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     

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.     

Expression Pattern of Markers in the Canine Ovarian Cycle

Although hormonal changes during different phases of the oestrous cycle of bitches are well-described, knowledge about the luteal phase and anoestrus is incomplete. Furthermore, which paracrine and autocrine critical factors that differentiate between follicles destined for atresia and those that continue to develop are unknown. In this study, ovarian tissue was collected from 39 healthy bitches that were subject to ovariectomy or ovariohysterectomy for surgical neutering or medical purposes such as unwanted pregnancy. Bitches were allocated to different groups depending on the stage of the oestrous cycle. Serum progesterone, LH, FSH and 17β-estradiol (E₂) -levels were determined and immunhistochemistry was performed for a variety of receptor antigens; Ki-67, vimentin, pan cytokeratin antibody, p53 and oestrogen receptor (ER) α antigens. Marked differences were found in progesterone concentration between pregnant and non-pregnant animals. Oestrogen concentration was significantly lower in pro-oestrus and ovulation than during the luteal phase. Although progesterone could be detected in cytoplasm of ovarian cells at each stage, its presence was restricted to follicular cells during anoestrus. A strong presence of AE1/AE3, vimentin and p53 was found in each oestrous stage, in contrast with Ki67. The localization of ERα appeared to vary during the oestrous cycle, a phenomenon that suggests a switch between target cells of oestrogen; while as a proliferation marker, the mild reaction of p53 during parturition suggests an apoptotic process at this stage of the cycle.     

Immunolocalization of sex steroid receptors in the epididymis and ductus deferens of immature and mature Japanese Quail, Coturnix Japonica

The goal of this study was to determine whether in the Japanese quail the male genital tract contains receptors for progesterone, androgen and estrogen (PR, AR and ER, respectively), which have significant roles in reproductive functions, and whether their localization changes during sexual maturation. The epididymis and ductus deferens (middle and ampulla regions) of immature (approximately 30-day-old) and mature male Japanese quail were collected and frozen sections of them were immunostained for PR, AR and ER. The immunoreaction products for AR and PR were found in the nuclei of epithelial cells in the efferent ductules, epididymal duct, and the middle and ampulla regions of the ductus deferens of mature and immature birds. In the mature birds, the epithelial cells of the efferent ductules, epididymal duct, and the middle and ampulla regions of the ductus deferens were positive for ER, although some of the cells in the ductus deferens were negative. The epithelial cells of the ductules in the epididymis stained positive for ER, but the immunoreactions were negligible in the ductus deferens of immature birds. These results suggest that the epididymis and ductus deferens in quail possesses PR, AR and ER receptors. Each receptor is expressed before sexual maturation, although enhancement of ER expression may occur during maturation.     

Comparison of steroid receptor expression in normal, dysplastic, and neoplastic canine and feline mammary tissues

Steroid receptor expression was assessed by immunohistochemistry in neoplastic, hyperplastic/dysplastic, and normal mammary tissue samples removed from 68 queens and 47 bitches, using monoclonal antibodies against human oestrogen-alpha (ER) and progesterone receptors (PR). Mammary lesions were classified according to World Health Organization (WHO) criteria, and all animals with invasive carcinomas were clinically followed for 2 years. Stromal and/or lymphatic invasion and histological grading were also recorded. In both species, ER expression was significantly higher in healthy tissues, hyperplastic/dysplastic lesions, and benign tumours than in carcinomas. The loss of ER expression was more marked in feline than in canine carcinomas. In queens, PR expression increased in dysplastic lesions and "in situ" carcinomas and decreased in invasive carcinomas, even if parts of these tumours were still PR-positive. In bitches no significant variation in PR expression was observed between normal tissue, dysplasias, and benign neoplasms, but was significantly lower in carcinomas. In both species ER and PR expression in invasive carcinomas did not correlate either with histological parameters or overall survival time. This study demonstrates several differences in steroid hormone dependency between the two species. The percentage of PR-positive feline carcinomas suggests a possible role of progesterone in promoting early tumour cell growth in queens. The low percentage of ER-positive invasive carcinomas further demonstrated the aggressive phenotype and behaviour of feline mammary tumours.     

Immunohistochemical study of hormonal receptors and cell proliferation in normal canine mammary glands and spontaneous mammary tumours

The expression of hormone receptors and their relationship to cell proliferation in six samples of normal canine mammary tissue, and 11 benign and 10 malignant mammary neoplasms from female dogs were assessed by immunohistochemistry in formalin-fixed paraffin-embedded samples, by using monoclonal antibodies against progesterone and oestrogen receptors, and nuclear antigen Ki-67 (MIB-1). Malignant tumours negative for progesterone receptors proliferated at higher rates than progesterone receptor-positive tumours, suggesting that the progression towards malignancy in spontaneous mammary tumours is accompanied by a decrease in hormonal steroid dependency. Only one malignant tumour was positive for oestrogen receptors.     

Expression of estrogen receptor 1 and progesterone receptor in primary goat mammary epithelial cells

The exact role and sensitivity of cells to estrogen and progesterone mediated through the steroid receptors during lactation is not known. Expression of estrogen receptor 1 (ESR1) and progesterone receptor (PGR) was quantified in mammary tissue‐derived primary goat mammary epithelial cells (pgMECs) to determine the influence of donor tissue physiology (lactating and juvenile) and cell culture growth conditions (basal and lactogenic) on ESR1 and PGR expression in the derived cells. Relative messenger RNA (mRNA) levels for both receptors were the highest in cell lines derived from mammary tissue of juvenile goats. Maintaining pgMECs in lactogenic conditions resulted in up‐regulation of ESR1 (1.36‐ to 12.35‐fold) and in down‐regulation of PGR (‐2.53‐ to ‐3.62‐fold), compared to basal conditions. Based on Western blotting analysis we suggest that the differences in mRNA expression are translated to the protein level. We suggest that differential expression in lactating conditions is correlated with terminal differentiation of the pgMECs. Double immunostainings showed that estrogen receptor alpha (ER‐α) positive cells do not exclusively belong to the luminal lineage and that ER‐α and PGR can be expressed individually or co‐expressed in the pgMECs. The derived primary cultures/lines in early passages are hormone‐responsive and represent a useful surrogate for mammary tissue in research experiments.     

Colostrogenesis: candidate genes for IgG1 transcytosis mechanisms in primary bovine mammary epithelial cells

Bovine colostrogenesis is distinguished by the specific transfer of IgG₁ from the blood to mammary secretions. The process has been shown to be initiated by hormones and occurs during the last weeks of pregnancy when steroid concentrations of estradiol (E₂) and progesterone (P₄) are highly elevated. Rodent intestinal uptake of immunoglobulin G is mediated by a receptor termed Fc fragment of IgG, Receptor, Transporter, alpha (FcGRT) and supported by light chain Beta‐2‐Microglobulin (β2M). We hypothesized that steroid hormone treatments (E₂ and P₄) of bovine mammary epithelial cells in vitro would induce up‐regulation of IgG₁ transcytosis candidate gene mRNA expression suggesting involvement in IgG₁ transcytosis. Two different primary bovine mammary epithelial cell cultures were cultured on plastic and rat tail collagen and treated with hormonal combinations (steroids/lactogenic hormones). Evaluated mRNA components were bLactoferrin (bLf: a control), bFcGRT, β2M, and various small GTPases; the latter components are reported to direct endosomal movements in eukaryotic cells. All tested transcytosis components showed strong expression of mRNA in the cells. Expression of bFcGRT, bRab25 and bRhoB were significantly up‐regulated (p < 0.05) by steroid hormones. bRab25 and bRhoB showed increased expression by steroid treatments, but also with lactogenic hormones. Analysis for the oestrogen receptor (ER) mRNA was mostly negative, but 25% of the cultures tested exhibited weak expression, while the progesterone receptor (PR) mRNA was always detected. bRab25 and bRhoB and likely bFcGRT are potential candidate genes for IgG₁ transcytosis in bovine mammary cells.     

Expression of progesterone receptor membrane component 1, serpine mRNA binding protein 1 and nuclear progesterone receptor isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy

The aim of this study was to investigate the (1) expression of progesterone membrane component 1 (PGRMC1), serpine mRNA binding protein 1 (SERBP1) and progesterone receptor (PR) mRNA and (2) protein expression levels of PGRMC1, SERBP1 and PR isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy. Uteri from cows on days 1-5, 6-10, 11-16 and 17-21 of the estrous cycle and weeks 3-5, 6-8 and 9-12 of pregnancy were used (n=5-6 per period). There were no changes (P>0.05) in PGRMC1 mRNA expression during the estrous cycle, while expression of SERBP1 and PR mRNA was the lowest (P<0.05) on days 11-16 relative to other days of the cycle. The highest mRNA expression of PGRMC1, SERBP1 and PR was found during pregnancy. There were no changes (P>0.05) in SERBP1 protein expression in cycling and pregnant cows, while the highest (P<0.05) PGRMC1 protein expression was found during weeks 3-5 of pregnancy. Similar protein expression profiles for PRA and PRB were found, and protein levels were highest on days 1-5 of the estrous cycle. From day 6 of the cycle, PRA and PRB protein expression decreased and were maintained at this lower level during pregnancy. In conclusion, our study assessed mRNA and protein expression levels of PGRMC1, SERBP1 and PR in the bovine myometrium during the estrous cycle and the first trimester of pregnancy. It is possible that progesterone (P4) affects myometrial function in a genomic and nongenomic manner.     

Progesterone receptor mRNA levels during pregnancy, labor, lactation and the estrous cycle in rat uterus

Progesterone plays important roles in the regulation of female reproduction. In this study, progesterone receptor (PR) mRNA levels in rat uterus during pregnancy, labor, lactation and the estrous cycle were examined by competitive RT-PCR. During pregnancy and lactation, PR mRNA levels had decreased on day 20 of pregnancy (P20) and P21 compared with P15 but increased during labor. After a decline on day 1 of lactation (L1), PR mRNA levels had increased again on L3 and L14 compared with P15, P18, P20, P21 and P21pm (at 2200-2300 h on P21). There was no significant change in the PR mRNA level during the estrous cycle. The PR mRNA level did not change during 1 week of progesterone treatment or afterwards. Injection of 17beta-estradiol did not affect PR mRNA levels in rats treated with progesterone or those without any injections. In rats on P18, 17beta-estradiol injection did not change PR mRNA levels after sham-operation but induced an increase in PR mRNA levels of rats ovariectomized 6 h before the treatment. These results suggest that uterine PR mRNA levels are differently regulated during late pregnancy, labor and lactation, and during labor estrogen is one of the essential factors for the increase in PR mRNA levels.     

Pseudo-placentational Endometrial Cysts in a Bitch

Cystic alterations of the canine endometrium compromise reproduction and fertility of the bitch and may lead to life-threatening diseases, such as pyometra. Even without clinical evidence, reduction of the uterine lumen by cysts implicates disturbances during migration, nidation and development of the embryo. Several studies point to the high variability of morphology of uterine endometrial cysts but they lack detailed analyses of alterations. In the present study, immunohistochemistry was used to investigate the expression of steroid hormone receptors (oestrogen, progesterone), proliferation activity, inflammation and infection in the cystic affected tissue regions in contrast to the normal endometrium. Oestrogen receptor expression showed a high density of receptors throughout the surface epithelial cells, crypt epithelial cells, glandular epithelial cells and stromal cells of the normal endometrium as well as the cystic affected regions. Proliferation in the cysts was verified in the middle and basal cells of the crypts. Neither in the endometrium nor in the cysts inflammatory processes or evidence of infection could be detected. Furthermore, lectin histochemistry and electron microscopic methods showed that lectin binding patterns and cell morphology of internal epithelial lining and surface epithelium of the cysts can be used to characterize and distinguish different types of cystic alterations. Analogies between epithelial cells of the glandular chambers of the canine placenta and the cystic cellular morphology, steroid hormone receptor distribution as well as lectin binding patterns of the endometrial cysts, as observed in this study, suggest to introduce the term 'pseudo-placentational endometrial cysts'.     

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.     

Studies on canine mammary tumours. II. Oestradiol and progesterone receptor binding capacity and histological type.

Tissue samples taken from the mammary gland of 42 dogs (age: 6 to 12 years) were examined. Thirty-eight samples showed neoplasia: 36 were epithelial while the remaining 2 proved to be connective tissue tumours. Thirty-four % of the neoplasms were new benign tumours (most frequently adenoma and fibroadenoma) and 66% were malignant ones (mainly adenocarcinoma). The oestrogen receptor (ER) and progesterone receptor (PR) binding capacity was determined on 21 tissue samples using the method of EORTC (1980). The connective tissue tumours and non-tumourous tissues contained no sexual steroid receptor. 71.4% of all tissue samples contained receptors. 61.9% of the samples was ER+, 42.8% was PR+, 33.3% contained both receptors, 28.6% was only ER+ and 9.5% only PR+. The average ER and PR binding capacity was 120.3 (5.0-622.8) and 266.7 (92.3-475.0) fmol/mg cytosol protein, respectively. No difference in receptor positivity was demonstrable between the benign and the malignant tumours. PR negativity accompanied by ER positivity was more common in the case of benign tumours. ER binding capacity tended to be correlated with age: this correlation could be described with a hyperboloid regression curve (r = -0.5931; 0.06 > p > 0.05).     

The effect of interferon-tau and ovarian steroids on the proliferation of bovine endometrial cells in vitro

The goal of this study was to evaluate the effect of various concentrations of interferon-tau (IFN-tau) with or without steroid hormones, 171 estradiol or progesterone, on the proliferation of bovine endometrial cells in vitro. Endometrial epithelial and stromal cells were isolated from the uterus of cows during the early estrus cycle (2-3 days) and incubated with different doses of IFN-tau with or without steroid hormones. The proliferation was determined by the MTT test in 48, 96, and 144 h of incubation. An antiproliferative activity of IFN-tau was observed both in epithelial and stromal cells cultured in RPMI 1640 medium supplemented with 10% FBS or. serum replacement. However, epithelial cells were more sensitive to antiproliferative action of interferon-tau. It;s activity was dose-and time-dependent. The inhibition of epithelial cell proliferation by 50% (ED50) was achieved at concentrations of 500 U/ml, 340 U/ml, and 8.8 U/ml of IFN-tau after 48, 96, and 144 h of incubation, respectively. None of the doses of IFN-tau (10-10.000 U/ml) used inhibited stromal cell proliferation in 50%. The most effective dose of IFN-tau inhibiting stromal cell proliferation was 10.000 U/ml, which decreased cell growth by 17.08%, 22.87%, and 2.6% after 48, 96, and 144 h of incubation, respectively. Steroid hormones, 17beta estradiol and progesterone, added to the culture of stromal cells with or without IFN-tau did not significantly modulate stromal cell growth. In contrast, a high concentration of progesterone (10(-5) M) alone significantly enhanced stromal cell growth. Progesterone at low, physiological concentrations (10(-7)- 10(-9) M) ameliorated the antiproliferative activity of IFN-tau, especially at the 10(-9 )M concentration. At this concentration, the stimulatory effect on stromal cell growth was observed. The mechanisms of such response are not entirely clear but may arise from the influence of IFN-tau on progesterone down regulation of its own receptor. Depicted activity of IFN-tau may find usefulness in therapy of neoplastic disorders.     

Relationship between cell proliferation and tenascin-C expression in canine gastrointestinal tumours and normal mucosa

Tenascin-C is an extracellular matrix glycoprotein that has been implicated in cell proliferation and adhesion by in vitro experiments. Its expression is known to be increased in canine and human gastrointestinal tumours. The aim of this study was to investigate the possible relationship between cell proliferation and tenascin expression in these tumours. In tissue sections of normal stomach, small intestine and colon, and gastrointestinal epithelial tumours, the monoclonal antibody Ki-67, which is directed against a proliferation-associated nuclear antigen, was used to identify proliferating cells. Serial sections were also stained for tenascin. Serial sections stained for tenascin and Ki-67 were compared to determine whether there is a correlation between tenascin expression and tumour cell proliferation. In the normal gastric mucosa, Ki-67 positive cells were confined to the neck region and in the normal small intestinal mucosa positive cells were confined to the lower parts of the crypts. In adenomas and carcinomas, the frequency of positive cells was increased at the edges of adenomas and invasive tumour margins of carcinomas and there was inter- and intra-tumoural heterogeneity. Carcinomas with lymphatic invasion showed a high Ki-67-index. There was no relation between cell proliferation and tenascin expression in both normal tissues and tumours studied. The absence of a correlation between tenascin and Ki-67 expression suggests that the main function of tenascin in both normal tissues and tumours of the canine gastrointestinal tract is antiadhesion rather than proliferation.     

Oestrogen,Progesterone and Oxytocin Receptors and COX‐2 Expression in Endometrial Biopsy Samples from the Induction of Ovulation to Luteolysis in Llamas (Lama glama)

Endometrial expression of oestrogen (ERα), progesterone (PR) and oxytocin receptor (OR) and cyclooxygenase‐2 (COX‐2) was evaluated from the induction of ovulation to luteolysis in llamas. Ovarian activity was daily assessed by ultrasonography in five females. Ovulation was induced immediately after the detection of an ovulatory follicle by a GnRH injection (Day 0). Endometrial samples were obtained by transcervical biopsies from the left and right horns on day 0 and days 4, 8, 10 and 12 post‐GnRH. Blood samples were collected daily for progesterone and estradiol‐17β determinations by RIA. An immunohistochemical technique was used to study receptors population and COX‐2 expression which were then evaluated by two independent observers. The expression of ERα and PR was highest on day 0 in the luminal epithelium and stroma in association with high plasma estradiol‐17β concentrations. Thereafter, a decrease in ERα population was registered on day 4 and a new increase of its expression was observed between days 8 and 12 in those cell types. Conversely, PR population was gradually down‐regulated until its lowest expression was reached on day 10 post‐GnRH in the luminal epithelium. Content of OR was similar throughout the study in all cell types. The expression of COX‐2 was highest from day 8 to 12 post‐GnRH in the luminal epithelium, in relation to the time of maximal PGF_2α release. Both steroid receptors populations and COX‐2 expression were similar between horns. Meanwhile, OR expression was higher in the right than in the left uterine horn. In summary, this study showed that the loss of endometrium sensitivity to progesterone by days 8–10 post‐induction of ovulation and the concomitant increase of COX‐2 expression could play a key role in the mechanism of luteolysis and somehow be related to the short corpus luteum lifespan of llamas.     

Cell-specific Distribution of Oestrogen Receptor-alpha in the Bovine Ovary

In this study, the expression of estrogen receptor alpha (ERα) in the bovine ovary is described. ERα was visualized by immunohistochemistry on paraffin sections of ovaries obtained from 11 non‐pregnant and 2 pregnant animals. In general, ERα was not observed in cells of primordial, primary and secondary follicles, whereas weak expression was noticed in cells of healthy and arteric tertiary follicles. In corpora lutea cells the expression of ERα was obvious. Intermediate to high ERα expression was present in thecal cells and in cells of the superficial and deep stroma, tunica albuginea and surface epithelium. Furthermore, the expression of ERα in stroma and tunica albuginea cells was in general, highest in cows with the lowest plasma progesterone levels, and lowest in cows with the highest plasma progesterone levels. Remarkably, the ERα expression in pregnant cows was in general, lower than in non‐pregnant cows with similar plasma progesterone levels. The relatively high expression of ERα in thecal and stromal cells in comparison with that in follicle cells suggests an indirect effect of estrogen on the follicular development. However, the exact function of ERα in the bovine ovary together with the cycle‐dependent variations in ERα expression remain to be elucidated.