Regulation by gonadal steroids of estrogen and progesterone receptors along the reproductive tract in female lambs.

The regulation of estrogen and progesterone receptor (ER, PR) expression by estradiol (E2) and progesterone (P4) in the oviduct, uterus and cervix of female lambs was studied. The animals received three intramuscular injections of E2, P4 or vehicle with an interval of 24 h and they were slaugthered 24 h after the third injection. Determinations of ER and PR were performed by binding assays and mRNAs of ER alpha and PR by solution hybridization. High levels of ER and PR in both cervix and oviduct were found in the female lamb, differing from other mammalian species. No significant effects by either E2 or P4 treatment on ER and PR levels in the cervix and oviduct could be observed. E2 treatment increased the mRNA levels of ERa and PR more than 3-fold in the cervix, while P4 treatment increased the mRNA levels of ERa and PR in the uterus. The results show differential effects of gonadal steroids on sex steroid receptor expression along the reproductive tract in female lambs, suggesting that steroid target tissues can modulate responses to the same circulating levels of steroid hormones.     

Localisation and expression of TRPV6 in all intestinal segments and kidney of laying hens

1. The aim of this study was to investigate the localisation and expression of the epithelial Ca2+ channel TRPV6 (transient receptor potential vanilloid channel type 6) in different intestinal segments and kidney of laying hens during peak lay. 2. Immunohistochemical analysis of the intestine indicated that TRPV6 was localised to the brush-border membranes of the duodenum, jejunum, ileum, caecum, and rectum. Expression was weaker in the rectum, and little or no expression was found in crypt and goblet cells. In addition, TRPV6 mRNA was quantified amongst different intestinal segments, and expression was highest in the duodenum and jejunum. Furthermore, Western blotting indicated that the duodenum expressed the greatest amount of TRPV6 and the rectum the least with the other segments expressing intermediate levels. 3. In the kidney, distinct immunopositive staining for TRPV6 was detected at the apical domain of the distal convoluted tubules (DCT) and medullary connecting tubules (CNT). Interestingly, distribution of TRPV6 extended to the proximal convoluted tubules (PCT). Furthermore, the kidney expressed lower TRPV6 mRNA and protein levels compared with that in the duodenum. 4. In conclusion, the epithelial Ca2+ channel TRPV6 is strongly expressed in the apical cells of the entire intestine and the renal tubules, suggesting that active Ca2+ transcellular transport plays a crucial role in dietary calcium (re)absorption in laying hens.     

Oestradiol enhances plasma growth hormone and insulin-like growth factor-I concentrations and increased the expression of their receptors mRNAs in the liver of ovariectomized cows

Many metabolic hormones, growth hormone (GH), insulin-like growth factor-I (IGF-I) and insulin affect ovarian functions. However, whether ovarian steroid hormones affect metabolic hormones in cattle remains unknown. This study aimed to determine the effect of sex steroids on the plasma profiles of GH, IGF-I and insulin and their receptors in the liver and adipose tissues of dairy cows. Ovariectomized cows (n = 14) were randomly divided into four groups: control group (n = 3) was treated with saline on Day 0; oestradiol (E2) group (n = 3), with saline and 1 mg oestradiol benzoate (EB) on Day 0 and 5, respectively; progesterone (P4) group (n = 4) with two CIDRs (Pfizer Inc., Tokyo, Japan) from Day 0; and E2 + P4 group (n = 4) with two CIDRs on Day 0 that were removed on Day 6 and were immediately injected with 1 mg EB. The animals were euthanized after the experiment, and liver and adipose tissues samples were quantitatively analysed using real-time PCR for the expression of mRNA for the GH (GHR), IGF-I (IGFR-I) and insulin (IR) receptor mRNAs. Oestradiol benzoate significantly increased the number of peaks (p < 0.05), pulse amplitude (p < 0.05) and area under the curve (AUC; p < 0.01) for plasma GH; moreover, it increased plasma IGF-I concentration (p < 0.05), but it had no effect on the plasma insulin profile. P4 significantly decreased the AUC (p < 0.01), compared with the control group, whereas it did not affect the number of peaks and the amplitude of GH pulses. P4 + E2 did not affect the GH pulse profile. E2 increased the mRNA expression of GHR, IGFR-I and IR in the liver (p < 0.05), whereas both P4 and E2 + P4 did not change their expressions. Our results provide evidence that the metabolic and reproductive endocrine axes may regulate each other to ensure optimal reproductive and metabolic function.     

蛋鸡TRPV6基因的原核表达、纯化及抗体制备

为制备蛋鸡TRPV6蛋白多克隆抗体,根据其基因序列,设计1对特异性引物,以卵巢组织中提取的总RNA为模板,扩增蛋鸡TRPV6基因1 801～2 176nt的375bp序列,构建原核表达质粒pET-32a（＋）-TRPV6;将重组质粒转化BL21（DE3）,经IPTG诱导表达TRPV6融合蛋白,通过镍离子螯合柱纯化后免疫新西兰大白兔,获得兔抗鸡TRPV6多克隆抗体,分别通过酶联免疫吸附试验（ELISA）法和Western blot检测抗体的效价和抗体特异性。结果表明,试验成功构建原核表达载体pET-32a（＋）-TRPV6,SDS-PAGE蛋白电泳检测发现目的蛋白大小约35 000;Western blot分析显示,表达的TRPV6融合蛋白具有良好的免疫原性,其抗体可与大肠杆菌表达的产物特异性结合;ELISA显示其抗体效价达1∶100 000。获得纯化的融合蛋白和多克隆抗体对研究TRPV6钙离子通道在蛋鸡髓质骨形成的作用机理具有重要作用。     

Estrogen inhibits interleukin-18 mRNA expression in the mouse uterus

Interleukin-18 (IL-18) is a proinflammatory cytokine expressed in female reproductive organs in humans, rats and mice. The physiological roles of uterine IL-18 and the regulatory mechanisms of IL-18 gene expression are unclear. The present study aimed to clarify the effects of estradiol-17beta (E2) and progesterone (P4) on IL-18 mRNA expression in the mouse uterus. Distribution and expression levels of IL-18 mRNA were studied using an RNase protection assay. Expression of IL-18 mRNA was observed in all organs studied, including testes, ovaries and uteri. The uterine IL-18 mRNA level of estrous mice was higher than that of diestrous mice. E2 treatment (1, 5, 25 or 250 ng/mouse) decreased uterine IL-18 mRNA levels in ovariectomized mice dose-dependently. E2 treatment acutely decreased IL-18 mRNA levels 3 h after injection, but these levels returned to the initial level after 48 h. P4 treatment (1 mg/mouse) decreased uterine IL-18 mRNA levels after 12 h, but levels returned to the initial level after 48 h. Both uterine IL-18 and IL-18Ralpha mRNAs were detected in cultured endometrial epithelial and stromal cells. These results suggest that uterine IL-18 expression is reduced by sex steroid hormones and that IL-18 acts on endometrial cells in a paracrine or autocrine manner.     

Steroid hormone-regulated let-7b mediates cell proliferation and basigin expression in the mouse endometrium

Let-7b, one of the let-7 family members, was studied for its regulative role in endometrial cells during early pregnancy in mice. According to real-time RT-PCR analysis, the expression of let-7b in epithelial cells increased gradually from day 1 to day 4 of preimplantation stages and reached the highest level on day 4. On the other hand, the highest level of let-7b in stromal cells was observed on day 1, although the expression was decreased on day 2 and increased significantly on day 4. By in situ hybridization, let-7b was also found to express in uteri during days 6-8 of pregnancy. Endometrial cells isolated from prepubertal mice were treated with steroid hormones, progesterone (P4), estradiol (E2) and P4 plus E2. After 96 h of culture in the presence of steroid hormones, the expression levels of let-7b were increased in the endometrial cells, although significant differences were only observed after P4 treatment in stromal cells and after individual E2 and P4 treatments in the epithelial cells. In association with the increased let-7b expression, the cell proliferation slope, measured by a MTT assay, significantly decreased in the presence of P4 and P4 plus E2 compared with the nonhormone and E2 treatment groups during 72-108 h of culture. Furthermore, results from transfection of let-7b into stromal cells isolated from day 4 pregnant mice or prepubertal mice demonstrated that let-7b attenuated the proliferation during the periods of time examined. After transfection of let-7b into mouse stromal cells isolated from day 7 of pregnancy, the expression of Basigin (Bsg), a matrix metalloproteinase (MMP) inducer, was suppressed, as well as that of MMP-9. In conclusion, this study clarifies the expression pattern of let-7b in uterine epithelial and stromal cells during preimplantation stages in mice, as well as the inhibitory effect of let-7b associated with steroid hormones on stromal cell proliferation and on the expression of MMP-9.     

性腺激素对山羊子宫内膜细胞TNF-α与TGF-β分泌活性的影响

为探讨性腺激素对山羊子宫内膜细胞分泌活性的影响,基于套皿培养技术构建永生化山羊子宫内膜上皮细胞(EEC)与基质细胞(ESC)共培养体外研究模型,通过ELISA检测性腺激素E2和/或P4对单独培养EEC中TNF-α与TGF-β分泌活性的调节作用以及ESC对该培养体系的影响.结果显示:与未加激素对照组相比,单独或混合添加E2和P4均可显著增加单独培养EEC向顶端分泌TNF-α水平(P＜0.05),而对细胞基底端TNF-α分泌活性影响不显著;E2单独作用较对照组显著降低EEC向细胞顶端分泌TGF-β水平(P＜0.05),P4单独或与E2共同作用下则主要对单独培养EEC顶端及基底端分泌TGF-p水平具有显著增强作用(P＜0.05).对于EEC-ESC共培养细胞模式,E2和/或P4显著降低EEC顶端和基底端TNF-α分泌水平(P＜0.05);EEC顶端TGF-β分泌水平在E2和/或P4作用下显著降低(P＜0.05),而EEC基底端TGF-β分泌水平不受激素作用所影响.激素对单独培养及与ESC共培养模式下的极化EEC顶端及基底端TNF-α与TGF-β分泌水平的差异,提示ESC对激素作用下山羊子宫内膜上皮细胞分泌方向及分泌水平的重要调节作用.     

Hormonal profile and morphological changes in pig ovaries after intraovarian infusions of Escherichia coli endotoxin

The purpose of this study was to estimate morphological changes in the ovary and size of the production of steroid hormones during the luteal phase of the estrous cycle in pigs after intraovarian infusions of Escherichia coli endotoxin. Polish Large White gilts (n = 15) of similar age (7-8 months) and body weight (90-110 kg) with two controlled subsequent estrous cycles were used. The animals were randomly divided into two groups: control (n = 9, the 10th day of the estrous cycle,) and treated with Escherichia coli endotoxin (n = 6, the same day of the estrous cycle). The gilts were infused with Escherichia coli endotoxin at a dose of 1 mg three times a day during six consecutive days, from the 14th to the 19th day of the estrous cycle. Plasma concentrations of progesterone (P4), androstenedione (A4), testosterone (T), estrone (E1) and estradiol-17 beta (E2) were determined by radioimmunoassay method. Infusions of Escherichia coli endotoxin resulted in a significant (p < 0.001) decrease in the production of P4, A4, T, E1 and E2 in the luteal phase as compared with the levels found in the control animals. Plasma level of P4, A4 and T was decreased by 84.6%, 86.0% and 73.0%, respectively. Plasma concentrations of E1 and E2 in some cases exceeded 5 pg/ml, nevertheless in the majority of the samples they were under sensitivity of the method. Escherichia coli endotoxin infusions resulted in a considerable decrease in the size of the ovaries, and morphological changes characteristic for acute and chronic inflammation were observed.     

Serum concentrations of cortisol, sex hormones of adrenal origin, and adrenocortical steroid intermediates in healthy dogs following stimulation with two doses of cosyntropin

OBJECTIVE: To compare the effects of 2 doses of cosyntropin (5 microg/kg vs 250 microg, IV) on serum concentrations of cortisol, sex hormones of adrenal origin, and adrenocortical steroid intermediates and determine the optimal sample collection time after adrenal stimulation with cosyntropin. ANIMALS: 10 healthy, privately owned, neutered dogs. PROCEDURE:Dogs were randomly assigned to initially receive cosyntropin at 5 microg/kg or as a total dose of 250 microg, IV. Dogs received the alternate dose 1 to 2 weeks later. Serum was obtained from blood samples collected before (0 minutes) and 30, 60, 90, and 120 minutes after cosyntropin administration. RESULTS:Maximum stimulation of cortisol, androstenedione, progesterone, and 17-hydroxyprogesterone production was achieved at 60 minutes following IV administration of cosyntropin at 5 microg/kg or as a total dose of 250 microg. Serum estradiol concentration did not increase in response to either cosyntropin dose. For all hormones, no significant difference in serum hormone concentrations was found among sample collection times of 0, 30, 60, and 90 minutes when comparing the 2 doses of cosyntropin. CONCLUSIONS AND CLINICAL RELEVANCE: Cosyntropin, when administered at 5 microg/kg, IV, effectively stimulated maximum production of cortisol, sex hormones of adrenal origin, and adrenocortical steroid intermediates at 1 hour after administration.     

miR-495-3p对山羊卵巢颗粒细胞功能的影响

旨在探究miR-495-3p对山羊卵巢颗粒细胞功能的影响及作用机制.本研究选取健康的3～4月龄大足黑山羊母羊,收集卵巢颗粒细胞,利用miR-495-3p模拟物(mimics)和抑制物(inhibitor)构建过表达和抑制模型,通过流式细胞术检测细胞凋亡和周期,ELISA分析颗粒细胞的雌二醇(E2)和孕酮(P4)分泌,采...     

Serum and Tissue Steroid Hormone Levels in Canine Mammary Tumours: Clinical and Prognostic Implications

Hormonal dependency of canine mammary tumours (CMT) has been studied over the last few decades. However, studies assessing the prognostic and predictive potential of serum and/or tissue steroid hormone levels are still scarce in CMT. To the best of our knowledge, this is the first report relating serum and tissue levels of steroid hormones and prognosis in dogs. Serum and tumour tissue from 45 female dogs with spontaneous CMT were included in the study. Moreover, serum and normal mammary tissue from 13 healthy female dogs were also included as controls. Steroid hormones were determined by competitive enzyme immunoassay. Overall, levels of steroid hormones in serum and tissue homogenates were significantly different between malignant and benign mammary tumours (p < 0.01), except for progesterone (P4) serum levels that revealed no statistical differences between groups. In malignant tumours, oestrone sulphate (SO4E1), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T) and P4 elevated tissue concentrations were significantly associated with tumour relapse and/or distant metastasis during follow‐up. A significant association was found between elevated tissue SO4E1 (p = 0.003), 17β‐oestradiol (E2) (p = 0.036), DHEA (p = 0.022), A4 (p = 0.001) and P4 (p = 0.013) concentrations and shorter disease‐free survival and overall survival in female dogs with malignant mammary tumours. The high levels of tissue steroids found in cases of poor prognosis open the possibility of additional new therapeutic approaches. Future clinical trials will be needed to clarify the usefulness of targeting steroid hormones in the treatment of this neoplastic disease.     

Consumption of bee pollen affects rat ovarian functions

The aim of this study was to examine possible effects of bee pollen added to the feed mixture (FM) on rat ovarian functions (secretion activity and apoptosis). We evaluated the bee pollen effect on the release of insulin‐like growth factor I (IGF‐I) and steroid hormones (progesterone and estradiol), as well as on the expression of markers of apoptosis (Bcl‐2, Bax and caspase‐3) in rat ovarian fragments. Female rats (n = 15) were fed during 90 days by FM without or with rape seed bee pollen in dose either 3 kg/1000 kg FM or 5 kg/1000 kg FM. Fragments of ovaries isolated from rats of each group (totally 72 pieces) were incubated for 24 h. Hormonal secretion into the culture medium was detected by RIA. The markers of apoptosis were evaluated by Western blotting. It was observed that IGF‐I release by rat ovarian fragments was significantly (p < 0.05) decreased; on the other hand, progesterone and estradiol secretion was increased after bee pollen treatment at dose 5 kg/1000 kg FM but not at 3 kg/1000 FM. Accumulation of Bcl‐2 was increased by bee pollen added at 3 kg/1000 kg FM, but not at higher dose. Accumulation of Bax was increased in ovaries of rats fed by bee pollen at doses either 3 or 5 kg/1000 kg FM, whilst accumulation of caspase‐3 increased after feeding with bee pollen at dose 5 kg/1000 kg FM, but not at 3 kg/1000 kg FM. Our results contribute to new insights regarding the effect of bee pollen on both secretion activity (release of growth factor IGF‐I and steroid hormones progesterone and estradiol) and apoptosis (anti‐ and pro‐apoptotic markers Bcl‐2, Bax and caspase‐3). Bee pollen is shown to be a potent regulator of rat ovarian functions.     

A role for the adrenal cortex in the onset of cystic ovarian follicles in the sow.

The corticotrophin (ACTH)-adrenal cortical axis has previously been implicated in the onset of cystic ovaries in the sow. In view of the role of the ACTH-adrenal cortical axis in stress, two sows were subjected to an elevated environmental temperature of 32 degrees C for three hours daily during the follicular phase of the estrous cycle. Plasma concentrations of glucocorticosteroids and progesterone fluctuated markedly in one sow that developed cystic ovaries. Concentrations of these hormones did not vary greatly in the other sow that did not develop cystic follicles. Exposure to an environmental temperature of 32 degrees C for three hours or injection of 1 IU/kg bodyweight of ACTH for each of two ovariectomized sows resulted in an elevation in progesterone values to 5-7 ng/ml plasma from basal levels of 1-2 ng/ml and a rise in total glucocorticosteroids from basal levels of 1 or 2 microgram/100 ml plasma to 4-10 microgram/100 ml. Injection of 2 mg/kg bodyweight of progesterone and 4 mg/kg bodyweight of cortisol into the ovariectomized sows was found to approximate these elevations in plasma steroid values. When either progesterone or cortisol was injected daily during the follicular phase into two intact sows in two successive experiments at these dosage levels, similarly elevated plasma steroid concentrations were seen and cystic ovarian follicles resulted. The results suggest that glucocorticosteroids and progesterone of adrenal origin may be involved in the onset of cystic ovaries in the sow.     

N-methyl-d,l-aspartate stimulates growth hormone and prolactin but inhibits luteinizing hormone secretion in the pig.

The effects of n-methyl-d,l-aspartate (NMA), a neuroexcitatory amino acid agonist, on luteinizing hormone (LH), prolactin (PRL) and growth hormone (GH) secretion in gilts treated with ovarian steroids was studied. Mature gilts which had displayed one or more estrous cycles of 18 to 22 d were ovariectomized and assigned to one of three treatments administered i.m.: corn oil vehicle (V; n = 6); 10 micrograms estradiol-17 b/kg BW given 33 hr before NMA (E; n = 6); .85 mg progesterone/kg BW given twice daily for 6 d prior to NMA (P4; n = 6). Blood was collected via jugular cannulae every 15 min for 6 hr. Pigs received 10 mg NMA/kg BW i.v. 2 hr after blood collection began and a combined synthetic [Ala15]-h GH releasing factor (1-29)-NH2 (GRF; 1 micrograms/kg BW) and gonadotropin releasing hormone (GnRH; .2 micrograms/kg BW) challenge given i.v. 3 hr after NMA. NMA did not alter LH secretion in E gilts. However, NMA decreased (P < .02) serum LH concentrations in V and P4 gilts. Serum LH concentrations increased (P < .01) after GnRH in all gilts. NMA did not alter PRL secretion in P4 pigs, but increased (P < .01) serum PRL concentrations in V and E animals. Treatment with NMA increased (P < .01) GH secretion in all animals while the GRF challenge increased (P < .01) serum GH concentrations in all animals except in V treated pigs. NMA increased (P < .05) cortisol secretion in all treatment groups. These results indicate that NMA inhibits LH secretion and is a secretagogue of PRL, GH and cortisol secretion with ovarian steroids modulating the LH and PRL response to NMA.     

Histological and sex steroid hormone receptor changes in testes of immature, mature, and aged chickens

Sex steroid hormone receptors play a central role in the regulation of reproduction in male chickens. In this work, we evaluated by histomorphometric methods and Western blot analysis changes in the number of the different cell populations and in the content of sex steroid hormone receptors in testes from immature (1.5-month-old), mature (12-month-old), and aged (48-month-old) chickens. The number of Sertoli cells, germ cells, and Leydig cells per area of testicular tissue markedly changed according to chicken age. The highest number of Sertoli and Leydig cells was found in testes of immature chickens, with a dramatic decrease in those of mature chickens; however, the number of germ cells was the highest in mature chickens in comparison with other ages. The content of androgen receptor diminished in testes of mature and aged animals in comparison with that of immature chickens. In contrast, the content of estrogen receptor alpha and progesterone receptor was higher in testes of mature animals than in other ages. Both progesterone receptor isoforms were expressed in a similar proportion in testes of immature and mature animals. Interestingly, progesterone receptor isoform A was the predominant isoform in aged animals. These results suggest that there are marked age-dependent changes in chicken testes histology and in sex steroid hormone receptors content that should contribute to sex steroid hormone actions, in this tissue throughout the lifespan of chickens.