[Show abstract][Hide abstract] ABSTRACT: In contrast to other domestic animal species, in vitro maturation (IVM) of oocytes in the horse is still not successful. Oocytes for IVM are obtained either from slaughterhouse ovaries or via ovum pick-up from living mares. Both situations may be associated with a stress-induced glucocorticoid release. So far, neither an involvement of glucocorticoids in follicle and oocyte maturation nor the presence of glucocorticoid receptors (GCR) in ovarian tissue has been investigated in the horse. We hypothesised that GCR are expressed in equine ovarian tissue independent of the animal's age and stage of the oestrous cycle. Ovaries (n=40) were collected from killed newborn female foals (n=10) and killed or slaughtered adult mares (n=10). For assessment of GCR mRNA expression, ovarian samples were fixed in Tissue-Tek O.C.T. Compound (Sakura Finetek, Zoeterwoude, the Netherlands) and stored at -80°C. Various cell populations were isolated using laser capture microdissection on cryosections. After RNA extraction, samples were analysed by qualitative RT-PCR and real time-PCR. For analysis of GCR protein, tissue was fixed in Bouin's solution and histological slides immunostained using a monoclonal antibody for GCR (Ab2768, Abcam, Cambridge, UK), followed by visualisation with diaminobenzidine. One tertiary follicle per slide (40×; light microscopy) was analysed and percentages of cells staining positive for GCR calculated. Statistical analysis was done with the SPSS Statistics 21 software (SPSS Inc., Chicago, IL, USA). Expression of mRNA for GCR was detected in oocytes, cumulus cells, granulosa, and theca cells, independent of age and stage of the oestrous cycle. In both neonates and adults, nuclei of the oocytes and cumulus cells stained positive for GCR regardless of stage of folliculogenesis. Also, GCR were constantly expressed in granulosa cells from both preantral and antral follicles. Percentage of granulosa cells staining positive for GCR (adult: 73.6±3.2, fillies: 72.4±1.9%) was higher (P<0.001) than of theca cells (adult: 56.8±3.9, fillies: 57.2±1.9%), but not affected by age. GCR were lacking in ovarian stroma of adults but not of neonates. In periovulatory follicles from adult mares, GCR were abundant in developing luteal cells. GCR were also detected in the nuclei of luteal cells in corpora haemorrhagica and corpora lutea. Follicular atresia was associated with a decrease of GCR independent of cell type and age. This study describes for the first time the expression of GCR in horse ovaries, which are present independent of age of the animal, stage of folliculogenesis, and oestrous cycle stage. Results suggest that glucocorticoids are involved in follicular and oocyte maturation, ovulation, and luteal function in the horse. Presence of GCR in the ovaries of newborn horses suggests a role of glucocorticoids in ovarian tissue maturation. Nevertheless, detrimental effects of excess glucocorticoid secretion due to stress on follicular development, oocyte maturation, and luteal function cannot be excluded in the mare.
Reproduction Fertility and Development 12/2014; 27(1):142. DOI:10.1071/RDv27n1Ab99 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: In mares, mating-induced persistent endometritis contributes to low fertility. The condition is in part related to delayed clearance of mucus accumulated within the uterine lumen. The objective of this study was to investigate the endometrial response of healthy mares to intrauterine (i.u.) treatment with N-acetylcysteine (NAC). Oestrous mares (n = 12) were randomly assigned to a treatment (TM) or control (C) group and received an i.u. infusion of 5% NAC and saline (total volume 140 ml), respectively. Endometrial biopsies were collected in five of the mares 24 h after treatment, in the remaining seven mares 72 h after treatment. Endometrial biopsies were evaluated for integrity of the luminal epithelium, number of polymorphonuclear neutrophils (PMN), staining for cyclooxygenase 2 (COX2), staining with Kiel 67 antigen (Ki-67), lectins and periodic acid-Schiff (PAS). The integrity of endometrial epithelial cells was not affected by treatment (no statistical differences between groups or times). At 24 h after treatment, the mean number of PMN in endometrial biopsies from NAC- and C-mares did not differ, but at 72 h after treatment, number of PMN was significantly higher (p < 0.05) in C (3.9 ± 0.6 PMN/field) compared with NAC-treated mares (2.3 ± 0.2 PMN/field). At 72 h after treatment, the intensity of staining for COX2 was significantly higher after saline than after NAC treatment (p < 0.05). In the epithelium, no differences in staining for the proliferation marker Ki-67 were seen with respect to time and treatment. Score for the lectin wheat germ agglutinin (WGA) was slightly higher in NAC-treated mares than in C-mares 72 h after treatment (p < 0.05). Score for PAS staining of mucus in deep uterine glands differed significantly between groups at 24 h after treatment (p < 0.05). The present study demonstrates that NAC does not adversely affect the endometrial function. Moreover, an anti-inflammatory effect on the equine endometrium was observed.
[Show abstract][Hide abstract] ABSTRACT: In most mammalian species, progestins have a major function in maintaining pregnancy. In humans, the physiologic initiation of parturition bears similarities with inflammatory processes and anti-inflammatory effects of progestins have been suggested to postpone birth until term. To examine if comparable effects exist in the horse, mares were treated with the synthetic progestin altrenogest from day 280 of gestation until parturition (N = 5) or were left untreated as controls (N = 7). Tissue from the amnion (AMN), allantochorion (AC), and endometrium (EM) was collected at foaling and mRNA expression of interleukin (IL)-6 and -8, cyclooxygenase 2 (COX2), estrogen receptor (ER) α, progesterone receptor, and oxytocin receptor (OTR) was analyzed. Leukocytes, steroid receptors, COX2, and OTR were also investigated by histology and immunohistochemistry. Expression of mRNA for IL-6 was higher in AMN and EM versus AC (P < 0.01). Expression of IL-8 was higher in AMN than AC and EM (P < 0.001). Steroid receptors and OTR were highly expressed in EM but not in AMN and AC (P < 0.001). Expression of COX2 was most pronounced in AC whereas IL expression was not upregulated in AC. No differences in mRNA expression existed between altrenogest-treated and control animals. Endometrial polymorphonuclear leukocytes were increased in altrenogest-treated mares. Epithelial cells of all tissues, except AC chorionic villi stained progesterone receptor-positive. Staining for ER was more pronounced in the amnion facing epithelium of the AC in altrenogest-treated versus control animals (P < 0.01). In conclusion, COX2 is highly expressed in the AC. The fetal membranes thus might play a role in the onset of labor in the horse. Altrenogest did not affect gene expression in the AMN, AC, and EM but had localized effects on inflammatory cells and ER expression. No anti-inflammatory effects of altrenogest in healthy, late pregnant pony mares could be detected.
[Show abstract][Hide abstract] ABSTRACT: An adjustment of sex ratio of offspring to the conditions present at conception is seen in many mammals including horses. This depends on preferential survival of male embryos under conditions of high energy intake. In several species, growth factors including insulin like growth factor (IGF)-1 have been shown to promote embryonic development by decreasing apoptosis and increasing cell proliferation. We hypothesized that sex-related differences in IGF-1 expression in equine embryos during the phase of maternal recognition of pregnancy might exist and thus contribute to preferential survival of embryos from either of both sexes under specific environmental conditions. Insulin like growth factor-1 mRNA expression of in vivo-produced equine embryos on different days of pregnancy (Day 8, N = 6; Day 10, N = 8; Day 12, N = 14) was analyzed. Insulin like growth factor-1 mRNA expression was evaluated by reverse transcription quantitative polymerase chain reaction. The sex of the embryo was determined by detection of X-inactivation specific transcript (Xist) RNA and equine sex determining region of the Y chromosome DNA. Embryos positive for Xist expression were classified as female, and Xist negative and equine sex determining region of the Y chromosome positive embryos were classified as male. From 28 embryos tested, 15 (54%) showed positive Xist expression and were thus classified as female. Insulin like growth factor-1 mRNA expression was influenced by sex (P = 0.01) but not by day of pregnancy (relative expression of IGF-1 in relation to β-actin, Day 8: male 5.1 ± 2.1, female 11.4; Day 10: male 5.2 ± 1.6, female 17.4 ± 6.7; Day 12: male 2.6 ± 0.3, female 11.6 ± 2.4). Results demonstrate an increased expression of IGF-1 in female equine embryos. Sex-related influences on expression of the IGF system are probably related to a gradual X chromosome inactivation.
[Show abstract][Hide abstract] ABSTRACT: Persistent breeding-induced endometritis is ranked as the third most common medical problem in the adult mare and leads to enormous economic loss in horse breeding. In mares suffering from persistent breeding-induced endometritis, increased amounts of intrauterine (i.u.) fluid or viscous mucus in estrus or after breeding may act as a barrier for sperm and can contribute to low fertility. Current therapies of these mares aim to eliminate i.u. fluid and mucus by uterine lavage and/or administration of ecbolic drugs. Recently, i.u. administration of N-acetylcysteine (NAC) has been shown to support therapy in mares with endometritis. It was the objective of the present study to investigate effects of an oral administration of NAC on the viscosity of i.u. fluid in estrous mares. It was hypothesized that oral treatment with NAC reduces the viscosity of i.u. fluid and has a positive effect on the inflammatory response of the endometrium. Mares (n = 12) were included in the study as soon as estrus was detected (ovarian follicle >3.0 cm and endometrial edema), which was defined as Day 1. They were randomly assigned to a treatment (10 mg/kg NAC on Days 1-4) or a control group (no treatment). On days 1 and 5 i.u. mucus was collected and its rheologic properties were accessed. On Day 5, endometrial biopsies were obtained and evaluated for integrity of the luminal epithelium, number of polymorphonuclear neutrophils (PMN), staining for cyclooxygenase 2 (COX2), staining with Kiel 67 antigen (Ki-67), lectins and periodic acid Schiff (PAS). In the treatment group, viscosity of i.u. mucus increased significantly between Days 1 and 5 (P < 0.05), while no differences were found in control mares (n.s.). At no time were significant differences between treated and control mares seen. Integrity of epithelium was not affected. After NAC treatment the mean number of PMN in endometrial biopsies was significantly lower compared to mares of the control group (1.9 ± 0.3 vs. 4.8 ± 0.4; P < 0.05). Nuclear immunostaining for COX2 was significantly lower after NAC treatment compared to control mares (P < 0.05). Score for PAS and Alcain staining of mucus in deep uterine glands differed significantly between groups (both P < 0.05). We conclude that oral NAC treatment does not reduce viscosity of uterine mucus but has an antiinflammatory effect on the equine endometrium.
[Show abstract][Hide abstract] ABSTRACT: Glucocorticoids (GCs) as mediators of the stress response may affect Leydig cell function by inhibiting either luteinizing hormone receptor expression or testosterone biosynthesis. The isozymes 11β-hydroxysteroid dehydrogenase (11βHSD) 1 and 11βHSD2 control the intracellular cortisol levels. Little is known about the effects of stress on fertility in the equine. The objective of the present study was to determine the presence and cellular localization of glucocorticoid receptors (GCR) and glucocorticoid-metabolizing enzymes (11βHSD1 and 11βHSD2) in equine epididymal and testicular tissue with special regard to sexual maturation. Testicular and epididymal tissue was collected from 21 healthy stallions, and four age groups were designed: pre-pubertal, young, mature and older horses. Immunohistochemistry (IHC) analysis and quantitative real-time PCR (qRT-PCR) were used. Pre-pubertal horses showed higher testicular gene expression of 11βHSD1, 11βHSD2 and GCR than horses of all other groups (p < 0.05). A positive intranuclear immunoreaction for GCR was seen in epithelial cells of caput, corpus and cauda epididymidis and in Leydig cells. Significant differences (p < 0.05) between age groups occurred. The number of Leydig cells staining positive for GCR was highest in immature stallions (p < 0.05). The enzyme 11βHSD1 was localized in epithelial cells of the caput and corpus epididymidis and in Leydig cells. As determined by enzyme assay, nicotinamide adenine dinucleotide (NAD)-dependant dehydrogenase (oxidation) activity was not detected in testicular tissue from immature stallions but in all other age groups (n = 3 per group). Results of this study suggest a contribution of GCs to maturation of male reproductive tissue in horses. In mature stallions, expression of 11βHSD enzymes and the oxidative 11βHSD activity in Leydig cells and epididymal basal and principal cells suggest a protective role on these tissues contributing to physiological intracellular glucocorticoid concentrations.
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis, which is essential for malignancies to progress, depends on various signalling proteins including vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors 1 and 2 (VEGFR-1 and VEGFR-2). Microvessel density (MVD) is frequently used to evaluate angiogenesis. This study assessed the relationship between expression of VEGF, VEGFR-1 and VEGFR-2, MVD and the survival time in dogs with lymphoma. VEGF, VEGFR-1 and VEGFR-2 expression was evaluated immunohistochemically and microvessel profiles were counted in 34 lymphoma samples. Seventy-nine percent of the samples showed high VEGF expression and 62% were highly positive for VEGFR-1; VEGFR-2 immunoreactivity was mostly negative. Dogs treated with chemotherapy had a median survival time of 266days, but no significant relationships were found between overall survival time, MVD and expression of VEGF, VEGFR-1 or VEGFR-2. In this study, VEGF its receptors and the MVD were no prognostic factors in dogs with lymphoma.
Research in Veterinary Science 05/2011; 92(3):444-50. DOI:10.1016/j.rvsc.2011.04.018 · 1.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aim of this study was to determine the intrauterine activity of matrix metalloproteinases (MMP)-2 and -9 after cessation of the local effect of progesterone. For this purpose, pregnancy was terminated in 10 bitches at mid-gestation with the progesterone receptor antagonist aglepristone (10 mg/kg body weight, sc, Alizine®; Virbac, France) at two subsequent days (group IRA = induced resorption/abortion). The IRA group was divided into two subgroups (Group I, n = 5, days 25-35 of pregnancy; group II, n = 5, days 36-45). Five further bitches were introduced with beginning abortion (group SRA = spontaneous resorption/abortion). Seven healthy bitches between day 25 and 45 of gestation served as controls. After ovariohysterectomy at the end of abortion and between days 25 and 45 of gestation, respectively, the distribution and activity of collagenases were investigated by immunohistochemistry and gelatin zymography. At placental sites, MMP-2 activity in the endometrium was significantly lower in IRA groups than in the SRA group (33.7 ± 11.8% and 39.3 ± 5.4% vs 52.2 ± 10.2%, p < 0.05); however, MMP-2 expression was lowest in the control group (control: 21.4 ± 6.3%; p < 0.01) and similarly in the myometrium (controls: 13.1 ± 2.5%; p < 0.05). MMP-9 activity was also lower in the endometrium and myometrium of the control group in comparison to SRA and IRA groups (11.8 ± 3.2%; p < 0.01 and 28.4 ± 32.8%; p < 0.05). At interplacental sites, the amount of active collagenases in the myometrium was significantly lower in the control group. It is concluded that the blockade of the biological progesterone effect was associated with an increase in activity of both collagenases.
[Show abstract][Hide abstract] ABSTRACT: A positive influence of altrenogest treatment on a retarded development of the conceptus around the beginning of placentation in mares older than 8 years could be recently demonstrated. In the present study, effects of altrenogest treatment in early-pregnant mares on conceptus development and expression of endometrial and embryonic genes were investigated. Genes were chosen according to a possible involvement in embryo-maternal interaction and embryonic development in the equine species. Mares were treated with altrenogest (0.044 mg/kg bodyweight) or sunflower oil (placebo) from day 5 to 11 after ovulation. Embryos (altrenogest n = 13, placebo n = 12) and biopsies were collected on day 11. Pregnancy rate and embryonic size were not influenced by treatment (embryonic diameter: altrenogest 7.0 ± 2.5, placebo 6.5 ± 1.7 mm, n.s.). The percentage of luminal epithelial cells, superficial glandular epithelial cells and interstitial cells with nuclei staining positively for the progesterone receptor was significantly lower (P < 0.05) in samples collected from altrenogest-treated than from placebo-treated mares (e.g., luminal epithelium: altrenogest 1.9 ± 1.7%, placebo 23.0 ± 10.5%, P < 0.05). Staining for COX2 (cyclooxygenase-2) was not affected by treatment. In the endometrium a slight but significant increase in the number of PMN (polymorph nuclear neutrophils) was seen in response to treatment (altrenogest 0.8 ± 0.5 PMN/field, placebo 0.3 ± 0.3 PMN/field; P < 0.05). No differences in the relative gene expression of COX2, the receptors for progesterone, estrogens and growth hormone as well as for IGF (insulin-like growth factor) 1 and 2 were detected. The relative gene expression of aquaporin 3 in relation to β-actin differed significantly (P < 0.05) between embryos from altrenogest (3.2 ± 0.8) and placebo-treated mares (1.3 ± 0.2), but no other genes were affected. The study demonstrates down-regulation of progesterone receptors in the endometrium of early pregnant mares by treatment with the progestin altrenogest. This increased expression of aquaporin 3 in the conceptus was not related to changes in embryonic size or development.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Endometrial bleeding during proestrus is a well-known phenomenon in the bitch. However, the exact events on the cellular level have not been studied. In the present investigation, immunohistochemical methods and transmission electron microscopy were employed to obtain more information about this cyclic event in canines. Long, stretched blood vessels were seen in H&E stained sections during proestrus. These vessels showed mitotic activity, as evidenced by Ki67 immunostaining. Although the endothelial lining and basement membrane of endometrial blood vessels seemed continuous, as indicated by immunohistochemical staining for laminin and Von Willebrand factor, transmission electron microscopy showed an extreme thinning and even interruption of the vascular wall in endometrial venules. Platelets were frequently seen in those areas, and also detected by immunohistochemistry. Interestingly, all endometrial capillaries examined by electron microscopy had an intact wall. We therefore postulate the endometrial venules to be the blood vessels that are mainly responsible for proestrus endometrial bleeding, rather than subepithelial capillaries.