Jadwiga Przala

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.

Expression of orexin receptors 1 (OX1R) and 2 (OX2R) in the porcine pituitary during the oestrous cycle.

Orexin A and B, also termed hypocretin 1 and 2, are associated with the stimulation of food intake and arousal. The biological actions of the hormones are mediated via two distinct G protein-coupled receptors, termed orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). OX1R is selective for orexin A and OX2R binds orexin A and orexin B with similar affinity. The present study analyzed mRNA and protein expressions of OX1R and OX2R in adenohypophysis (AP) and neurohypophysis (NP) of cycling pigs. The tissue samples were harvested on days 2-3, 10-12, 14-16, and 17-19 of the oestrous cycle. Using quantitative real-time PCR higher OX1R gene expression was detected in AP on days 2-3 relative to days 10-12, 14-16 and 17-19 (p<0.05). In NP the OX1R mRNA level was elevated on days 10-12 compared to the remaining stages (p<0.05). OX2R gene expression in AP was the lowest on days 10-12 (p<0.05 compared to days 2-3 and 17-19) and the expression peak occurred on days 17-19 (p<0.05 vs. the all studied stages). In NP the highest (p<0.05) expression of OX2R mRNA was noted on days 17-19 in relation to the remaining periods. OX1R protein content in AP was greatest on days 10-12 (p<0.05), whereas in NP it was greatest on days 2-3 and 14-16 (p<0.05 vs. days 10-12 and 17-19). In both cases the lowest OX1R protein expression was observed during follicular phase (p<0.05 in relation to three remaining studied stages). OX2R protein in AP was lower (p<0.05) on days 2-3 and 14-16 compared to days 10-12 and 17-19. In NP the lowest (p<0.05) expression of this protein was on days 17-19 and the highest on days 10-12 (p<0.05 compared to days 2-3 and 17-19). In summary, the present findings provide the first evidence that OX1R and OX2R mRNAs and proteins occur in the pituitary of the pig and indicate the dependence of orexin receptor expression on the endocrine reproductive state.

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.

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.

Porcine theca cells produce immunoreactive β-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.

Leptin Gene and Protein Expression in the Ovary During the Oestrous Cycle and Early Pregnancy in Pigs

Leptin, the product of the obese gene, is the hormone originally identified in adipocytes. It is involved in the control of satiety and energy metabolism. More recent observations suggest that leptin plays an important role in reproduction. Leptin mRNA and protein have been found in the human and the murine ovary. However, the expression of leptin in the porcine ovary has not been examined. Therefore, the aim of the present work was to compare the expression levels of porcine leptin mRNA by semiquantitative RT-PCR and in situ hybridization, as well as leptin protein by Western blotting in the corpus luteum (CL) and ovarian stroma (OS) during mid- and late-luteal phase of the oestrous cycle as well as during days 14-16 and 30-32 of pregnancy. Leptin gene and protein expression in CL was increased on days 14-16 of the cycle compared with pregnant animals. Leptin gene expression in OS was higher during the late-luteal phase of the cycle than on days 30-32 after conception. However, comparison of leptin protein expression in OS between days 14-16 of the cycle and days 30-32 of pregnancy indicates a higher protein expression during pregnancy. Moreover, leptin gene expression was higher in porcine CL and OS on days 14-16 of pregnancy in comparison to days 30-32. Contrary to leptin mRNA expression, a higher leptin protein expression was observed on days 30-32 compared with days 14-16 after conception. In summary, the present study provides the first evidence that leptin mRNA and protein occur in porcine ovary and vary during the oestrous cycle and pregnancy. Moreover, the obtained results indicate that also locally synthesized leptin may participate in the control of pig reproduction by exercising its action at the ovarian level.

The influence of GnRH, oxytocin and vasoactive intestinal peptide on the secretion of β-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 McCoys 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 McCoys 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.

In vivo modulation of follicle-stimulating hormone release and β 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: activins 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.

Expression of leptin and its receptor genes in the ovarian follicles of cycling and early pregnant pigs.

Leptin is a polypeptide hormone produced primarily by adipocytes. It has been implicated in the regulation of satiety and energy homeostasis. Leptin has been suggested to play a role in reproduction based on its involvement in the regulation of the hypothalamic-pituitary-gonadal axis via endocrine, paracrine and/or autocrine pathways. The aim of the present study was to localize the cellular distribution of leptin and the long isoform of leptin receptor (OB-Rb) genes in porcine ovarian antral follicles and to compare the expression levels of leptin and OB-Rb mRNAs in porcine granulosa cells (GC), theca interna (TIC) and theca externa (TEC) cells during the luteal phase of the estrous cycle and in early pregnancy. The expression of leptin and OB-Rb genes was detected in GC, TIC and TEC. Significantly higher levels of leptin gene expression in GC were observed during the mid- and late-luteal phases of the cycle than on days 30 to 32 of pregnancy. On days 14 to 16 of pregnancy, leptin mRNA expression was higher than that on days 14 to 16 of the cycle. The expression of the OB-Rb gene in GC and TEC increased during pregnancy in comparison with the analyzed luteal phases of the cycle. Our results validate the hypothesis that locally produced leptin plays a role in the regulation of porcine reproduction at the ovarian level and exerts a direct effect on porcine follicles. The differences in OB-Rb gene expression in porcine GC and theca cells also suggest that their sensitivity to leptin varies in the ovaries of pregnant and cyclic pigs.

Expression of Leptin and Long‐form Leptin‐receptor Proteins in Porcine Hypothalamus during Oestrous Cycle and Pregnancy

In this study, we examined the levels of leptin and OB-Rb protein expression in the discrete areas of the porcine hypothalamus (mediobasal hypothalamus--MBH, pre-optic area--POA, stalk median eminence--SME) during mid- and late-luteal phases of the oestrous cycle (days 10-12 and 14-16) as well as two stages of pregnancy (days 14-16 and 30-32). The analysis showed that during the cycle, leptin protein expression in MBH was higher in the mid-luteal phase than late-luteal phase. In the case of OB-Rb protein expression, a higher level was observed in MBH during the late-luteal phase in comparison to the mid-luteal phase, whereas in POA and SME the opposite dependence was noticed. In turn, during pregnancy, leptin protein expression in MBH and POA, and OB-Rb protein expression in POA were more pronounced on days 14-16 than on days 30-32. In contrast, leptin protein content in SME as well as OB-Rb protein in MBH and SME was higher on days 30-32 than during the earlier stage of pregnancy. Comparison of leptin and OB-Rb protein expression between the cycle (days 10-12) and pregnancy showed a higher level of leptin and OB-Rb protein contents in POA as well as in SME during pregnancy (on days 14-16 and 30-32, respectively). Yet, OB-Rb protein expression in POA on days 30-32 of pregnancy was lower in comparison to days 10-12 of the cycle. Furthermore, during pregnancy, leptin protein expression in MBH was lower (days 14-16 and 30-32), whereas OB-Rb protein expression in that area of hypothalamus was higher (days 30-32) in comparison to the mid-luteal phase. Our results indicate that both leptin and OB-Rb are synthesized in the porcine hypothalamus and suggest the participation of leptin in auto/paracrine regulation of these brain areas functions, including control of reproduction during the oestrous cycle and early pregnancy.