summary, the obtained results indicate that leptin may participate in the control of pig reproduction at the ovarian level and have a direct effect on the ovary during both, the luteal phase of the cycle and early pregnancy. Moreover, changes in OB-Rb gene and protein expression in ovarian structures strongly suggest that their sensitivity to leptin varies throughout the luteal phase of the cycle and early pregnancy.

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.

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

Previous studies have demonstrated the presence of endogenous opioid peptides (EOP) in the ovary and suggested their implication in local interactions within ovarian structures. Nevertheless, data pertaining to the expression of genes, coding for the opioid precursors, in ovarian cells are still rudimentary and not available for the pig. The study was undertaken to test whether genes of the opioid precursors - proopiomelanocortin (POMC), proenkephalin (PENK) and prodynorphin (PDYN) - are expressed in non-treated and gonadotropin-treated theca and granulosa cells isolated from ovarian follicles of the pig. The cells were isolated from small (days 15-16 of the estrous cycle) and large (days 19-20) porcine follicles. Dispersed cells were cultured in Eagle's medium under the water saturated atmosphere of 95% air and 5% CO(2), in the presence or absence of respective gonadotropin; theca cells with LH (100 ng/ml) and granulosa cells with FSH (100 ng/ml). Following 24h-incubation, the cells were harvested and the total RNA was isolated. The expression of genes coding for opioid precursors was estimated by the semi-quantitative RT-PCR technique involving co-amplification of the target cDNA (POMC, PENK or PDYN) and control cDNA (beta-actin or 18S rRNA). Specificities of PCR products were confirmed by Southern analysis and sequencing. In theca cells the expression of opioid precursors appeared to be gonadotropin-dependent except for PENK in the cells isolated from large follicles. In turn, granulosa cells exhibited the expression of POMC and PENK genes independently on treatment with FSH. This gonadotropin induced the expression of PDYN gene in granulosa cells isolated from small and large follicles and significantly increased POMC mRNA content in the cells from the large ones. The present studies indicate that porcine follicular cells (especially granulosa cells) may produce opioid peptides and that gonadotropins may modulate gene expression of their precursors in these cells. Moreover, our results support a participation of opioid peptides in the local regulations within ovarian follicle.

Effects of oxytocin alone and in combination with selected hypothalamic hormones on ACTH, beta-endorphin, LH and PRL secretion by anterior pituitary cells of cyclic pigs.

Oxytocin (OT) is involved in the stimulation of secretion of anterior pituitary hormones in females during the periovulatory and periparturient periods. In the present study we examined the role of OT in control of ACTH, beta-endorphin, LH and PRL secretion in vitro from dispersed anterior pituitary cells collected from gilts during the luteal (Days 10-12; n=6) and follicular (Days 18-20; n=5) phases of the estrous cycle. Isolated anterior pituitary cells (1 x 10(6)/ml) were transferred into 24-well plates, separately for each animal, and were pre-incubated for three days at 37 degrees C in atmosphere of 5% CO(2) and 95% air. The cells which attached to the dishes were incubated (3.5 h, 37 degrees C) in McCoy's medium in the absence (control) or in the presence of the following factors: CRH alone (10(-10), 10(-9), 10(-8), 10(-7) M), OT alone (10(-8), 10(-7), 10(-6) M), LVP alone (10(-7) M), OT (10(-7) M) plus CRH (10(-9) M) and LVP (10(-7) M) plus CRH (10(-9) M) for studying ACTH and beta-endorphin secretion; OT alone (10(-8), 10(-7), 10(-6) M), GnRH alone (100 ng/ml), CRH alone (10(-9) M), OT (10(-7) M) plus GnRH (100 ng/ml) and OT (10(-7) M) plus CRH (10(-9) M) for studying LH and PRL secretion. Concentrations of the studied hormones in media were analyzed by RIA. Oxytocin alone increased ACTH (at doses 10(-7), 10(-6) M), beta-endorphin (at dose 10(-8) M), LH (at dose 10(-8) M) and PRL (at doses 10(-7), 10(-6) M) secretion by pituitary cells isolated only from luteal-phase gilts. None of the studied hormone concentrations in the medium was increased in response to OT when pituitary cells of follicular-phase gilts were examined. Oxytocin in combination with CRH exerted an additive effect on beta-endorphin secretion during the luteal phase. Summarizing, in the present study the stimulatory effect of oxytocin on ACTH, beta-endorphin, LH and PRL secretion by pituitary cells isolated from gilts during the luteal phase was demonstrated. However, the cells collected from follicular-phase gilts appeared to be unresponsive to OT. Moreover, interaction between OT and CRH in affecting beta-endorphin secretion was shown. These results suggest that OT may be transiently involved in the modulation of anterior pituitary hormone secretion in cyclic pigs.

Mechanisms ensuring optimal conditions of implantation and embryo development in the pig.

The paper summarizes results of a series of studies concerning luteolysis and early pregnancy in pigs. The involvement of the oxytocin (OT)/OT receptor system in the mechanism of corpus luteum (CL) protection during early pregnancy as well as the implication of luteinizing hormone (LH) in the endometrial prostaglandin (PG) release and synthesis are described. In addition, the role of leptin in the regulation of ovarian steroidogenesis and the expression of leptin and its receptor (OB-Rb) genes in hypothalamus, pituitary and reproductive tissues are reported. Moreover, a strong emphasis was placed on the mechanism of PGE2 participation in the local endocrine regulations of reproductive processes occurring in the utero-ovarian area as well as on the vascular endothelial growth factor (VEGF) ligand-receptor system in the ovary and uterus.

Different signaling in pig anterior pituitary cells by GnRH and its complexes with copper and nickel.

Gonadotropin releasing hormone (GnRH) is an essential factor in the regulation of synthesis and release of pituitary gonadotropins. After binding to specific receptors and coupling with G proteins, it triggers the intracellular signaling involving the synthesis of inositol phosphates and diacylglycerol. Previously we have showed that certain metal complexes with GnRH, i.e. copper (Cu-GnRH) and nickel (Ni-GnRH) are able to bind to the GnRH receptors. The intracellular signalling of these complexes, however, has not been yet elucidated. In this experiment, the ability of the Cu-GnRH and Ni-GnRH complexes to modulate cAMP synthesis and phosphoinositols formation in the pig anterior pituitary cells in vitro was studied. The native GnRH and its metal complexes stimulated the luteinizing hormone (LH) release, but only the effect of Cu-GnRH was found to be a dose-dependent. The metal complexes did not significantly influence inositol phosphates accumulation, while their effect on cAMP synthesis was significantly more potent than that of GnRH alone. We conclude that the Cu-GnRH and Ni-GnRH complexes increase LH release in the porcine pituitary cells although their intracellular signaling is different from that of the native GnRH. It seems that metal complexes with GnRH deserve more attention in further studies.

Long form leptin receptor mRNA expression in the hypothalamus and pituitary during early pregnancy in the pig.

OBJECTIVE: The aim of this study was the detection and location of long form leptin receptor (OB-Rb) in different area of hypothalamus and pituitary in the pig during early pregnancy. SETTINGS AND DESIGN: Expression of OB-Rb was examined by RT-PCR in the different area of hypothalamus: medial basal hypothalamus (MBH), preoptic area (POA), stalk median eminence (SME), as well as pituitary: the anterior (AP) and posterior (NP) lobe collected from gilts at days 14-16 (n=4) and 30-32 (n=4) of pregnancy. RESULTS: The results showed that OB-Rb mRNA was expressed in the hypothalamus (MBH, POA and SME), pituitary (AP, NP) and adipose tissue in the pig during early pregnancy (at days 14-16 and 30-32). CONCLUSION: These findings support the idea that leptin might play a role in the regulation of the hypothalamic-pituitary axis activity, and consequently in the control of pregnancy during critical period of embryo implantation in the pig.

Leptin gene expression in the hypothalamus and pituitary of pregnant pigs.

BACKGROUND: Leptin, the 16-kDa peptide hormone product of the ob gene, is a regulatory hormone secreted mainly by adipose tissue. Recent studies have shown leptin production by other tissues, including rat hypothalamus, rat and human pituitary, rat skeletal muscle, kidney and stomach, human and porcine placenta, human mammary epithelial cells as well as endometrial tissues. This hormone is a central modulator of food intake, metabolism and neuroendocrine functions. OBJECTIVES: The aim of the study was to detect and locate porcine leptin gene expression in the different areas of the hypothalamus and pituitary on days 14-16 and 30-32 of pregnancy in pigs. METHOD: Leptin gene expression was analysed by RT-PCR method. PCR products were subjected to sequencing analysis. RESULTS: Leptin mRNA was expressed in the medial basal hypothalamus, preoptic area, stalk median eminence, anterior pituitary, posterior pituitary and adipose tissue on days 14-16 and 30-32 of pregnancy. Sequence analysis of the 258 bp product from the hypothalamus and pituitary confirmed 99% homology with the corresponding region of porcine leptin cDNA sequence. CONCLUSION: Leptin mRNA expression in the porcine hypothalamus and pituitary gland implies its paracrine and/or autocrine role in the regulation of hypothalamic-pituitary axis activity.

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.

In vivo modulation of follicle-stimulating hormone release and beta subunit gene expression by activin A and the GnRH agonist buserelin in female rats.

The effects of separate and simultaneous recombinant bovine (rb) activin A and buserelin administration on the FSH release and pituitary FSH beta subunit gene expression in vivo were examined in ovariectomised, estradiol pretreated rats. The animals received a single injection of either rb activin A (50 ng), buserelin (1 micro g) or activin/buserelin (50 ng+1 micro g/0.1 ml PBS) into the jugular vein and were killed 30 min, 1, 3 and 5h later. Activin A stimulated FSH release and effect appeared 1h after injection (168% increase of controls) reaching a maximum at 3h (437% of controls). Activin A and buserelin exerted their effects with a distinct time courses: activin's stimulation was not so rapid when compared with buserelin. The simultaneous administration of rb activin A and buserelin amplified FSH release (118, 309, 1006 and 779% of controls). The low dose of activin A was sufficient to elevate FSH beta mRNA level as early as 3 and 5h after administration (170 and 140%, respectively). Activin plus buserelin stimulation resulted in a higher (340 and 360% of controls) FSH beta gene expression than after their separate administration. These results suggest that activin and buserelin may act independently and synergistically in the regulation of FSH release and beta subunit mRNA level.