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.     

Divergent Roles for Adiponectin Receptor 1 (AdipoR1) and AdipoR2 in Mediating Revascularization and Metabolic Dysfunction in Vivo

Adiponectin is a well described anti-inflammatory adipokine that is highly abundant in serum. Previous reports have found that adiponectin deficiency promotes cardiovascular and metabolic dysfunction in murine models, whereas its overexpression is protective. Two candidate adiponectin receptors, AdipoR1 and AdipoR2, are uncharacterized with regard to cardiovascular tissue homeostasis, and their in vivo metabolic functions remain controversial. Here we subjected AdipoR1- and AdipoR2-deficient mice to chronic hind limb ischemic surgery. Blood flow recovery in AdipoR1-deficient mice was similar to wild-type; however, revascularization in AdipoR2-deficient mice was severely attenuated. Treatment with adiponectin enhanced the recovery of wild-type mice but failed to rescue the impairment observed in AdipoR2-deficient mice. In view of this divergent receptor function in the hind limb ischemia model, AdipoR1- and AdipoR2-deficient mice were also evaluated in a model of diet-induced obesity. Strikingly, AdipoR1-deficient mice developed severe metabolic dysfunction compared with wild type, whereas AdipoR2-deficient mice were protected from diet-induced weight gain and metabolic perturbations. These data show that AdipoR2, but not AdipoR1, is functionally important in an in vivo model of ischemia-induced revascularization and that its expression is essential for the revascularization actions of adiponectin. These data also show that, in contrast to revascularization responses, AdipoR1, but not AdipoR2 deficiency, leads to diet-induced metabolic dysfunction, revealing that these receptors have highly divergent roles in vascular and metabolic homeostasis.     

Fine structure of the porcine myometrium during the oestrous cycle

The ultrastructure of the porcine myometrium was studied at well-defined stages of the oestrous cycle by transmission electron microscopy. The fine structure of the myometrium in the pig was found to be similar to that observed in other genital organs and species and did not show obvious variations through the oestrous cycle. The cell-to-cell contacts between smooth-muscle cells most consistently found were intermediate junctions and simple appositions, whereas interdigitations of muscle cell processes were more sparse. Gap junctions were few and small. Nerve terminals possessing presynaptic vesicles typical of the adrenergic and cholinergic type were found, though sparsely, both within and close to muscle bundles.     

The unoccupied nuclear oestradiol receptor in the rat uterus and hypothalamus during the oestrous cycle.

The nuclear oestrogen receptor population in the rat uterus contained an unoccupied receptor component that bound oestradiol with the high affinity (Kd congruent to 0.5 nM) characteristic of oestrogen receptors. This unoccupied receptor was present at all phases of the oestrous cycle. Its content changed in parallel with that of the total nuclear receptor during the cycle. Oestradiol administration to the immature rat resulted in increases in the uterine content of long-term nuclear receptors (i.e., those still present 8 h after administration); these increases were due to occupied oestrogen receptors, since the content of unoccupied receptor was unchanged. Our previous experiments [White & Lim (1980) Biochem. J. 190, 833-837] have shown in contrast, that oestradiol administration results in an increase in the content of unoccupied nuclear receptor in the hypothalamus. However, as in the uterus, similar cyclic changes in the content of unoccupied nuclear receptor occurred in parallel with those of the total nuclear receptor population in the hypothalamus. Differences and similarities between the unoccupied nuclear receptor of the uterus and hypothalamus are briefly discussed.     

Intracellular relationships of the oestrogen receptor in the rat uterus and hypothalamus during the oestrous cycle.

Simultaneous measurements were made of the specific oestrogen receptor in the nuclear and cytosol fractions prepared from the uterus and hypothalamus of 50--81-day-old female rats undergoing a 4-day oestrous cycle. In the uterus, the content of nuclear receptor fluctuated in concert with known cyclic changes in the secretion of oestrogen, being maximal at pro-oestrus. Over the period of 50--81 days, the nuclear content at all phases increased with age, again corresponding to known age-related increases in ovarian secretion of oestrogen. This age-related increase in nuclear content, averaged from the values of the different phases in each age group, was related to equivalent increases in uterine wet weight, an increase of 1 pmol of receptor being accompanied by an increase of 80--90 mg. The concentration of cytosol receptor was maintained constant, with respect to wet weight, throughout the cycle and with age, irrespective of changes in nuclear content. In the uterus of normal mature females, translocation of receptor into the nucleus did not lead to depletion of cytosol receptor, suggesting a process of continuous replenishment/synthesis. In the hypothalamus, the nuclear content of oestrogen receptor was also maximal at pro-oestrus. In contrast with the uterus, the content of hypothalamic cytosol receptor was minimal at this phase and reflects depletion of the cytosol receptor, possibly as a result of translocation. The extent of translocation was low compared with that in the uterus and did not alter with age during the age-period studied. This low nuclear binding of the receptor in vivo is discussed in relation to the presence of a cytosol factor, present in limiting amounts, which in vitro mediates the binding of cytosol receptor to oligo(dT)-cellulose. The difference in the physiological response of the uterus and of the hypothalamus to oestrogens may be related to the extent of nuclear binding of receptor.     

The expression of chemerin and its receptors (CMKLR1, GPR1, CCRL2) in the porcine uterus during the oestrous cycle and early pregnancy and in trophoblasts and conceptuses

Recent research has demonstrated that chemerin may take part in the regulation of reproduction. The aim of this study was to determine the expression of chemerin system – chemerin and its receptors, chemokine-like receptor 1 (CMKLR1), G protein-coupled receptor 1 (GPR1) and C-C chemokine receptor-like 2 (CCRL2) – in the porcine uterus during the oestrous cycle and early pregnancy, and in trophoblasts and conceptuses by real-time PCR and western blotting. Chemerin concentrations in uterine luminal flushings (ULF) were determined using ELISA test. In the endometrium, the highest expression of chemerin and GPR1 proteins was observed during the mid-luteal phase; CMKLR1, during the late luteal phase; and CCRL2, during the follicular phase of the cycle. In the myometrium, chemerin protein expression was enhanced during the early luteal phase, and chemerin receptor proteins were highly expressed during the follicular phase. In the endometrium of pregnant pigs, the highest expression of chemerin and CCRL2 protein was observed during implantation; CMKLR1, during placentation; and GPR1, during embryo migration. In the myometrium, chemerin and CCRL2 protein expression increased at the end of implantation, and the expression of CMKLR1 and GPR1 protein was enhanced during implantation. In the conceptuses and trophoblasts, the highest expression of chemerin system proteins was observed during placentation, with the exception of GPR1 protein in the trophoblasts. The highest concentrations of the analysed adipokine were observed in ULF during the luteal phase of the cycle and during maternal recognition of pregnancy. This is the first study to demonstrate that the expression of the chemerin system in the porcine uterus, conceptuses and trophoblasts, and chemerin concentrations in ULF are influenced by the hormonal milieu in different stages of the oestrous cycle and in early pregnancy. The present results also suggest that chemerin is implicated in the regulation of reproductive functions in pigs.     

The role of AdipoR1 and AdipoR2 in liver fibrosis

Activation of the adiponectin (APN) signaling axis retards liver fibrosis. However, understanding of the role of AdipoR1 and AdipoR2 in mediating this response is still rudimentary. Here, we sought to elucidate the APN receptor responsible for limiting liver fibrosis by employing AdipoR1 and AdipoR2 knock-out mice in the carbon tetrachloride (CCl₄) model of liver fibrosis. In addition, we knocked down receptor function in primary hepatic stellate cells (HSCs) in vitro. Following the development of fibrosis, AdipoR1 and AdipoR2 KO mice had no quantitative difference in fibrosis by Sirius red staining. However, AdipoR2 KO mice had an enhanced fibrotic signature with increased Col1-α1, TGFß-1, TIMP-1, IL-10, MMP-2 and MMP-9. Knockdown of AdipoR1 or AdipoR2 in HSCs followed by APN treatment demonstrated that AdipoR1 and AdipoR2 did not affect proliferation or TIMP-1 gene expression, while AdipoR2 modulated Col1-α1 and α-SMA gene expression, HSC migration, and AMPK activity. These finding suggest that AdipoR2 is the major APN receptor on HSCs responsible for mediating its anti-fibrotic effects.     

Immunophenotype of porcine oviduct epithelial cells during the oestrous cycle: a double-labelling immunohistochemical study

The luminal epithelium of the porcine oviduct is composed of ciliated cells and secretory cells, but it is assumed for several species that under the control of steroid hormones secretory cells are able to be transformed into ciliated cells. In order to better understand such physiological changes during the different stages of the oestrous cycle, we evaluated epithelial cell proliferation together with oestrogen receptor (ER) expression of porcine ampullary oviducts. To identify the immunophenotype of proliferating cells, double immunohistochemistry was performed using anti-chromogranin A antibody (anti-CgA) as the second primary antibody. Anti-CgA, recently shown to be an immunocytochemical marker of ciliated cells of the cow, also labelled specifically the luminal surface of ciliated cells of the pig. Double labelling of sections with the monoclonal antibody MIB-1 against the proliferation-associated nuclear epitope Ki-67 and anti-CgA clearly demonstrates that MIB-1 was selectively localised in the nuclei of secretory cells. Proliferative activity was not observed in CgA-positive ciliated cells in all examined stages of the oestrous cycle. The percentage of Ki-67-positive epithelial cells was higher at pro-oestrus, compared with the other stages of the oestrous cycle. Furthermore, ER immunoreactivity was exclusively detected in the nuclei of the epithelial cells, which were negative for CgA. We conclude, therefore, that oestrogen may induce the initial proliferation of secretory cells and promote the differentiation into ciliated cells.     

Concentration of steroids in bovine peripheral plasma during the oestrous cycle and the effect of betamethasone treatment.

Testosterone, oestradiol and progesterone were measured in peripheral plasma during the oestrous cycle of 6 heifers. Oestradiol and progesterone results confirmed earlier reports. Concentration of testosterone on the day of oestrus was 40+/-3 pg/ml (mean+/-S.E.M.), and two peaks were detected during the cycle, one 7 days before oestrus (1809+/-603 pg/ml) and the other (78+/- 7 pg/ml) on the day before the onset of oestrus. The concentration of progesterone declined in most cases 1 day after the maximum concentration of testosterone. Betamethasone treatment in 5 heifers extended luteal function by an average of 10 days: plasma androstenedione and oestradiol concentrations were unaltered; cortisol values were depressed for at least 16 days after treatment; testosterone concentrations were lowered by 13+/-2-4% during treatment, and except in one heifer the peak on Day -7 was abolished.     

Expression of prostaglandin synthesis pathway enzymes in the porcine corpus luteum during the oestrous cycle and early pregnancy

Prostaglandins (PGs) of luteal origin may have paracrine and/or autocrine actions on the functions of the corpus luteum (CL). Previously, we have shown that enzymes of PG synthesis pathway such as prostaglandin E synthase (mPGES-1), prostaglandin F synthase (PGFS) and prostaglandin 9-ketoreductase (CBR1) are important in regulation of PG production in the conceptuses and endometrium of cyclic and pregnant pigs. Therefore, localization and expression patterns of these enzymes were determinated in porcine CL. The PGFS protein content was lower in metestrus and higher around luteolysis, and then decreased in late regressing CL. PGFS protein levels were lower on days 5-8 of pregnancy and did not differ between days 10 and 25. Elevated expression of mPGES-1 mRNA was found in early luteal phase. The mPGES-1 protein content, similarly to PGFS, was higher during luteolysis. mPGES-1 mRNA and protein levels were constant between days 5 and 25 of pregnancy. PGFS and mPGES-1 expression was down-regulated on days 16-17 of the oestrous cycle when compared to the corresponding days of pregnancy. Enhanced mPGES-1/PGFS ratio occurred during early luteal phase and days 5-8 of pregnancy. Expression of CBR1 mRNA and protein was constant during the cycle and pregnancy. Our studies revealed higher mPGES-1/PGFS ratios in the CL during early luteal phase and corresponding days of pregnancy that could favor PGE(2) synthesis and may be important in the control of luteal development. However, PG synthesis in the endometrium/conceptus rather than in the CL could be involved in luteolysis and maternal recognition of pregnancy in pigs.     

Follicular growth and kinetics in the Indian gerbil (Tatera indica) ovary during oestrous cycle

The growth of the follicle and oocyte in the Indian gerbil (Tatera indica) was a continuous process. The relationship between follicle and oocyte or its nucleus was log linear, represented by the equation log Y =a +b logX.A linear relationship (Y =a +bX)existed between the oocyte and its nucleus. The number of stages I and II follicles varied significantly during the oestrous cycle. Maximum percentage of stage I follicles was observed during oestrus and metoestrus, while stage II follicles were abundant during dioestrus, metoestrus and pro-oestrus. These follicles were significantly more in number than other types of the follicles. The occurrence of comparatively larger follicles during pro-oestrus and the presence of newly formed corpora lutea at oestrus, indicated ovulation in the early oestrus.     

Cellular proliferation in the lymphoid tissues of an inbred (RT1a) rat strain during the oestrous cycle

Changes in body weight, uterine weight and tissue weight, cell content and cellular proliferation of the thymus and uterine regional and popliteal lymph nodes were examined at daily intervals during the oestrous cycle of the DA (RT1a) inbred rat strain. Thirty-nine sexually mature virgin animals, aged between 13 and 15 weeks were used in this investigation. During dioestrus body weight and uterine weight fell significantly, while intrathymic and intranodal cellular proliferation increased significantly. These findings are discussed in relation to ovarian hormone secretion and it is suggested that increased thymocyte and lymphocyte proliferation occurs in response to rising oestrogen levels. This proliferative response prepares the female for the immunological challenge with allogeneic spermatozoa should mating occur during the subsequent oestrous phase.     

Immunoglobulins in the mouse uterus during the oestrous cycle

The distribution of IgA, IgG and IgM was studied by an immunoperoxidase technique on sections of mouse uteri at each stage of the oestrous cycle. Staining for IgG and IgA was highest at pro-oestrus, declined at oestrus and was very low during the other stages. At pro-oestrus IgG was found throughout the stroma, in the uterine lumen, and in 10% of glandular lumina; very few IgG-containing plasma cells were present. At pro-oestrus, IgA was found in the uterine lumen, and in most of the uterine glands, both in the lumen and in the epithelium; little IgA was present in the stroma. IgA-plasma cells were detected at each stage of the cycle and were particularly numerous at pro-oestrus and oestrus. These results suggest that IgA is secreted locally from plasma cells into the uterine gland through the glandular epithelium, but that IgG enters the stroma from the local capillaries. The obvious increase in IgG and IgA secretion at pro-oestrus, when plasma oestradiol levels are highest, supports the hypothesis that, during the oestrous cycle, the humoral immune response is regulated in the uterus by ovarian hormones.