Expression of the orexin system in the porcine uterus,conceptus and trophoblast during early pregnancy

Orexin A and B are hypothalamic peptides derived from the prepro-orexin (PPO) precursor. Orexins stimulate food intake and arousal. Those peptides bind and activate two G protein-coupled receptors: orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). Numerous authors have suggested that orexins play an important role in the regulation of the reproductive functions. The objective of the present study was to analyse the presence of and changes in the gene and protein expression pattern of the orexin system in the porcine uterus, conceptus and trophoblast (chorioallantois) during early pregnancy. In the endometrium, the highest PPO and OX1R gene expression was detected on days 15 to 16 of gestation. The OX2R mRNA content in the endometrium was higher on days 10 to 11 and 15 to 16 than on days 12 to 13 and 27 to 28. In the trophoblasts, PPO gene expression was higher on days 30 to 32 than on days 27 to 28. The highest PPO protein content in the endometrium was noted on days 12 to 13. The highest OX1R protein content in the endometrium was detected on days 10 to 11, whereas OX2R protein on days 15 to 16. In the trophoblasts, PPO and OX1R protein levels were more pronounced on days 27 to 28 than on days 30 to 32, but OX2R expression was higher on days 30 to 32. The expression of PPO, OX1R and OX2R was different in the conceptuses and trophoblasts during early pregnancy. Local orexin production and the presence of the specific orexin receptors suggest that the orexin system may participate in the control of porcine reproductive functions by exerting endocrine and auto/paracrine effects on the uterus, conceptuses and trophoblasts during early pregnancy. This study provides the first evidence for the presence of orexins and their receptors in the uteri, conceptuses and trophoblasts in pigs during early pregnancy. The local orexin system is dependent on the stage of pregnancy.     

Effect of orexin B on CYP17A1 and CYP19A3 expression and oestradiol,oestrone and testosterone secretion in the porcine uterus during early pregnancy and the oestrous cycle

Orexin A (OXA) and B (OXB) are hypothalamic neuropeptides identified as regulators of food intake, energy homoeostasis, sleep–wake cycle and arousal. They also create an integrative link between energy homoeostasis and reproduction. Although their functions in the ovaries and testes have been partially explored, to date, less attention has been focused on the role of the peptides in the uterus. The aim of this study was to investigate the effect of one of orexins – orexin B on oestradiol (E₂), oestrone (E₁) and testosterone (T) secretion by porcine endometrial and myometrial slices as well as the gene expression of key steroidogenic enzymes responsible for steroid production (CYP17A1, CYP19A3) during the luteal phase of the oestrous cycle (days 10 to 11) and early pregnancy (days 10 to 11, 12 to 13, 15 to 16, 27 to 28). Orexin B suppressed E₂ secretion by endometrial slices on days 10 to 11 and 15 to 16 of pregnancy, and days 10 to 11 of the cycle. In the myometrium, OXB inhibited E₂ production on days 10 to 11 of pregnancy, whereas on days 12 to 13 it enhanced steroid output. Endometrial E₁ release was potentiated by the peptide during all studied periods of the cycle and pregnancy, with the exception of days 12 to 13, when an inhibitory effect was observed. Myometrial secretion of E₁ was increased, except on days 27 to 28. Testosterone secretion by endometrial slices was increased on days 12 to 13 and 27 to 28 of pregnancy. On days 10 to 11 of the cycle, T release was stimulated in response to the lowest and decreased under the influence of the highest dose of OXB. In the myometrium, T production was inhibited by OXB on days 10 to 11 of pregnancy and during the corresponding period of the cycle. On days 27 to 28 of pregnancy, T release was potentiated by the lowest dose of OXB. Expression of both genes was modified by OXB depending on the period of pregnancy and the type of examined uterine tissues. Our findings suggest that OXB, through modulation of uterine steroidogenesis, may have a regulatory role in the uterus.     

Changes in plasma orexin A and orexin B concentrations during the estrous cycle of the pig

Orexin A (OXA) and orexin B (OXB) are neuropeptides synthesized mainly in the lateral hypothalamus, which are involved in the control of various physiological functions such as energy homeostasis, sleep, wakefulness and feeding behavior. The present study analyzes orexins A and B levels in the porcine plasma during the estrous cycle. The highest plasma concentrations of orexin A were observed on days 2–3 of the estrous cycle (p < 0.05 relative to days 10–12 and 14–16) and the lowest (p < 0.05) on days 14–16. The highest orexin B levels in the blood plasma were noted on days 17–19 (p < 0.05 vs. days 14–16). We demonstrated the presence of OXA and OXB in porcine blood plasma and the impact of the phase of the estrous cycle on the observed changes in plasma orexin levels.     

Localization and correlation between NADPH-diaphorase and nitric oxide synthase isoforms in the porcine uterus during the estrous cycle

Nitric oxide (NO), a highly reactive free radical is involved in vasodilation, neurotransmission, hormone secretion, and reproduction. Since all known nitric oxide synthase (NOS) isoforms possess NADPH-diaphorase (NADPH-d) activity, NADPH-d histochemistry was used as a commonly accepted procedure for NOS identification. The aim of our study was to determine the cellular localization of NADPH-d, eNOS, and iNOS in the porcine uterus and the correlation between NADPH-d and NOS activity in the early, middle, late luteal, and follicular phase of the estrous cycle. Light-microscopic observations of the sections revealed the differential expression of the NADPH-d in the analyzed stages of the estrous cycle. The most intense staining was observed in the luminal epithelium in the late luteal phase and in some groups of the endometrial glands in all studied stages. Positive reaction was also found in the endothelial cells of blood vessels and in the myometrium itself. Immunostaining for eNOS was observed in the luminal and glandular epithelium in all studied stages, but no clear fluctuations were observed. The endothelium of both endometrial and myometrial blood vessels displayed pronounced eNOS immunostaining. Strong iNOS staining was observed in the luminal epithelium in the late luteal and follicular phase and in selected groups of endometrial glands. Thus, only NADPH-d and iNOS undergo cyclic changes in the studied stages of the estrous cycle. The differential expression of NADPH-d/NOS in the porcine uterine horn during the estrous cycle suggests a role for NO in modulating uterine function.     

Localization and effects of orexin on fasting motility in the rat duodenum

The orexins [orexin A (OXA) and orexin B (OXB)] are novel neuropeptides that increase food intake in rodents. The aim of this study was to determine the distribution of orexin and orexin receptors (OX1R and OX2R) in the rat duodenum and examine the effects of intravenous orexin on fasting gut motility. OXA-like immunoreactivity was found in varicose nerve fibers in myenteric and submucosal ganglia, the circular muscle, the mucosa, submucosal and myenteric neurons, and numerous endocrine cells of the mucosa. OXA neurons displayed choline acetyltransferase immunoreactivity, and a subset contained vasoactive intestinal peptide. OXA-containing endocrine cells were identified as enterochromaffin (EC) cells based on the presence of 5-hydroxytryptamine immunoreactivity. OX1R was expressed by neural elements of the gut, and EC cells expressed OX2R. OXA at 100 and 500 pmol x kg(-1) x min(-1) significantly increased the myoelectric motor complex (MMC) cycle length compared with saline. Similarly, OXB increased the MMC cycle length at 100 pmol x kg(-1) x min(-1), but there was no further effect at 500 pmol x kg(-1) x min(-1). We postulate that orexins may affect the MMC through actions on enteric neurotransmission after being released from EC cells and/or enteric neurons.     

The effect of the estrous cycle on the expression of prepro-orexin gene and protein and the levels of orexin A and B in the porcine pituitary

Hypothalamic peptides orexin A (OXA) and orexin B (OXB) are derived from the proteolytic cleavage of a common precursor molecule, prepro-orexin (PPO). They act via two orexin receptors (OX1R and OX2R), which belong to the G-protein coupled receptor superfamily. Orexins are implicated in the regulation of arousal states, energy homeostasis and reproductive neuroendocrine function. The objective of this study was to investigate the presence and changes in orexin expression in the porcine pituitary during the estrous cycle. Adenohypophysis (AP) and neurohypophysis (NP) tissue samples were harvested on days 2 to 3, 10 to 12, 14 to 16, and 17 to 19 of the estrous cycle. The expression of the PPO gene increased in AP and NP during the estrous cycle. The highest PPO protein concentrations in AP were reported on days 2 to 3 (P<0.05), and in NP – on days 10 to 12 and 17 to 19 (P<0.05). The expression of PPO mRNA was lower in AP than in NP, but PPO protein levels were higher in AP. In AP, OXA immunoreactivity was higher (P<0.05) on days 10 to 12 and 14 to 16. In NP, the highest (P<0.05) content of the analyzed protein was observed on days 10 to 12 and the lowest (P<0.05) – on days 14 to 16 and 17 to 19. OXB immunoreactivity in AP reached the highest level (P<0.05) on days 2 to 3, and the lowest level (P<0.05) was determined on days 10 to 12 and 17 to 19. OXB protein concentrations in NP peaked (P<0.05) on days 10 to 12 of the cycle. Our study was the first experiment to demonstrate the expression of the orexin gene and orexin proteins in the porcine pituitary and the correlations between expression levels and the phase of the estrous cycle.     

Localization of neurokinin A and chromogranin A immunoreactivity in the developing porcine adrenal medulla

Summary The presence of neurokinin A immunoreactivity was studied in the chromaffin cells of the porcine adrenal medulla and in the nerve fibres innervating the adrenal gland during ontogenic development. For comparison, chromogranin A immunoreactivity was used as a marker for chromaffin cells.Whereas chromogranin A was found in chromaffin cells through all steps in embryonic development, three developmental stages of neurokinin A immunoreactivity could be distinguished. In the first and second trimester of gestation, neurokinin A was observed in some groups of chromaffin cells, but no neurokinin-immunoreactive nerve fibres could be detected. In the last trimester of gestation, neurokinin A-reactive chromaffin cells and nerve fibres were both found in adrenal glands. However, in adrenal glands of neonatal piglets, neurokinin A was found only in nerve fibres and not in chromaffin cells. From these results a hypothesis is proposed that neurokinin A might act as a neurotrophic factor in the early stages of the developing porcine chromaffin cells. Biochemical studies are being performed in order to confirm these morphological results and to study the possible role of neurokinin A as a neurotrophic factor in the adrenal gland.On leave from Xian Medical University of China.     

Effects of orexin on cultured porcine adrenal medullary and cortex cells

New orexigenic peptides called orexins have recently been described in the neurons of the lateral hypothalamus and perifornical area. No orexins have been found in the adipose tissues or visceral organs, including the adrenal gland. However, expression of the orexin receptor (OXR) in the rat adrenal gland has been reported. With regard to the effects of orexins on peripheral organs, we previously reported that orexins suppress catecholamine synthesis and secretion in the rat pheochromocytoma cell line PC12. To further clarify the pharmacological effects of orexins on peripheral organs, we examined the effects of orexin-A on catecholamine, cortisol, and aldosterone secretion, using cultured porcine adrenal glands. We initially confirmed the expression of the orexin receptor (OXR-1) in cultured porcine adrenal medulla and cortex. Orexin-A (1000 nM) significantly increased the release of both epinephrine (E) and norepinephrine (NE) from porcine adrenal medullary cells. Similarly, orexin-A (> or = 100 nM) significantly increased the release of both cortisol and aldosterone from porcine adrenal cortex cells. Orexin-A (100 nM) significantly inhibited basal and the PACAP-induced increase in cAMP levels in adrenal medullary cells. Conversely, orexin-A (>o = 100 nM) significantly increased the cAMP level in adrenal cortex cells. These results indicate that orexin-A induces the release of catecholamine from porcine adrenal medullary cells, and aldosterone and cortisol from the cortex cells and has opposite effects on cAMP levels in adrenal medulla and cortex.     

Localization of 3H-estradiol in the uterus of pregnant and non-pregnant armadillos

Summary The uptake and retention of radiolabeled estradiol by the uterus was examined in the armadillo. One pregnant and two non-pregnant armadillos were treated with 1.4 g/kg body weight of ³H-estradiol (E₂) by injection into the left ventricle, and one non-pregnant animal was injected with both the labeled hormone and 140 g/kg body weight of unlabeled E₂. One and a half hour after injection, the animals were sacrificed and the uteri were removed and processed for autoradiography. In the non-pregnant animals, nuclear localization was observed in the interstitial cells and glandular epithelium of the endometrium and the connective tissue cells and smooth muscle of the myometrium. Additionally, there was a gradation of uptake in the epithelial cells of the endometrium in that the glandular cells of the basal region were heavily labeled, while those cells in the sinusoidal, and luminal regions contained successively less label. The luminal cells were poorly labeled. In the pregnant female, the smooth muscle and glandular cells hypertrophied and their nuclei contained less label than was observed in the non-pregnant animals. The arteries of the myometrium were more easily distinguished in the pregnant animals and the nuclei of the endothelial cells and smooth muscle were more consistently labeled than those of the non-pregnant armadillos.     

Localization of follicle regulatory protein in the porcine ovary

The granulosa cell secretes a protein (follicle regulatory protein: FRP) that affects the responsiveness of other follicles to gonadotropin stimulation. This protein was purified, partially characterized, and rabbit antisera as well as monoclonal antibodies were prepared against FRP. Fixed sections of porcine ovaries were prepared on slides and then incubated with the monoclonal antibody or polyclonal antisera and then incubated with either biotinylated mouse IgM or rabbit IgG antisera, respectively. These sections were then incubated with avidin conjugated to horseradish peroxidase, followed by substrate. Staining with both the monoclonal antibody and the antisera was present in the cytoplasm of granulosa cells of small- or medium-sized antral follicles. Staining distribution was localized preferentially to cells near the basal lamina; the antral granulosa cells of viable follicles did not stain. Neither primordial follicles nor pre-antral follicles (less than 300 microns in diameter) showed any positive staining. Thecal cells were not stained in follicles less than 5 mm in diameter, whereas some large follicles (greater than 5 mm) contained staining in the theca. In the latter, specific granulosa staining was only weakly positive with the polyclonal antibody and negative with the monoclonal antibody. Atretic follicles contained significant staining of all epithelial cells adjacent to the basal lamina by both the monoclonal and polyclonal antibody preparations. Staining of the luteal ovary by the monoclonal antibody was limited to the large luteal cells. These findings suggest that FRP is produced by the granulosa cells of porcine follicles at the stage of maturation corresponding to 0.5 mm in diameter. As the viable follicle increases in size, production of FRP in the granulosa is reduced below the detectable level when the follicle exceeds 5 mm in diameter. The main source of FRP during the luteal phase is the large cell of the corpus luteum.     

Progesterone induction of lysozyme and peptidase activities in the porcine uterus.

When nonpregnant ovariectomized gilts were treated daily for 15 days with progesterone or progesterone plus estradiol, there was a tenfold increase in the amount of secreted protein that could be flushed from their uteri compared with control animals administered either no steroid or only estradiol. This increase was due to the appearance of a number of new proteins not present in the controls. In addition to a purple-colored protein with acid phosphatase activity, which has been described previously, there were large increases in lysozyme and leucine aminopeptidase activity. All three enzymes appeared to be induced by progesterone. By continuing the progesterone treatments for periods up to 60 days, it was possible to recover very high levels of each of these enzymes from the uterine flushings of the pigs. Cathepsin activities (B₁, D, and E) were also found to increase as progesterone treatment was prolonged. The levels of the peptidases does not seem to be coordinated since their activities change relative to each other when different animals are examined. Acid phosphatase (due entirely to the purple protein), lysozyme, and leucine aminopeptidase activities are also detectable in allantoic fluid after Day 30 of pregnancy and reach a maximum between Days 60 and 80. It is suggested that these enzymes may be maternal in origin.     

Localization of prostaglandin F in the ovine uterus during early pregnancy

As part of a study on the anti-luteolytic action of the sheep conceptus, the distribution of prostaglandin F (PGF) within the uteri of 19 pregnant and 14 non-pregnant ewes was studied using three antisera to PGF_2α and fluorescent antibody tracing. In the uteri of seven non-pregnant ewes up to Day 11 after estrus (Day 0) and in uteri of $\text{18}\text{19}$ pregnant ewes up to Day 50, PGF was localized mainly in the lamina propria, with very little on epithelial cells lining the uterine lumen. After Day 11, in the uteri of seven non-pregnant ewes, PGF was localized on the surface of luminal epithelial cells and throughout their cytoplasm, and to a lesser extent in the lamina propria. The distribution of PGF on Days 14 and 17 in the gravid and non-gravid horns of the uteri of 13 ewes made unilaterally pregnant (conceptus confined to one uterine horn) was similar to that described above for normal pregnant and non-pregnant ewes after Day 11 respectively.These results are interpreted to indicate a change in the distribution of PGF in sheep uteri due to the presence of a conceptus.     

Localization of the murine cholecystokinin A and B receptor genes

We have determined the chromosomal locations of the two cholecystokinin (CCK) receptor genes in the mouse. Genetic localization utilized an interspecific backcross panel formed from the cross (C57BL/6J x Mus spretus) F₁ x Mus spretus. Genomic DNAs from 94 individuals in the backcross were analyzed by Southern hybridization with rat CCK_A and CCK_B receptor cDNA probes. Unique map positions were determined by haplotype analysis with 650 previously mapped loci in the mouse backcross. The CCK_A receptor gene (Cckar) mapped to mouse Chromosome (Chr) 5, in tight linkage with the DNA marker D5Bir8. The CCK_B receptor gene (Cckbr) mapped to mouse Chr 7, tightly linked to the -hemoglobin locus (Hbb). This localization places Cckbr in the same region as the mouse obesity mutation tubby (tub), which also maps near Hbb (2.4±1.4 cM). Since CCK can function as a satiety factor when administered to rodents, localization of Cckbr near the tub mutation identifies this receptor as a possible candidate gene for this obesity mutation.     

Localization of relaxin binding sites in the rat uterus and cervix by autoradiography

125I-labeled porcine relaxin was injected into 27-day-old rats treated with pregnant mare's serum gonadotropin (PMSG) and known target tissues for relaxin, the myometrium, endometrium and cervix, and putative control tissues, heart, thigh muscle and duodenum, examined for binding by autoradiography. Specific binding in the target tissues was demonstrated by simultaneous injection of excess unlabeled relaxin. Radioactivity was located and quantified by grain counts predominantly over the inner, circular muscle layer of the myometrium and the cervix and to a lesser extent over the outer longitudinal muscle layer of the myometrium and the endometrium. The route of injection, the circulation time, or counting grains in transverse or longitudinal sections of myometrium made little difference in these results. Ovariectomy decreased, but not significantly, the grain count in all of the target tissues studied and estrogen treatment of ovariectomized animals restored the numbers of grains to approximately that of intact PMSG-treated rats. The degree of binding of the cervix was approximately that of the circular myometrial muscle. This work confirms the presence of specific receptors for relaxin in the rat uterus and cervix of primed rats and it also suggests that the inhibitory action of relaxin upon the myometrium is primarily on the inner circular muscle layer.     

Normal murine and porcine embryos recruit NK cells to the uterus

Decidual NK cells, indistinguishable from those found in lymphoid tissues, are present in cell suspensions prepared from maternal decidua of random-bred mice between Days 6.5 and 10.5 of first gestation. The stringency of the correlation between NK cells and normal embryos during successful pregnancy is unknown. Our previous finding that active NK cells were unable to mediate lysis of fresh embryonic tissues at any stage during gestation suggests that if NK cells play a functional role in normal pregnancy it would be a noncytolytic role. Before studies on the function of uterine NK cells were undertaken, evidence that the association of NK cells with normal embryos is widespread was sought by assessing NK cell activity in cell suspensions from decidua of syngeneically mated mice, from decidua of multiparous, random-bred mice, and from the endometrium of pigs during first pregnancy. Neither parity nor maternal-fetal compatibility changed the pattern of high levels of decidual NK cell activity early in pregnancy followed by decline. Porcine NK cell activity was not detected in uterine cells isolated from cycling pigs but in pregnant animals it gradually increased during the preattachment period and reached levels greater than those in blood, postattachment (Day 28). Some of this activity was hormone dependent but sustained increases in NK cell activity required the presence of an embryo. These studies demonstrate that the association of functional NK cells with normal embryos is widespread during early pregnancy.     

The in vitro effect of progesterone on the orexin system in porcine uterine tissues during early pregnancy

Background

Orexin A (OXA) and orexin B (OXB) are hypothalamic-derived peptides that participate in the regulation of energy metabolism, food intake and reproductive function by influencing the hypothalamic-pituitary-ovarian axis. Orexins are also produced in the endometrium, myometrium and placenta, which suggests that they could act as a link between energy metabolism and the reproductive system. Changes in the expression of orexin and the orexin receptor genes and proteins during the oestrous cycle and early gestation in pigs imply that orexin activity may be regulated by local factors within the uterus. The aim of this study was to investigate the influence of progesterone (P₄) on the expression of orexin system genes, and proteins in the porcine uterus during early gestation. Gene expression was analyzed by real-time PCR. Adiponectin secretion was determined by ELISA, and the receptors proteins content was defined using western blot analysis.

Results

In the endometrium, P₄ enhanced OXA secretion on days 10 to 11 of gestation and OXB secretion on days 12 to 13. In the myometrium, P₄ inhibited the secretion of both orexins on days 15 to 16 and OXB secretion also on days 12 to 13. In the endometrium, P₄ inhibited the expression of orexin receptor 1 (OX1R) protein at nearly all times analyzed, whereas the expression of orexin receptor 2 (OX2R) protein was inhibited only on days 15 to 16 of gestation. In the myometrium, P₄ stimulated OX1R protein expression on days 12 to 13 and 15 to 16 of gestation and inhibited OX1R protein expression on days 27 to 28. The expression of OX2R protein in the myometrium increased on days 12 to 13 and decreased on days 10 to 11 and 15 to 16.

Conclusions

The results indicate that P₄ could regulate the expression of the orexin system in the porcine uterus during early pregnancy, which suggests the presence of a local feedback loop that could play an important role in the regulation of maternal metabolism during pregnancy. The findings may contribute to the existing knowledge of the mechanisms linking maternal energy metabolism with the regulation of the reproductive system during pregnancy.

     

Localization of arylsulphatase A and B activities in rat kidney.

Very high arylsulphatase activity has been detected in rat kidney. It is the highest in renal cortex (19 U/g tissue), 3-30 times higher than in other rat organs. Histochemically, arylsulphatase B (N-acetylgalactosamine-4-sulphate sulphatase) activity is localized in large lysosomes of proximal convoluted tubules, where it accounts for over 90% of total arylsulphatase activity. This suggests that the enzyme plays an important role in the degradation of endocytosed sulphated oligosaccharides.