Expression of oestrogen receptor,androgen receptor and progesterone nuclear receptor in sheep uterus during the oestrous cycle

Oestrogen, androgen and progesterone are involved in the regulation of uterine physiological functions, with the participation of the following proteins: oestrogen receptor (ER), androgen receptor (AR) and progesterone nuclear receptor (PGR). In this study, we used immunohistochemistry to detect the localization of ERα, ERβ, AR and PGR in sheep uterus. Additionally, we used real‐time polymerase chain reaction (RT‐qPCR) and Western blot technique to analyse their expression profiles at different stages of sheep oestrous cycle in the endometrium and myometrium. Immunohistochemical analysis showed that ERα, ERβ, AR and PGR were present in sheep uterus in oestrus, mainly in the uterine luminal epithelium, stroma, gland and myometrium. Real‐time polymerase chain reaction results showed that in the endometrium, ERα expression level was highest in oestrus. ERβ and PGR, instead, were highly expressed in pro‐oestrus. In the myometrium, ERα was highly expressed in both oestrus and pro‐oestrus, and ERβ was highly expressed in oestrus and dioestrus. Progesterone nuclear receptor expression was highest in oestrus, followed by metoestrus. In the endometrium, both receptors ERα and ERβ were abundant in pro‐oestrus, while the maximum AR protein content was found in oestrus. At this stage of the oestrous cycle, PGR protein concentration in the myometrium was significantly lower than those observed in other stages. These results suggest that these receptors are important for sheep reproductive function, as their expression at mRNA and protein levels exhibits particular time‐ and tissue‐specific profiles along the oestrous cycle.     

Melatonin treatment prior to the normal breeding season increases fetal number in United Kingdom sheep flocks

Litter size determined ultrasonographically at 45–90 days after mating in eight groups of commercial sheep (n = 2609) treated with slow release melatonin implants immediately prior to the natural breeding season showed a 19% treatment effect averaged over the eight groups, from 148% to 167% (P < 0.01) compared with controls (n = 2499). Control groups had a litter size ranging from 124% to 194% thereby representing most commercial production systems in the UK sheep industry. This is the first large scale study of slow release melatonin implants in commercial UK flocks to demonstrate a significant positive effect on litter size when used before the natural breeding season. The data indicate that increased productivity and profitability could be achieved while maintaining desirable purebred characteristics.     

Effects of endocrine disrupting compounds on the pathology and oestrogen receptor alpha and beta distribution in the uterus and cervix of ewe lambs

A number of chemicals have been classed as endocrine disrupting compounds due to their ability to mimic the actions of endogenous hormones in vivo and in vitro. The objective of this experiment was to determine the pathological changes and oestrogen receptor (ER) distribution in the cervix and uterus of prepubertal ovariectomised ewe lambs following exposure to a range of compounds with a predominantly oestrogenic effect. Lambs were exposed to diethylstilbestrol (0.175 mg/kg biweekly), bisphenol-A (3.5mg/kg biweekly) or octylphenol (3.5mg/kg biweekly) for 6 weeks. Following sacrifice, uterine and cervical tissue pathology was assessed. The endometrial and myometrial areas were quantified and the distribution of ERalpha and ERbeta assessed by immunohistochemistry. No differences were observed between control and octylphenol-exposed lambs in uterine gross pathology and histopathology. Uteri from bisphenol-A- and diethylstilbestrol-exposed lambs were heavier than both control and octylphenol-exposed lambs. In the bisphenol-A-exposed lambs, endometrial oedema accounted for a significant increase in the endometrial cross-sectional area over the other groups. Uteri from animals exposed to diethylstilbestrol showed variable pathology including oedema and cellular proliferation. Keratinisation of the cervical epithelium was observed in both bisphenol-A- and diethylstilbestrol-exposed lambs. Exposure to diethylstilbestrol and bisphenol-A was associated with a diffuse intracellular distribution of ERalpha and ERbeta in the uterine endometrium. This was in addition to the strong cytoplasmic staining of uterine epithelial cells and nuclear staining of specific sub-epithelial cells observed in all groups. We conclude that a 6-week exposure of lambs to bisphenol-A and diethylstilbestrol altered the uterocervical environment and has the potential to disrupt subsequent reproductive function. Pathological changes could not be detected in the uterus or cervix of lambs exposed to octylphenol.     

Histological and immunohistochemical investigations of ovarian interstitial glands during non‐breeding season in camels (Camelus dromedarius)

The aim of this was to investigate the histology and immunohistochemistry of interstitial glands during non‐breeding season in camel ovaries. A total of 21 mature, non‐pregnant and apparently healthy camels aged between 8 and 12 years were slaughtered. The ovaries were removed within 15 min, cleaned from adipose tissue, weighted and examined grossly. The histological preparation was made, and then, the blocks were cut at 3–5 microns thickness and stained by H&E for histological examinations. Moreover, some sections were stained with Sudan Black for lipid detection. Immunohistochemical analysis of paraffin‐embedded ovarian tissues was performed to detect the localization of S‐100, vimentin, progesterone receptors (PR) and oestrogen receptors (ER). Immunoreactive signals were detected using UltraVision Detection System. The results revealed that the interstitial glands were located in the cortical region and they were arranged in various arrangements either single, in couple or in groups rich in lipid droplet. All interstitial gland arrangements were enclosed by connective tissue capsules containing fibroblasts and collagenous fibres separated them from the surrounding ovarian structures. Both interstitial glands and their surrounding CT were penetrated by several blood vessels. There was a strong immunoreactive signal for S‐100 in the nuclei of interstitial cells, and no signals were detected either in cells of the interstitial glands or their connective tissue with PR. We could conclude that the interstitial gland is distinct in ovary of camel and further studies are needed to elucidate its rule in steroid synthesis.     

Effects of gestation and lactation on vitamin D receptor amounts in goats and sheep

During gestation and lactation, an increased demand for calcium (Ca) due to the development of fetal skeleton and excretion via milk is observed. The higher need for Ca is met by an augmented mobilisation of Ca from bones and by an increased absorption from the intestines. The main influence on this physiological process of active absorption has Vitamin D, acting through Vitamin D receptors (VDR) located in the mucosal wall of the intestines, thus increasing Ca absorption. As a consequence of inadequate Ca absorption, metabolic diseases like milk fever can develop. In this study immunohistochemical procedures were applied to colon mucosa biopsies of pregnant and lactating goats and sheep, to study the effect of late gestation, parturition and lactation on VDR amount. Colon mucosa biopsies were collected 2 weeks before parturition, 1 and 4 weeks post partum (pp), 2, 3, 4, and 5 months pp from 11 dairy goats and 11 sheep. Immunohistochemistry was performed employing a biotinylated monoclonal rat anti-VDR antibody and streptavidin peroxidase techniques. Nuclei and cytoplasm of enterocytes stained positively for VDRs. Strongest immunoreactions were observed in intermediate and superficial glandular cells. The biopsy samples taken during early lactation revealed a lower immunoreaction for VDR compared with samples taken during later stages of lactation. In conclusion, immunochemistry and biopsy technology are useful tools to assess changes in VDR expression in relation to varying demands for Ca in the process of a reproductive cycle. These results show that in dairy goats and sheep, an influence of gestation and lactation on VDR is obvious.     

发育各期奶牛乳腺肥大细胞类胰蛋白酶的表达

为了研究奶牛乳腺肥大细胞的组织化学特点和分布特征,对21头发育各期奶牛乳腺进行取样,应用免疫组织化学方法（SP）,观察奶牛乳腺肥大细胞类胰蛋白酶的表达。结果显示,奶牛乳腺肥大细胞表达类胰蛋白酶,肥大细胞数量在各期有显著差异,泌乳期的乳腺肥大细胞数量明显减少（P〈0.01）,分娩后60d奶牛乳腺肥大细胞的数量最少。结果表明,肥大细胞数量随生理周期的改变而改变,静止期奶牛的局部免疫能力增强。     

Ultrasonographic measurements of adrenal glands in cats with hyperthyroidism

Feline hyperthyroidism is potentially associated with exaggerated responsiveness of the adrenal gland cortex. The adrenal glands of 23 hyperthyroid cats were examined ultrasonographically and compared to the adrenal glands of 30 control cats. Ten hyperthyroid cats had received antithyroid drugs until 2 weeks before sonography, the other 13 were untreated. There was no difference in adrenal gland shape between healthy and hyperthyroid cats: bean-shaped, well-defined, hypoechoic structures surrounded by a hyperechoic halo in 43/60 (71.6%) healthy cats and 34/46 (73.9%) hyperthyroid cats; more ovoid in 13/60 (21.6%) healthy cats and 9/46 (19.6%) hyperthyroid cats while more elongated in 4/60 (6.7%) healthy cats, 3/46 (6.5%) hyperthyroid cats. Hyperechoic foci were present in 9/23 (39.1%) hyperthyroid cats and 2/30 (6.7%) healthy cats. The adrenal glands were significantly larger in hyperthyroid cats, although there was overlap in size range. The mean difference between hyperthyroid cats and healthy cats was 1.6 and 1.7 mm in left and right adrenal gland length, 0.8 and 0.9 mm in left and right cranial adrenal gland height, and 0.4 and 0.9 mm in left and right caudal adrenal gland height. There was no significant difference between the adrenal gland measurements in treated and untreated hyperthyroid cats. The adrenomegaly was most likely associated with the hypersecretion of the adrenal cortex documented in hyperthyroid cats. Hyperthyroidism should be an alternative to hyperadrenocorticism, hyperaldosteronism, and acromegaly in cats with bilateral moderate adrenomegaly.     

Clinical and ultrasonographic findings in the mammary gland of sheep

AIM: To compare clinical and ultrasonographic findings and assess the value of ultrasonography for evaluating the structure and condition of the teat and teat canal of sheep.

METHODS: The mammary glands of 30 lactating ewes were examined clinically and using ultrasonography. The distance from the teat tip to the ground was measured and diagnostic images of the body of the gland, gland cistern and teat cistern were obtained using an 8.5 MHz frequency ultrasonic transducer. A California mastitis test (CMT) and bacteriological culture were performed on milk samples collected from each half of each udder.

RESULTS: Udder parenchyma and teats were palpably normal in 27/30 (90%) ewes. Milk clots or abnormalities were evident in 4/30 (13%) udders, lesions in the glandular parenchyma and/or teats were evident by palpation in 3/30 (10%), and pathogenic bacteria were cultured from 2/30 (7%). In both of the latter cases, mastitis was diagnosed based on clotted and discoloured appearance of milk and highly positive CMT reactions. Ultrasonographic examination in these udders revealed non-homogenous regions in the glandular tissue and alternating areas of hyperechogenic and hypoechogenic tissue. Overall, highly positive CMT reactions were evident in milk from 14/30 (47%) left halves and 9/30 (30%) right halves (p=0.02). No relationship between teat-to-ground distances and CMT results was evident (p>0.05). Teat canal lengths ranged from 5.7–10.3 mm (mean=8.6; SD=1.3 mm) and the widths from 1.8–3.1 mm (mean=2.3; SD=0.4 mm). Milk sampled from ewes that had long (≥9 mm) or wide (≥2 mm) teat canals was highly positive to the CMT in 75% and 83% of cases, respectively.

CONCLUSIONS: Ultrasonographic measurements of the length and width of the teat canal appeared to correlate with CMT results. The use of ultrasonography in evaluating the health of the udder of small ruminants holds promise for the future.     

发情旺季母水牛生殖激素水平变化的研究

为了研究发情旺季母水牛生殖激素水平的变化规律,试验采用酶联免疫分析方法(ELISA)测定了发情周期血样中的促卵泡素(FSH)、促黄体素(LH)、雌二醇(E2)和孕酮(P4)4种激素,并分析了这些生殖激素在发情周期中的变化规律。结果表明:血清中FSH和LH的含量均由排卵前的第4天开始缓慢上升,排卵前达到峰值,在排卵后又明显下降;E2的含量在排卵前1天出现峰值,在排卵后第9天又出现1个小波峰;P4的含量在排卵后的第5天明显上升,第13天时达峰值,在排卵前3天出现最低水平。     

Prion protein genetic polymorphisms and breeding strategies in Portuguese breeds of sheep

Polymorphisms at the prion protein locus were studied in 965 animals, representative of 13 native and 3 exotic sheep breeds in Portugal. In general, ARQ was the predominant allele in most breeds, but the desirable ARR allele was present in all breeds studied. A different pattern was found in Ile de France, where ARR was predominant and VRQ had the highest frequency among the breeds analysed. In most of the breeds, especially in those of the coarse-wool type, the major hurdle in selecting for genetic resistance to clinical scrapie was the elimination of ARQ rather than VRQ alleles. Breeding schemes aiming at the creation of ARR-homozygous selection nuclei were simulated for representative breeds, and results indicate that, with intense selection, it would take between 4 years in Ile de France and 11 years in Mondegueira to obtain homozygosity in the nucleus, and the length of time needed for fixation was not affected by the type of mating (random or assortative) used. Nevertheless, intense selection for the PrP genotype alone would have undesirable consequences in terms of inbreeding, and correlated responses in production and adaptation traits should be evaluated before such a scheme is adopted.     

Androgen receptor expression in normal, hyperplastic and neoplastic hepatoid glands in the dog

Neoplasms of the perianal glands are common in the dog, particularly in the male. The occurrence of these tumours appears to be hormone related and castration, without excision of the tumour, has sometimes resulted in regression of the tumour. The aim of this study was to investigate the expression of androgen receptors (AR) in normal, hyperplastic and neoplastic hepatoid glands in the dog. Thirty-one samples of canine hepatoid gland tissues were investigated. The lesions, classified according to WHO criteria, were comprised of 19 hyperplastic tissues, 10 benign lesions (2 hepatoid gland epithelioma and 8 hepatoid adenomas), and 19 carcinomas. Five samples from normal hepatoid glands were also investigated. The AR expression was evaluated by immunohistochemistry using a streptavidin-biotin peroxidase method. The immunoexpression was scored by two pathologists as the percentage of positive nuclei. The intensity of staining was also considered. AR expression was detected in all normal and abnormal glands. However, in hyperplastic tissues the percentage of positive nuclei was significantly higher than in normal tissue and especially in reserve basaloid cells. A similar increase in the percent of positive nuclei was also observed in hepatoid epitheliomas, while in hepatoid adenoma the percent of AR-immunolabelling was only slightly increased compared to normal tissue. In hepatoid carcinomas the percent of AR-positive cells was similar to that observed in benign tumours. The grade of differentiation of hepatoid carcinomas did not affect AR expression. These results demonstrate that increased AR expression is maintained throughout perianal gland cancer progression and that hepatoid gland carcinomas still express AR. Although further studies may be required to evaluate the hormonal background of these diseases, dogs bearing those carcinomas might benefit from castration or anti hormonal therapy.     

Changes in oestrogen,progesterone and epidermal growth factor receptor concentrations and affinities during the oestrous cycle in the normal mammary gland and uterus of dogs

Changes in the concentrations and affinities of receptors for oestrogen (ER), progesterone (PR) and epidermal growth factor (EGF-R) were studied in mammary glands of healthy bitches with regard to age, the location in the mammary chain and the stage of the oestrous cycle. Uterus was used as the reference tissue for the evaluation of steroid receptors. Mammary and uterine samples from 7 healthy bitches were taken at five stages of the oestrous cycle in such a way that all the locations in the mammary chain were represented at each stage of the cycle (10 samples/dog). ER, PR and EGF-R were detected by biochemical assays using increasing concentrations of tritiated (steroids) or iodinated (EGF) ligands. A significant direct correlation was found between the ER and PR concentrations for mammary and uterine samples. No significant correlation was found between the steroid receptors and EGF-R concentrations. Mammary ER concentrations were significantly higher in bitches of 5 years of age or older than in younger ones; in posterior glands (4th and 5th pairs) than in anterior glands; and in the mid-luteal phase. Mammary PR did not vary significantly with age or location but was significantly lower in the early luteal phase than in other phases. A similar decrease in PR concentrations was observed in the uterus during the early luteal phase and uterine ER and PR concentrations were very low in the mid-luteal phase. Mammary EGF-R were not significantly higher in the early or mid-luteal phase than in pro-oestrus or anoestrus.The differences observed between the uterine and mammary steroid receptor concentrations during the oestrous cycle could be due to different mechanisms for regulating steroid receptor expression in the two tissues. Mammary EGF-R concentrations may be linked, as in other species, to cellular proliferation and/or to the serum progesterone concentrations.Abbreviations BSA bovine serum albumin - EGF epidermal growth factor - EGF-R receptor for epidermal growth factor - ER oestrogen receptor - K _d dissociation constant - LH luteinizing hormone - p probability of error - PR progesterone receptor     

Changes in estrogen receptor expression in the chick thymus during late embryonic development

In chickens, although estrogen receptors (ER) are reported to be associated with the immunological processes, detailed information about the differences in ER expression in the tissues related to the development of lymphocytes is not fully known, especially during the developmental stage. To learn more about this immunological relationship, we used semi‐quantitative polymerase chain reaction method to detect the ER expression levels in the thymus tissues of chicks during the developmental stage. Furthermore, ER‐expressing cells were detected by immunohistochemistry. The results of this study show that the expression level of ER increased on embryonic day 16 and decreased on day 20. Furthermore, ER expression was significantly higher in male than in female chickens at day 16. The increased expression on day 16 and decreased level on day 20 were also reproduced in the incidence of immunoreactive cells, although there was a 1‐day delay in the elevated incidence of the cells. This study revealed the changes in ER expression and the incidence of ER‐positive cells in the thymus of chickens during the developmental stage.     

哺乳动物性别决定机理及性别鉴定方法研究进展

哺乳动物的性别决定是由SRY基因为调控中心、多基因参与的级联调控过程。本文综述了性别决定基因及其功能、性别决定的分子模型及性别鉴定的方法等方面的研究进展。