Plasma follicle stimulating hormone and luteinizing hormone patterns during the estrous cycle of ewes

Fifteen Suffolk ewes were used in three experiments to compare plasma follicle stimulating hormone (FSH) and luteinizing hormone (LH) patterns during the estrous cycle and to determine whether FSH levels undergo changes in pulse frequency. Luteinizing hormone changed inversely with progesterone levels whereas FSH and progesterone concentrations revealed no obvious relationship. Unlike LH, FSH levels did not pulsate during the follicular phase. Higher FSH levels were detected on days 1, 6 and 12 and lower levels on days 0, 4 and 16. Coincident preovulatory LH and FSH surges were observed and this was the only time FSH and LH levels appeared to be jointly controlled.     

Characterization of alpha-melanocyte-stimulating hormone in rat pancreas

Reverse-phase high performance liquid chromatography and radioimmunoassay were used to characterize alpha-melanocyte-stimulating hormone (alpha-MSH)-like peptides in rat pancreas. Relative to synthetic alpha-MSH standards, serial dilutions of pancreas extracts showed parallel and concentration dependent displacement of (125I) alpha-MSH from alpha-MSH antibody. Chromatographic separation revealed immunoreactive material coeluting with synthetic N,O-diacetyl alpha-MSH, which accounted for 78% of total alpha-MSH materials in this tissue. The remainder of immunoreactive alpha-MSH coeluted with synthetic alpha-MSH, desacetyl alpha-MSH, or their methionine sulfoxides. In contrast with anterior pituitary, it appears that biosynthetic processing of alpha-MSH from pro-opiomelanocortin (POMC) may be similar in rat pancreas and pituitary intermediate lobe, since their relative alpha-MSH immunoreactive elution profiles were similar. These findings support the hypothesis of tissue specific regulation of biosynthetic processing of POMC.     

Effects of thymulin and GnRH on the release of gonadotropins by in vitro pituitary cells obtained from rats in each day of estrous cycle

The effects of thymulin and GnRH on FSH and LH release were studied in suspension cultures of anterior pituitary cells from female adult rats sacrificed on each day of the estrous cycle. The spontaneous release of gonadotropins by pituitaries, as well as their response to GnRH or thymulin addition, fluctuated during the estrous cycle. Adding thymulin to pituitary cells from rats in diestrus 1 increased the concentration of FSH; while in cells from rats in estrus, FSH level decreased. Thymulin had a stimulatory effect on the basal concentration of LH during most days of the estrous cycle. Adding GnRH increased FSH release in cells from rats in diestrus 1, diestrus 2, or proestrus, and resulted in higher LH levels in cells obtained from rats in all days of the estrous cycle. Compared to the GnRH treatment, the simultaneous addition of thymulin and GnRH to cells from rats in diestrus 1, diestrus 2, or proestrus resulted in lower FSH concentrations. Similar results were observed in the LH release by cells from rats in diestrus 1, while in cells from rats in proestrus or estrus, LH concentrations increased. A directly proportional relation between progesterone serum levels and the effects of thymulin on FSH release was observed. These data suggest that thymulin plays a dual role in the release of gonadotropins, and that its effects depend on the hormonal status of the donor's pituitary.     

Gonadotropin-inhibitory hormone (GnIH) and its receptor in the female pig: cDNA cloning, expression in tissues and expression pattern in the reproductive axis during the estrous cycle

Since its discovery, gonadotropin-inhibitory hormone (GnIH) has appeared to act as a key neuropeptide in the control of vertebrate reproduction. GnIH acts via the novel G protein-coupled receptor 147 (GPR147) to inhibit gonadotropin release and synthesis. To determine the physiological functions of GnIH in the pig, a study was conducted to clone and sequence the cDNA of the GnIH precursor and GPR147. Our results demonstrated that the cloned pig GnIH precursor cDNA encoded three LPXRF and that its receptor possessed typical transmembrane features. Subsequently, tissue expression studies revealed that GnIH was mainly expressed in the brain, corresponding largely with the tissue expression patterns of GPR147 in the pig. The expression patterns in the reproductive axis of the female pig across the estrous cycle were also systemically investigated. The hypothalamic levels of both GnIH and its receptor mRNA were lowest in estrus and peaked in the proestrus and diestrus phases. The highest pituitary GnIH mRNA level was detected in the metestrus, and its receptor displayed a somewhat similar pattern of expression to that of the ligand. However, the expression patterns of GnIH and GPR147 were negatively correlated in the ovary. Immunolocalization in the ovary during the estrous cycle revealed that the immunoreactivities of GnIH and GPR147 were mainly localized in the granulosa and theca cells of the antral follicles during proestrus and estrus and in the luteal cells during metestrus and diestrus. Taken together, this research provided molecular and morphological data for further study of GnIH in the pig.     

Oxytocin secretion induced by osmotic stimulation in rats during the estrous cycle and after ovariectomy and hormone replacement therapy

Hyperosmolality is a potent stimulus for the secretion of oxytocin. Oxytocinergic neurons are modulated by estrogen and oxytocin secretion in rats varies according to the phase of the estrous cycle, with higher activity during proestrus. We investigated the oxytocin secretion induced by an osmotic stimulus (0.5 M NaCl) in female rats. Plasma oxytocin and the oxytocin contents in the neurohypophysis and the paraventricular and supraoptic nuclei were determined during the morning (8-9 h) and afternoon (17-18 h) of the estrous cycle and after ovariectomy followed or not by hormone replacement. Plasma oxytocin peaked in control animals during proestrus. Oxytocin content decreased in the paraventricular and supraoptic nuclei during proestrus and estrus compared to diestrus and increased in the neurohypophysis during proestrus morning. No significant difference was observed in the oxytocin content of the neurohypophysis, nuclei or plasma between ovariectomized animals and ovariectomized animals treated with estrogen or estrogen plus progesterone. Therefore, any ovarian factor other than estrogen or progesterone seems to play a direct or indirect role in the increase in oxytocin secretion. The osmotic stimulus caused an increase in plasma oxytocin throughout the estrous cycle. A reduction in oxytocin content during diestrus and an increase during proestrus were observed in the paraventricular nuclei. In ovariectomized animals, the treatment with estrogen potentiated the response of oxytocin to the osmotic stimulus, with the response being even stronger in the case of estrogen plus progesterone. In conclusion, the ovarian steroids estrogen plus progesterone could modulate the osmoreceptor mechanisms related to oxytocin secretion.     

Nitric oxide enhances the sensitivity of alpaca melanocytes to respond to α-melanocyte-stimulating hormone by up-regulating melanocortin-1 receptor

Nitric oxide (NO) and α-melanocyte-stimulating hormone (α-MSH) have been correlated with the synthesis of melanin. The NO-dependent signaling of cellular response to activate the hypothalamopituitary proopiomelanocortin system, thereby enhances the hypophysial secretion of α-MSH to stimulate α-MSH-receptor responsive cells. In this study we investigated whether an NO-induced pathway can enhance the ability of the melanocyte to respond to α-MSH on melanogenesis in alpaca skin melanocytes in vitro. It is important for us to know how to enhance the coat color of alpaca. We set up three groups for experiments using the third passage number of alpaca melanocytes: the control cultures were allowed a total of 5 days growth; the UV group cultures like the control group but the melanocytes were then irradiated everyday (once) with 312 mJ/cm² of UVB; the UV + L-NAME group is the same as group UV but has the addition of 300 μM L-NAME (every 6 h). To determine the inhibited effect of NO produce, NO produces were measured. To determine the effect of the NO to the key protein and gene of α-MSH pathway on melanogenesis, the key gene and protein of the α-MSH pathway were measured by quantitative real-time PCR and Western immunoblotting. The results provide exciting new evidence that NO can enhance α-MSH pathway in alpaca skin melanocytes by elevated MC1R. And we suggest that the NO pathway may more rapidly cause the synthesis of melanin in alpaca skin under UV, which at that time elevates the expression of MC1R and stimulates the keratinocytes to secrete α-MSH to enhance the α-MSH pathway on melanogenesis. This process will be of considerable interest in future studies.     

Effect of the season on some aspects of the estrous cycle in Martina Franca donkey

The Martina Franca (MF) donkey breed, with 48 jackasses and 515 jennies, is considered an endangered breed according to the data from the Monitoring Institute for Rare Breeds and Seeds in Europe. The knowledge of the estrous cycle characteristics has a great impact for assisted reproduction, especially in endangered species. In this study, the estrous cycle characteristics were investigated in 12 MF jennies throughout the year. Estrous cycle, estrous and diestrous lengths, follicular growth and ovulation, and estradiol-17β (E2) and progesterone (P4) plasma concentrations were monitored in MF jennies and compared in different seasons. In all jennies (100%) estrous cycle was detected during the whole year, with no differences in the estrous cycle length among seasons. However, a significant increase of estrous length in spring and summer compared with autumn and winter was found. Diestrus was shorter in summer than in the other seasons. Estrous behavior was always shown and characterized by rhythmic eversion of the vulvar labia (winking) with exhibition of the clitoris, urination, male receptivity and clapping, with sialorrhoea, neck and head extension, and back ears. Estrus was characterized by the ovulation of a larger follicle in spring and summer than in autumn and winter. The pattern of E2 and P4 plasma concentrations during the estrous cycle were similar to that reported for the mare, but without differences among the four seasons, so that a negligible effect of environmental conditions on ovarian E2 and P4 secretion was hypothesized, despite the larger diameter of the ovulating follicle in spring and summer.     

Molecular mechanism of cell proliferation in rodent uterus during the estrous cycle

The rodent uterus is a widely studied target tissue for sexual steroid hormone action. The aim of the present study was to assess the molecular mechanism that participates in the initiation of cell proliferation of the rat uterine epithelial cells during the estrus (E)–metestrus (M) transition. Cell proliferation, ERα, c-fos, cyclin D1 and D3, cdk4, and cdk6 proteins were assessed in these animals by immunohistochemistry. Estradiol (E₂) and progesterone (P₄) plasma levels were assessed by RIA. The results indicate that the glandular epithelium starts to proliferate at 21:00 h on estrus day, and initiates at least 3 h before the luminal epithelium does. Fos expression was markedly increased during the afternoon of estrus day, and its increase was in parallel to ERα expression. Interestingly, both, cyclin D1 and D3 were abundantly expressed in the luminal and glandular epithelia, and nuclear immunolabelling of cyclin D1 and D3 precedes BrdU incorporation in the cell. cdk4 and cdk6 were localized in the nuclei in both epithelia throughout the studied time course. In addition, cdk4 was more abundant throughout estrus and metestrus days than cdk6. The overall results indicate that ERα, Fos and cyclins D1 and D3, cdk4 and cdk6 are expressed in both glandular and luminal epithelia of the rat uterus during the E–M transition. In conclusion, there is a good correlation between sequential expression of these proteins and cell cycle progression in the rat uterine epithelial cells during the estrous cycle. However, the differences observed in the cellular localization, time course of expression and the cellular types that express both cyclins between physiological and pharmacological conditions, demonstrated different mechanisms of regulation and should be due to the complex hormonal milieu during the estrous cycle.     

Pivotal roles of alpha-melanocyte-stimulating hormone and the melanocortin 4 receptor in leptin stimulation of prolactin secretion in rats

Leptin, the obese gene product, was reported to stimulate prolactin (PRL) secretion, but the neuroendocrine mechanism underlying this hormonal response is largely unknown. Thus, in this study we examined the involvement of several important PRL regulators in the leptin-induced PRL secretion in male rats. Compared with the values in normally fed rats, food deprivation for 3 days significantly decreased both PRL and leptin levels in the plasma. These changes were reverted to normal by a 3-day constant infusion of 75 microg/kg/day of leptin to the fasted rats, while 225 microg/kg/day of leptin further elevated both PRL and leptin levels. These four groups of animals were used for the following experiments. Results of dopamine and serotonin turnover studies in the brain and the pituitary indicated that neither of these biogenic amines plays a primary role in mediating leptin's effects on PRL. Repeated intracerebroventricular injections over 72 h of neutralizing antibodies against vasoactive intestinal peptide, PRL-releasing peptide, or beta-endorphin, did not significantly suppress the leptin actions. However, both the blockade of the melanocortin (MC) 4 receptor (R) and the immunoquenching of brain alpha-melanocyte-stimulating hormone (alpha-MSH) completely abolished the leptin-induced PRL release, and the stimulation of the MC4-R, but not the MC3-R, significantly elevated PRL levels in the fasted rats. These results suggest that alpha-MSH, a cleaved peptide from pro-opiomelanocortin of which synthesis is stimulated by leptin, may be the pivotal neuropeptide in the brain mediating the leptin's stimulatory influence on PRL secretion. It was also suggested that the MC4-R may be the primary subtype of the MC-Rs mediating this action of alpha-MSH.     

Effects of corticotropin-releasing hormone on proopiomelanocortin derivatives and monocytic HLA-DR expression in patients with septic shock

Little is known about interactions between immune and neuro-endocrine systems in patients with septic shock. We therefore evaluated whether the corticotropin-releasing hormone (CRH) and/or proopiomelanocortin (POMC) derivatives [ACTH, β-endorphin (β-END), β-lipotropin (β-LPH), α-melanocyte stimulating hormone (α-MSH) or N-acetyl-β-END (Nac-β-END)] have any influences on monocyte deactivation as a major factor of immunosuppression under septic shock conditions. Sixteen patients with septic shock were enrolled in a double-blind, cross-over and placebo controlled clinical study; 0.5 μg/(kg_bodyweight h) CRH (or placebo) were intravenously administered for 24 h. Using flow cytometry we investigated the immunosuppression in patients as far as related to the loss of leukocyte surface antigen-DR expression on circulating monocytes (mHLA-DR). ACTH, β-END immunoreacive material (IRM), β-LPH IRM, α-MSH and Nac-β-END IRM as well as TNF-α and mHLA-DR expression were determined before, during and after treatment with CRH (or placebo). A significant correlation between plasma concentration of α-MSH and mHLA-DR expression and an inverse correlation between mHLA-DR expression and TNF-α plasma level were found. Additionally, a significant increase of mHLA-DR expression was observed 16 h after starting the CRH infusion; 8 h later, the mHLA-DR expression had decreased again. Our results indicate that the up-regulation of mHLA-DR expression after CRH infusion is not dependent on the release of POMC derivatives. From the correlation between plasma concentration of α-MSH and mHLA-DR expression, we conclude that in patients with septic shock the down-regulation of mHAL-DR expression is accompanied by the loss of monocytic release of α-MSH into the cardiovascular compartment.     

Effects of neonatal treatment with phytoestrogens, genistein and daidzein, on sex difference in female rat brain function: estrous cycle and lordosis

It is well known that neonatal exposure to estrogen induces masculinization or defeminization of the brain. In this study, the effects of neonatal treatment with two kinds of soybean isoflavone aglycone, genistein (GS) and daidzein (DZ), on the estrous cycle and lordosis behavior were investigated. Female rats were injected subcutaneously with 1 mg GS, 1 mg DZ, 100 microg estradiol (E2), or oil daily for 5 days from birth. As a result, vaginal opening was advanced in GS- or E2-treated females. A vaginal smear check indicated that oil- or DZ-treated females showed a constant 4- or 5-day estrous cycle, whereas GS- or E2-treated rats showed a persistent or prolonged estrus. Ovariectomy was performed in all females at 60 days of age. The ovaries in the GS- or E2-treated groups were smaller than those in the oil- and DZ-treated groups and contained no corpora lutea. In the DZ group, although corpora lutea were seen, ovaries were smaller than that of control females. Behavioral tests were carried out after implantation of E2-tubes. All of the oil- or DZ-treated females showed lordosis with a high lordosis quotient (LQ). On the other hand, as male rats, LQs were extremely low in the E2-treated group, when compared to the oil-treated group. In the GS-treated group, the mean LQ was lower than that in the oil-treated group, but higher than those in the E2-treated female or male groups. These results suggest that genistein acts as an estrogen in the sexual differentiation of the brain and causes defeminization of the brain in regulating lordosis and the estrous cycle in rats. In addition, neonatal daidzein also has some influence on ovarian function.     

The influence of steroids on vascular tension of isolated superficial veins of the nose and face during the estrous cycle of gilts

The arrangement of the superficial facial veins enables blood flow from the nasal cavity into the peripheral circulation by two pathways: through the frontal vein into the cavernous sinus and through the facial vein into the external jugular vein. The current study was designed to determine whether estradiol and progesterone affect the vascular tone of the superficial veins of the nose and face in cycling gilts (Sus scrofa f. domestica) and to analyze the immunolocalization of progesterone receptors and estradiol receptors in these veins. The influence of hormones on vascular tension differed depending on the type of vessel and the phase of the estrous cycle. Estradiol decreased vascular tension in the nasal vein during the follicular phase (P < 0.05) and increased tension in the frontal vein during the luteal phase (P < 0.05). Progesterone increased the vascular tension of the frontal vein (P < 0.05) and decreased the tension of the other veins (P < 0.05) in both phases of the cycle. Expression of estradiol receptor β but not of progesterone receptor was observed in the superficial veins of the nose and face. In conclusion, the effect of ovarian steroid hormones on the vascular tension of the superficial veins of the nose and face in female pigs as well as the reactivity of these veins to steroid boar pheromones can affect the blood supply from the nasal cavity to the venous cavernous sinus. We propose that the ovarian steroid hormones that modulate the vascular tension of the nasal and facial veins may also influence the action of boar pheromones absorbed into the nasal mucosa in gilts and may reach the brain via local destination transfer.     

The expression of angiogenic growth factors and their receptors in ovarian follicles throughout the estrous cycle in the ewe

Healthy follicles are highly vascularized whereas those undergoing atresia have poor vascularity, suggesting a relationship between follicular vascularization and follicular function. Vascularization is regulated by angiogenic factors, and among them vascular endothelial growth factor (VEGF) and angiopoietin-Tie (Ang-Tie) systems are of central importance. The objectives of this study were to determine if VEGF, VEGF receptor-2 (VEGFR-2), and components of the Ang-Tie system are expressed in ovarian follicles at both the protein and mRNA levels and to explore if their expression is related to the stage of the estrous cycle in the ewe. Ovaries from cyclic ewes were collected during the luteal phase (n = 5) or before (n = 5), during (n = 4), and after (n = 4) the preovulatory luteinizing hormone (LH) surge. After fixation, ovaries were wax-embedded, serially sectioned, and analyzed for both protein and mRNA expression of VEGF, VEGFR-2, angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), Tie-1 (mRNA only), and Tie-2. mRNA was studied by in situ hybridization using digoxigenin-11-UTP-labeled ovine riboprobes. A similar pattern of expression was observed for mRNA and protein for all of the factors. Both mRNA and protein expression of VEGF, VEGFR-2, Ang-1, Ang-2, Tie-1 (mRNA only), and Tie-2 in the granulosa and theca cells of follicles ≥2 mm in diameter was significantly different among the stages of the estrous cycle, with the highest expression detected at the post-LH surge stage. Theca cells expressed significantly greater levels of the six angiogenic factors compared with granulosa cells at all stages of the estrous cycle. Expression levels in granulosa and theca cells were comparable between small (2.0 to 2.5 mm) and medium (2.5 to 4.0 mm) follicles, but large follicles (>4.0 mm) expressed higher mRNA and protein levels (all P < 0.05) for all factors at all stages of the estrous cycle. These data show (i) that VEGF, VEGFR-2, and the Ang-Tie system are present in both granulosa and theca cells of the ovarian follicle, (ii) that thecal cells consistently express greater levels of all of these factors compared with granulosa cells, and (iii) that their levels of expression are related to the stage of the estrous cycle and to follicle size.     

Cervical changes in estrogen receptor alpha,oxytocin receptor,LH receptor,and cyclooxygenase-2 depending on the histologic compartment,longitudinal axis,and day of the ovine estrous cycle

The aim was to investigate the histologic distribution of estrogen receptor α (ERα), oxytocin receptor (OxR), LH receptor (LHR), and cyclooxygenase-2 (COX-2) in the cervix of the ewe during the estrous cycle. Immunohistochemistry was performed in the cranial and caudal cervix of Corriedale ewes on Day 1 (n = 6), 6 (n = 5), or 13 (n = 6) after estrous detection (Day 0). The ERα proportional score (%ERα nuclei) was lower in the cranial cervix than in the caudal cervix, whereas the OxR and COX-2 immunostaining areas (%areas) were greater in the cranial cervix than in the caudal cervix (P < 0.04). The %ERα nuclei decreased from Days 1 to 13 in luminal epithelia, but increased from Days 1 to 6 or remained unchanged in stromata (P < 0.003). The %OxR area was higher on Day 6 than on Days 1 and 13 in the superficial glandular epithelium, and increased from Days 1 to 13 in the deep glandular epithelium (P < 0.04). The %LHR area increased during the estrous cycle in luminal epithelia and fold stroma (P < 0.004). The %COX-2 area was restricted to epithelia, and it was lower on Day 1 than on Days 6 and 13 in luminal epithelia (P < 0.05). Differences in ERα, OxR, LHR, and COX-2 between cranial and caudal cervical zones indicate different physiological functions, and their cyclic variations in the cervical epithelia, in contrast to little or no variations in the stroma, suggest a hormone-responsive driving role of epithelia in cervical function.     

Studies on the relationship between paracoccidioidomycosis in ddY mice and their estrous cycle

The relationship between paracoccidioidomycosis in ddY mouse and its estrous cycle was studied. Adult ddY mice of both sexes were used as experimental animals. Estrous cycle of female mice was examined before inoculation of Paracoccidioides brasiliensis yeast cells and mice were divided into 5 groups such as proestrus, estrus, metestrus-I, metestrus-II and diestrus. Each mouse was inoculated intravenously with 10⁶ P. brasiliensis yeast cell units and sacrificed on day 28 after inoculation. Their internal organs were cultured, and in addition, their histopathologies were studied. As a result, there was no difference in the organ cultures among the male and the female mice of 5 groups. However, histopathologically, the female groups at estrus, metestrus-I and metestrus-II were affected more severely than the male group, and the susceptibility of the female mice to the fungus was closely related to their estrous cycles.Abbreviations BHI-D brain heart infusion agar supplemented with 1.0% of anhydrous dextrose - PAS periodic acid-Schiff techniques - PBS phosphate buffered saline solution - SD standard deviation     

促性腺激素释放激素及多巴胺对斜带石斑鱼生长激素分泌及其mRNA表达的调控

研究斜带石斑鱼生长激素分泌及其mRNA表达的调控规律对于性别分化的控制、临床药物的选择,以及石斑鱼的增养殖等均具有重要的理论意义和实践意义。本文应用静态孵育系统,采用放射免疫测定法和化学发光液相杂交实验,研究GnRH和DA对斜带石斑鱼GH分泌、GHmRNA合成的调控作用。100nmol／LsGnRH作用斜带石斑鱼脑垂体碎片1也4h,明显促进GH的释放和GHmRNA的合成,并具有时间依存性;10nmol／L～1μmol／LsGnRH作用1h能明显促进斜带石斑鱼脑垂体释放GH,促进GHmRNA的合成,表现出明显的剂量效应。100nmol／L、1μmol／LmGnRH作用1h以一定的剂量依存方式促进GH的释放、促进GHmRNA的合成,但mGnRH的效应比相应剂量的sGnRH的作用弱。APO为DA受体的非选择性激动剂,不同剂量APO对斜带石斑鱼脑垂体碎片的作用结果显示,10nmol／L-1μmol／L APO以剂量依存方式促进斜带石斑鱼脑垂体碎片释放GH、促进GHmRNA的合成：1μmol／LAPO作用12h以上明显促进GH的释放和GHmRNA的合成,并随时间的延长而增加。与sGnRH对斜带石斑鱼GH释放、GHmRNA合成的作用相比,APO的作用较弱。本文研究结果证实GnRH和DA能促进斜带石斑鱼脑垂体GH释放和GHmRNA合成。