Ectopic endometrial tissue in mesonephric duct remnants in bitches

Common congenital embryonic remnants of the canine female genital tract are Gartner cysts originating from mesonephric duct remnants. They can increase in size and lead to adverse effects in fertility and health. In the present study, three cases of mesonephric remnants in bitches were analysed. The mesonephric remnants featured an inner lining endometrium comprising surface epithelium, glands and stroma. This ectopic endometrium was further characterized by immunohistochemistry (oestrogen and progesterone receptors, proliferation activity, cytokeratin, alpha smooth muscle actin, and vimentin) and lectin histochemistry compared with normal uterine endometrium. Furthermore, hypertrophic cells at the serosal site of the uteri were detected and analysed in the same way compared with normal serosa. The ectopic endometrium of case no. 2 mesonephric remnant was comparable with normal endometrium whereas in nos 1 and 3 uteri the ectopic endometrium was reduced in thickness. In all mesonephric remnants, surface and glandular epithelial cells of the ectopic endometrium gave positive immunoreactions for cytokeratin, oestrogen and progesterone receptors and showed lectin-binding patterns comparable with normal endometrium. Some of the stromal cells of the ectopic endometria were smooth muscle actin and vimentin positive. Mitotic activity of the ectopic endometria was comparable with normal endometria. Hypertrophic epithelial cells of the serosal side showed positive reactions to anti-oestrogen receptor and anti-cytokeratin immunohistochemistry as well as lectin binding patterns and mitotic activity comparable with the normal canine serosa. The present study is the first considerable immunohistochemical characterization of canine mesonephric remnants and discusses the appearance of ectopic endometrium in mesonephric remnants.     

Lectin binding patterns of uterine glands in mares with chronic endometrial degeneration

OBJECTIVE: To evaluate changes of glycoconjugate in uterine glands of endometrial tissues obtained from mares. ANIMALS: adult mares. PROCEDURE: Uterine biopsy samples were collected during the breeding season and analyzed histologically for signs of chronic endometrial degeneration. Stage of the estrous cycle was established, using clinical examination and determination of hormonal status. Uterine tissue samples were analyzed, using lectin histochemical and immunohistochemical techniques (estrogen and progesterone receptors). Connective tissues were stained to determine alterations of ground substance in periglandular fibrosis. RESULTS: Of 50 mares, 30 (60%) were classified as normal or having modest alterations, and 20 (40%) were classified as having moderate or severe endometrial degeneration. In normal equine endometrium, several lectins (Helix pomatia agglutinin, Lotus tetragonolobus agglutinin, Ricinus communis I agglutinin, Ulex europaeus agglutinin, and wheat germ agglutinin) bound to glycoconjugates of the luminal epithelium and openings of uterine glands. Lectin binding patterns of cystic dilated glands or fibrotic glands in endometrial samples were remarkably strong, whereas normal surrounding cells remained unstained. Lotus tetragonolobus lectin was not suitable for detecting endometrial alterations. Connective tissues stained with Alcian blue and results of Hale colloidal-iron binding revealed acidic ground substance in periglandular fibrosis. Estrogen and progesterone receptors were evenly distributed in healthy and affected endometrial samples. CONCLUSIONS AND CLINICAL RELEVANCE: Glycoconjugate patterns of uterine glands were altered in mares with chronic endometrial degeneration. Therefore, uterine secretions are likely to be altered. These changes are not induced by changes in content of estrogen and progesterone receptors in endometrial tissues.     

Steroid receptor expression and HER-2/neu (c-erbB-2) oncoprotein in the uterus of cats with cystic endometrial hyperplasia-pyometra complex

The presence of oestrogen-alpha receptor (ER), progesterone receptor (PR), and HER-2/neu (c-erbB-2) oncoprotein in the uterine walls of 10 healthy cats and 20 subjects with cystic endometrial hyperplasia-pyometra (CEH-P) were evaluated. Lesions were graded according to the severity of cystic dilation, hyperplasia and inflammation, and were classified as normal, mild uterine hyperplasia and severe uterine hyperplasia. The ER, PR and c-erbB-2 expression in the endometrium, glandular epithelium, stromal fibroblasts and myometrial smooth muscle cells was quantified by immunohistochemistry. The ER, PR and c-erbB-2 staining patterns differed between normal uteri and uteri with CEH-P. The ER expression was tended to be higher in the endometrial surface and glandular epithelium in the severe hyperplasia group (P > 0.05) and significantly lower in the mild hyperplasia cases compared with normal endometrium (P < 0.05), whereas the PR expression in both severe and mild hyperplasia cases tended to be higher in stromal cells and glandular epithelium than those in the normal uteri. C-erbB-2 immunoreactivity was observed only in the endometrial surface and glandular epithelium of the uterine wall and immunostaining was found to be highest in cases with severe hyperplasia. As a conclusion, we suggest that c-erbB-2 oncoprotein may play a role in the pathogenesis of the CEH together with the ER and PR in cats, and that ER does not have a role in the mechanism of pyometra, whereas PR plays a role in the pathogenesis of both CEH and pyometra.     

Characterization of Foamy Epithelial Surface Cells in the Canine Endometrium

In mature bitches, endometrial epithelial surface cells modify function and corresponding morphology during the oestrous cycle. During late metoestrous, endometrial epithelial surface cells frequently accumulate fat and thereby adopt a foamy morphology. This cyclic appearance of foamy endometrial epithelial cells (fEECs) seems to be physiological in the dog, whereas in other species, it indicates pathological changes. Function of these fEECs has not been identified until now. Therefore, the aim of the study was to characterize the fEECs by means of transmission electron microscopy and immunohistochemistry. Different manifestations of fEECs were observed and analysed with regard to proliferative activity and presence of different epithelial adhesion molecules including PLEKHA7, β‐catenin and E‐cadherin. PLEKHA7 was restricted to the apical regions of the fEECs, whereas E‐cadherin and β‐catenin were demonstrated basolateral. The immunohistochemical detection of steroid hormone receptors demonstrated the responsiveness of the fEECs to steroid hormones. Intense progesterone receptor expression was observed in the fEECs indicating a high responsiveness to this hormone. Considering a potential function of the fEECs, we hypothesized that leptin, a hormone produced by other lipid‐accumulating cells and described to be involved in reproduction, in particular during implantation, might also originate from the fEECs which was confirmed by immunohistochemical methods. Moreover, leptin receptor was found in fEECs indicating the fEECs as both, source and target for leptin. Therefore, we conclude that fEECs in the canine uterus have a potential role in early pregnancy events and that the different observed manifestations might simply reflect the variations of signs of pseudopregnancy among bitches.     

Crb1在小鼠生后不同发育阶段子宫组织中的表达

应用HE染色方法和免疫荧光组织化学技术对小鼠生后不同发育阶段子宫组织结构的发育以及极性调控蛋白Crb1的定位表达进行了研究,结果显示,随个体发育,子宫管腔及子宫腺腔不断扩大,子宫内膜上皮持续增厚、固有层内子宫腺逐渐发达;4周龄时子宫腔面出现纤毛,随发育进程越来越发达;在各发育阶段的小鼠子宫组织中均有Crb1的表达,随个体发育表达呈现先增强后减弱的趋势,在8周龄时达到最强;Crb1主要定位于子宫内膜上皮细胞和子宫腺上皮细胞的胞膜和胞质,提示Crb1可能与小鼠子宫内膜上皮细胞和腺上皮细胞的极性建立和维持有关。     

Cyclical endometrial steroid hormone receptor expression and proliferation intensity in the mare

The aims of this study were to investigate the steroid hormone receptor expression and the proliferation intensity during the equine endometrial cycle by immunohistological methods, established for routine examination of formalin-fixed, paraplast-embedded specimens. Endometrial biopsy specimens were obtained during one cycle from 7 mares. In comparison with the blood steroid hormone levels the quantity and distribution of oestrogen (ER) and progesterone receptors (PR) and the proliferation marker Ki-67 antigen expression were investigated. Rising 17beta-oestradiol concentrations in preoestrus induce a synchronous expression of ER, PR and Ki-67 antigen in stromal cells. In the early dioestrus 17beta-oestradiol levels decrease and progesterone levels reach their maxima. This correlates with an intense proliferation activity and the highest hormone receptor expression in epithelial cells. In accordance to the morphological features of asynchronous glandular differentiation in fibrotic areas (endometrosis) their epithelial hormone receptor expression is out of phase.     

Immunity and inflammation in the uterus

Microbes often infect the uterus and particularly the endometrium of animals. Infections are most commonly associated with natural service, pregnancy and the post-partum period, leading to inflammation with the elaboration of cytokines, chemokines and prostaglandins. Clinical diseases such as metritis, endometritis and abortion are important causes of infertility. The adaptive immune response to infection has been characterized previously, so the present review aims to highlight the emerging role for innate immunity in the endometrium. The detection of microbes and the innate immune response depends on the detection of pathogen-associated molecular patterns by pattern recognition receptors. The main families of pattern recognition receptors are Toll-like receptors (TLRs), nucleotide oligomerization domain-like receptors, retinoic acid-inducible gene I-like receptors and C-type lectin receptors. These receptors are most often expressed by hematopoietic cells, but the epithelial and stromal cells of the endometrium also possess functional receptors. For example, endometrial cells express TLR4 for recognition of the lipopolysaccharide endotoxin of Gram-negative bacteria, leading to secretion of IL-6, IL-8 and prostaglandin E(2) . It is likely that the epithelial and stromal cells provide a first line of defence in the endometrium to alert hematopoietic cells to the presence of microbes within the uterus.     

Mineralised deposits in the uterine glands of mares with chronic endometrial degeneration

Chronic degenerative disease of the mare's endometrium is characterised by changes in the uterine glands, including cystic dilation, hyperplasia and periglandular fibrosis. Endometrial biopsies were taken from 23 mares with different grades of endometrial degeneration. Solid structures were identified within the lumina of the uterine glands and shown to be calcified by histochemical staining. Most of them were not homogenous but composed of a mixture of mineral and organic substances. Further examinations of these mineralised structures by immunohistochemical methods revealed the presence of the non-collagenous matrix proteins osteopontin, osteonectin and bone sialoprotein, which are known to be involved in calcification processes such as urolithiosis. Osteopontin and bone sialoprotein were identified within the calculi, frequently arranged in concentric layers. Osteonectin was the only matrix protein that was also present in the glandular epithelium. Osteocalcin was not found in either the calculi or the glandular epithelial cells.     

Distribution of androgen receptor in the porcine uterus throughout pregnancy.

The uterus is a well-known target of endocrine, paracrine and autocrine acting molecules among which steroid hormones (oestrogens, androgens and progesterone) are of special importance. The uterine tissues (endometrium and myometrium) undergo morphological and physiological changes which are associated with changes in expression of steroid hormone receptors. Androgen receptors (AR) that mediate the action of androgens have already been detected in porcine uteri during the oestrous cycle and early pregnancy. To evaluate the role of AR in uterine physiology, the presence of ARmRNA and AR protein localization in the porcine uterus from day 10 to day 90 of pregnancy and in the uterus from the foetus of day 90 postcoitum (p.c.) and from the neonatal 1-day-old piglet was studied. ARmRNA was detected in the porcine endometrium up to day 18 p.c., while AR protein was detectable in glandular epithelium and stromal cells as through day 90 of pregnancy. AR was also detected in the myometrium on all investigated days of pregnancy; however, on day 90, the immunostaining was present only in a limited number of cells. AR immunostaining was clearly demonstrated in the uterus of the female foetuses on day 90 as well as in the uterus of 1-day-old piglets. The physiological relevance of this finding needs further elucidation.     

Tumour necrosis factor in the canine endometrium: an immunohistochemical study

Tumour necrosis factor (TNF), a pleiotropic cytokine that regulates cell growth and differentiation as well as the synthesis of other cytokines, has been identified in the uterus of several species describing a cyclic pattern, eventually under ovarian steroid regulation. Information is yet limited on the presence of TNF protein in the canine endometrium during the oestrous cycle and early pregnancy. This study depicts the temporal immunolocalization of TNF in the bitch endometrium along the oestrous cycle and changes associated with the early steps of embryo invasion. TNF immunolabelling was found in both the stromal fibroblasts and epithelial components of the canine endometrium in all stages studied. Stromal immunostaining was more intense than that of the epithelia, in all the stages of the oestrous cycle. In addition, a tendency for a decrease in the surface epithelium intensity score was found in early dioestrus. A positive glandular content was only observed in anoestrus and proestrus stages. In early pregnancy (days 13-16), TNF immunolabelling was detected at the embryo-maternal surface, in the syncytium cords and the trophoblast, as well in the endometrial stroma and the basal endometrial glands, but not in the lacunar epithelium. The overall TNF immunoreactivity was higher in early pregnancy samples in comparison with those of the early dioestrus and dioestrus stages, suggesting it plays a role during implantation.     

Establishment and characterization of a coculture system of equine endometrial epithelial and stromal cells

To investigate the equine endometrium as close to the in vivo situation as possible, we established a coculture system for epithelial and stromal cells (ECs/SCs). ECs and SCs were isolated from nine endometrial tissue specimens. ECs obtained as glandular formations were cultivated on one side of the semipermeable membrane of a Millicell^® insert. After 2 days, SCs (2 × 10⁴ cells/membrane) were seeded onto the other side of the same membrane. During cocultivation, the low serum containing culture medium (Theuß et al., 2010) was supplemented with different concentrations and combinations of 17β‐estradiol (2.0–3.0 pg/ml medium) and progesterone (0.5–15.0 ng/ml medium). Once the cocultures formed continuous cell layers as determined by phase‐contrast microscopy, the membranes were fixed and processed for light microscopical examination. Cytokeratin 19, steroid hormone receptors and the uterine proteins uteroglobin and calbindin_D9k were detected using immunocytochemistry to determine the degree of culture purity and functional cellular differentiation. The culture purity of the EC layer averaged ≥95%. Uteroglobin and calbindin_D9k were consistently expressed in ECs, while hormone receptors were predominantly absent in both cell populations. An explicit cytomorphological epithelial differentiation with formation of round‐oval to polygonal cell forms was encountered in ≤50% of all ECs and independent of supplemented steroids. Based on the findings altogether, and despite the partly absent congruence to the in situ prerequisites, we established a standardized and reproducible coculture system, which offers a basic approach for studies of physiologic and pathophysiologic issues in the mare.     

Uterine diseases in cattle after parturition

Bacterial contamination of the uterine lumen is common in cattle after parturition, often leading to infection and uterine disease. Clinical disease can be diagnosed and scored by examination of the vaginal mucus, which reflects the presence of pathogenic bacteria such as Escherichia coli and Arcanobacterium pyogenes. Viruses may also cause uterine disease and bovine herpesvirus 4 (BoHV-4) is tropic for endometrial cells, causing a rapid cytopathic effect. The elimination of pathogens by the innate immune system is dependent on pattern recognition receptors binding pathogen-associated molecules. Uterine epithelial and stromal cells express receptors such as Toll-like Receptor 4 that binds E. coli lipopolysaccharide. The infertility associated with uterine disease is caused by damage to the endometrium and disruption of ovarian cyclic activity. Bacteria modulate endometrial prostaglandin secretion, and perturb ovarian follicle growth and function. Understanding the molecular basis of uterine disease will lead to novel approaches to treating infertility.     

犬正常发情期与患子宫蓄脓的子宫和卵巢组织结构及乳铁蛋白表达的对比

本研究旨在探明犬子宫和卵巢在正常发情期与患子宫蓄脓时，其组织结构及乳铁蛋白（lactoferrin，LF）表达的变化特征。应用Masson’s、VVG、PAS组织化学染色方法观察乏情期、发情期、患子宫蓄脓时犬子宫及卵巢的组织结构特点，用免疫组织化学SP法观察LF的分布特征。结果显示，正常发情期犬：乏情期、发情期子宫内膜肌层厚度比分别为0.762 0、0.924 3；乏情期子宫固有层中胶原纤维含量大于发情期，发情期卵巢中胶原纤维含量大于乏情期；子宫血管层及卵巢血管内弹性膜清晰完整；子宫腺在乏情期时浅层管腔小，深层较大、腺管上皮为单层柱状上皮、上皮细胞及管腔内PAS阳性反应较强，发情期子宫腺管腔变大、腺管上皮为单层立方上皮、上皮细胞及管腔内有PAS阳性反应；子宫黏膜上皮在乏情期和发情期均为单层柱状上皮，但胞核位置不同，乏情期胞核位于中央，发情期胞核位于顶部。患子宫蓄脓犬：子宫内膜肌层厚度比为1.615 0；子宫固有层和卵巢中胶原纤维含量少于正常发情期；子宫腺管腔大，且形状不规则，管腔内有炎性细胞浸润，腺管上皮为单层立方上皮，有淋巴细胞位于基膜，上皮细胞及管腔内PAS阳性反应较弱；子宫血管层及卵巢血管内弹性膜较正常发情期变薄，且有断裂现象；子宫黏膜上皮为单层柱状上皮，胞核位于基底。LF在乏情期子宫腺上皮和卵巢中的表达水平高于发情期，而在子宫黏膜上皮中发情期的表达水平高于乏情期。患子宫蓄脓时犬子宫和卵巢中LF的表达水平均较低。综上表明，犬正常发情期与患子宫蓄脓时的子宫和卵巢组织结构特点显著不同，LF的表达水平也存在差异。     

Infertility associated with the absence of endometrial progesterone receptors in a bitch

A three‐year‐old intact female Old English sheepdog was presented for evaluation of infertility. A uterine biopsy was performed during dioestrus, and the microscopic appearance was inconsistent with progesterone stimulation; the glands were sparse, simple and failed to show coiling, while the glandular epithelium was cuboidal instead of columnar. There was very little evidence of glandular activity. Due to the inappropriate appearance of the glands for the stage of the cycle, immunohistochemistry for progesterone receptors was performed. No progesterone receptor‐positive immunoreactivity was identified in the endometrial luminal epithelium, glandular epithelium or stroma. Weak intranuclear immunoreactivity was identified within the smooth muscle cells of the myometrium. The absence of progesterone receptors within the endometrial glands is the most likely explanation for the abnormal appearance of the endometrium and for this bitch's infertility. To our knowledge, this is the first report of endometrial progesterone receptor absence in a bitch.     

Uropathogenic virulence factor FimH facilitates binding of uteropathogenic Escherichia coli to canine endometrium

Pyometra is a potentially life-threatening condition in bitches and is often caused by Escherichia coli infection. Both pathogenic and non-pathogenic E. coli strains commonly carry the genes for type 1 fimbriae that mediate bacterial adhesion onto host epithelium. To investigate whether the type 1 fimbrial adhesin, FimH, facilitates the binding of uropathogenic E. coli to canine endometrium, the fimH gene was insertionally inactivated in a pathogenic E. coli strain. The ability of E. coli to bind to canine endometrial epithelial cells was determined in vitro using canine uterine biopsies. Binding of the fimH mutant was only 0.3% of that of the wild type. Complementation of the mutation restored the phenotype to that of the parent. This study has developed an in vitro model that allows quantitative and qualitative assessment of bacterial binding to canine endometrium and has demonstrated that the fimH gene plays a role in adherence of pathogenic E. coli to canine endometrium.     

Resident Macrophages and Lymphocytes in the Canine Endometrium

Resident immune cells play a major role in endometrial immunity and in tissue homoeostasis. This study aimed to analyse the distribution of macrophages, B and T lymphocytes (respectively, Mø, B‐Lym and T‐Lym) in the canine endometrium throughout the oestrous cycle and in late involution (at the proestrus stage post‐parturition). An immunohistochemistry technique was used on samples from 50 post‐pubertal healthy female dogs, of which five in late post‐partum. The distribution of resident immune cells was analysed in three endometrial layers (superficial, intermediate and basal areas). Mø, B‐Lym and T‐Lym were demonstrated to reside in the endometrium in all the stages of the canine cycle; their numbers being considerably higher during late involution. T‐Lym were scattered in the stroma or amidst the glandular epithelium, constituting the predominant immune cell population in anestrus and proestrus, but decreased in number at all other stages. Endometrial B‐Lym remained fairly constant during the canine cycle, although its numbers were higher in late involution. Mø counts were higher during anestrus compared to the other stages, the cells being displaced into the superficial endometrial layer. Mø demonstrated the highest level in late involution samples, forming small aggregates below the surface epithelium. The number of immune cells was not normally distributed, suggesting the influence of individual factors, such as age or parity, not explored herein due to limited sample availability. Still, this study provides important information for the interpretation of endometrial biopsies in dogs and for the understanding of the increased susceptibility to uterine infection during dioestrus found in the bitch.     

Structure and Steroidogenesis of the Placenta in the Antarctic Minke Whale (Balaenoptera bonaerensis)

There are few reports describing the structure and function of the whale placenta with the advance of pregnancy. In this study, therefore, the placenta and nonpregnant uterus of the Antarctic minke whale were observed morphologically and immunohistochemically. Placentas and nonpregnant uteri were collected from the 15th, 16th and 18th Japanese Whale Research Programme with Special Permit in the Antarctic (JARPA) and 1st JARPA II organized by the Institute of Cetacean Research in Tokyo, Japan. In the macro- and microscopic observations, the placenta of the Antarctic minke whale was a diffuse and epitheliochorial placenta. The chorion was interdigitated to the endometrium by primary, secondary and tertiary villi, which contained no specialized trophoblast cells such as binucleate cells, and the interdigitation became complicated with the progress of gestation. Furthermore, fetal and maternal blood vessels indented deeply into the trophoblast cells and endometrial epithelium respectively with fetal growth. The minke whale placenta showed a fold-like shape as opposed to a finger-like shape. In both nonpregnant and pregnant uteri, many uterine glands were distributed. The uterine glands in the superficial layer of the pregnant endometrium had a wide lumen and large epithelial cells as compared with those in the deep layer. On the other hand, in the nonpregnant endometrium, the uterine glands had a narrower lumen and smaller epithelial cells than in the pregnant endometrium. In immunohistochemical detection, immunoreactivity for P450scc was detected in most trophoblast cells, but not in nonpregnant uteri, suggesting that trophoblast epithelial cells synthesized and secreted the sex steroid hormones and/or their precursors to maintain the pregnancy in the Antarctic minke whale.     

Heat shock protein HSP90 immunoexpression in equine endometrium during oestrus,dioestrus and anoestrus

Heat shock proteins play a crucial role in cellular development, proliferation, differentiation and apoptosis. Heat shock protein 90 (HSP90) has been localised in the human endometrium, where its immunoexpression changes during the menstrual cycle. Similar studies have not been done for the equid species, so the present study aimed to describe endometrial HSP90 immunoexpression in mare endometrium. Endometrial biopsies were formalin-fixed and paraffin-embedded, and sections were stained with haematoxylin–eosin in preparation for HSP90 immunohistochemistry. Immunostaining and morphometric analyses were performed on the epithelial lining, endometrial glands and connective stroma during oestrus, dioestrus phase and anoestrus period (n = 7 per phase or period). Immunoexpression was localised in the basal region of the epithelial cells lining the lumen. Immunoexpression was greater during oestrus than during either dioestrus or anoestrus. During anoestrus, there was little immunostaining in the endometrium, suggesting that HSP90 is involved in the functional modulation of sex steroid receptors in cyclic mares. Indeed, the function of HSP90 as a chaperone in the folding of proteins, such as steroid receptors, might explain the greater intensity of immunostaining during the oestrus and dioestrus phases, compared the anoestrus period. We conclude that, in the mare, HSP90 plays a role in endometrial function and that further studies are needed to test whether it is important in pathological conditions as endometritis.