Effects of beta-OH butyrate on bovine granulosa and theca cell function in vitro.

In this study, the effects of beta-OH butyrate (BHB) levels, associated with a negative energy balance, on bovine granulosa and theca cell function were investigated in vitro. Granulosa and theca cells of healthy large follicles (>8 mm), obtained from slaughterhouse ovaries, were cultured in serum free medium containing 0, 0.5, 1 or 1.5 mm BHB and 3 mm glucose, to mimic the situation in the early postpartum dairy cow. Hormone concentrations (progesterone, oestradiol-17beta and/or androstenedione) in spent medium and cell numbers were measured after 48 h of culture. No effects of BHB on theca cell numbers or on steroid production were observed. In granulosa cells, all BHB treatments evenly increased cell numbers (p < 0.05), while they reduced progesterone and oestradiol-17beta production per cell (p < 0.05). These effects may be attributed to the use of BHB as energy source which is however differently metabolized than glucose. Conclusively, in the presence of physiological glucose concentrations BHB can modulate granulosa but not theca cell function in vitro.

Effect of non-esterified fatty acids on bovine theca cell steroidogenesis and proliferation in vitro.

Elevated serum non-esterified fatty acid (NEFA) levels associated with a negative energy balance (NEB) may affect ovarian function and hence reproductive performance in high-yielding dairy cows. We have investigated the individual and combined effects of the three major NEFAs on bovine theca cell proliferation and steroidogenesis in vitro. Theca cells from healthy large follicles (>8 mm) obtained from slaughterhouse ovaries were cultured in serum free medium in the presence of 0, 50, 150 and 200 microM of palmitic acid (PA; C16:0); 0, 50, 150 and 250 microM of stearic acid (SA; C18:0); and/or 0, 50, 150 and 250 microM of oleic acid (OA; C18:1). Progesterone and androstenedione concentrations were measured in spent medium after 48 h of culture and cell numbers were determined spectrophotometrically per culture well. Cell viability was assessed by annexin-V FITC/propidium iodide staining. Only the treatment with 200 microM of PA inhibited cell proliferation (P<0.001) when tested individually, both of the mixtures tested (M1=100 microM of PA, 130 microM of SA and 140 microM of OA; M2=200 microM PA, 260 microM of SA and 280 microM of OA) reduced cell numbers (P<0.001). Progesterone and androstenedione production, both per well and per 10(4) cells, were not affected by any of the treatments, with the exception of M2. This mixture reduced progesterone production per well and per 10(4) cells (P<0.05). The effects observed were most likely caused by the cytotoxic action of the NEFAs, as demonstrated by the increased percentage of early apoptotic (M1) and late apoptotic/necrotic cells (M1 and M2) in the combination treatments (P<0.05). When combined, elevated physiological concentrations of PA, SA and OA can modulate theca cell proliferation and steroidogenesis in vitro by reducing theca cell viability. These NEFAs may be one of the mediators through which NEB compromises ovarian functioning and thus fertility in high-yielding dairy cows.

Hormonal and metabolic profiles of high-yielding dairy cows prior to ovarian cyst formation or first ovulation post partum.

The present study aimed to investigate the pathogenesis of cystic ovarian disease (COD) in high-yielding dairy cows postpartum (pp). Hormonal and metabolic profiles during the first 3 weeks pp as well as during the final week prior to ovulation/cyst formation, were compared between dairy cows that developed either an ovulatory follicle (OV) or a cyst (CYST) < day 60 pp. Thirty-four lactations of 28 high-yielding (9500 kg/305 days) Holstein-Friesian dairy cows were studied. Ovaries of cows were scanned twice a week from day 10 pp on, until ovulation/cyst formation. Milk yield data, body condition scores and blood samples, for determination of oestradiol-17beta, insulin, beta-OH-butyrate and non-esterified fatty acids, were collected simultaneously. Milk samples for progesterone analysis were collected daily. Four lactations were excluded from further analysis because of irregular pp ovarian cyclicity, excluding COD. Eight lactations (26.7%) developed a cyst, while 22 lactations ovulated < days 60 pp. Ovulation and cyst formation occurred at similar times pp. Metabolic and hormonal profiles did not differ between CYST and OV lactations during the first 3 weeks pp. In the final week prior to cyst formation/ovulation, insulin concentrations were lower in CYST than in OV lactations while no differences were observed for any of the other parameters tested. In two lactations, cyst formation was preceded by suprabasal progesterone and increased oestradiol-17beta concentrations. These results suggest that cyst formation in high-yielding dairy cows pp is associated with lower insulin levels but not with other distinct hormonal and metabolic alterations. However from this study, we cannot exclude the involvement of subtle hormonal and metabolic changes in the pathogenesis of ovarian cysts. Suprabasal progesterone, and altered oestradiol-17beta concentrations, seem to play a minor role in cyst formation.

New techniques for the assessment of canine semen quality: a review.

Until recently, canine semen assessment was routinely performed by conventional light microscopic techniques. The limitations of these methods include subjectivity, variability, the small number of spermatozoa analyzed, and poor correlation with fertilizing potential. The last decade, several new in vitro techniques have been introduced for canine semen assessment that enable a more detailed evaluation of several sperm characteristics. Numerous fluorescent staining techniques have been developed for the evaluation of specific sperm characteristics and functions, including plasma membrane integrity, capacitation status and the acrosome reaction. By combining fluorescent stains, several functional sperm characteristics can be assessed simultaneously. Moreover, by means of flow cytometry, large numbers of fluorescently labelled spermatozoa can be analysed in a short interval. Following thorough standardization and validation, computer-assisted sperm analysis systems provide objective and detailed information on various motility characteristics and morphometric dimensions that cannot be identified by conventional light microscopic semen analysis. In vitro assays, evaluating the capacity of canine spermatozoa to bind to the zona pellucida or oviductal explants, or to penetrate the oocyte, provide additional information on canine gamete interaction that may be useful in predicting the fertilizing potential of spermatozoa. Although substantial improvements have been made in canine semen assessment, surprisingly few parameters were correlated with in vivo fertility. Therefore, further research is required to determine which sperm characteristics are of clinical value for predicting the in vivo fertility in dogs.

Effect of non-esterified fatty acids on bovine granulosa cell steroidogenesis and proliferation in vitro.

In high-yielding dairy cows, the negative energy balance (NEB) during the first weeks post partum may influence dominant follicle growth and steroidogenesis. Since non-esterified fatty acid (NEFA) concentrations are elevated during NEB and are shown to be toxic for several cell types, we investigated the individual and combined effects of the three main NEFA's on granulosa cell proliferation and steroidogenesis in vitro. Granulosa cells from large follicles were cultured for two days in serum free medium in the presence of palmitic (C16:0) (PA), stearic (C18:0) (SA) and/or oleic acid (C18:1) (OA). Addition of 150, 300 or 500 microM of PA and SA inhibited cell proliferation (P<0.05) while OA only elicited such an effect at 500 microM (P<0.01). In the combination treatment (150 microM of each fatty acid), cell numbers were also reduced (P<0.01). These inhibitory effects on cell number are partly due to the induction of apoptosis by these NEFA's, as was demonstrated by annexin V-FITC/propidium iodide staining of the granulosa cells. Oestradiol-17beta production was stimulated by all doses of PA, by 300 and 500 microM of SA and by 500 microM of OA (P<0.05). Combined treatment with 150 microM of each fatty acid also stimulated oestradiol-17beta production per 10(4) cells (P<0.05). We can conclude that PA, SA and to a lesser degree OA modulate granulosa cell proliferation and steroidogenesis in vitro. These effects may be involved in the occurrence of ovarian dysfunction during the postpartum period in high-yielding dairy cows.

Sperm distribution in the genital tract of the bitch following artificial insemination in relation to the time of ovulation.

In the present study, sperm distribution in the genital tract of the bitch following artificial insemination (AI) in relation to the time of ovulation was investigated by histology, scanning electron microscopy (SEM) and flushing. Ten bitches were inseminated intravaginally with 500 x 10(6) spermatozoa: three dogs before ovulation, four dogs during ovulation and three dogs after ovulation. Ovariohysterectomy was performed 24 h after AI. Half of the genital tract was divided into nine segments (cervix, corpus uteri, caudal, middle and cranial uterine horn (UTH), utero-tubal junction (UTJ), isthmus, ampulla and infundibulum), which were processed for histology and SEM. The contralateral UTH and uterine tube (UT) were flushed, and several sperm characteristics were assessed. Histology revealed that the spermatozoa were mainly located in the uterine glands and at the UTJ, while very few spermatozoa were detected in the UT. Insemination during ovulation resulted in higher percentages of glands with spermatozoa in the different parts of the uterus (P < 0.05). Evaluation by SEM showed higher numbers of spermatozoa in several parts of the uterus for bitches inseminated during ovulation (P < 0.05). The mean number of spermatozoa flushed from the UTH and the UT was low. No significant differences in the evaluated sperm quality parameters were found between the flushings of the UTH and the UT. In conclusion, based on our findings, the uterine glands and the UTJ might act as sperm reservoirs in the bitch and sperm transport in the genital tract is affected by the time of AI in relation to ovulation.

The use of epostane in an attempt to reproduce cystic endometrial hyperplasia in the bitch.

The aetiology and pathogenesis of spontaneous cystic endometrial hyperplasia (CEH) in the bitch is not yet completely understood. Recent research based on the expression of uterine sex hormone receptors in spontaneous cases of CEH suggested that a temporary progesterone deficiency during late oestrus-early metoestrus may be responsible for the onset of CEH development. In the present study a temporary progesterone deficiency during late oestrus-early metoestrus was experimentally created using an inhibitor of progesterone synthesis, epostane. At day 49 of metoestrus, there was a significant reduction in the size of the uterine wall, mainly due to endometrial atrophy, and there was also a significant increase in the mucus-filled uterine lumen in the bitches that had been treated with epostane compared to the control bitches. No significant differences in the expression of sex hormone receptors was observed between the two groups. As no CEH developed in the epostane-treated bitches, an additional oestrogenic stimulus may be required to stimulate endometrial proliferation. Therefore, it is suggested that deficient luteinization of the corpus luteum may be the trigger in the pathogenesis of CEH, as the secretion of varying amounts of sex steroids depends on the degree of luteinization.

Cell-specific distribution of progesterone receptors in the bovine ovary.

This study describes the localization of progesterone receptors (PR) in the bovine ovary. Ovaries were obtained from 11 non-pregnant and two pregnant cows. Progesterone receptors were visualized by immunohistochemistry on paraffin sections. Nuclear staining for PR was observed in cells of the follicles, corpora lutea, theca layers, surface epithelium, tunica albuginea, and in superficial and deep stroma cells. No staining was noticed in apoptotic bodies of atretic follicles. Expression of PR in follicle cells indicates an intrafollicular role of progesterone. The higher expression in thecal cells compared with follicle cells indicates that thecal cells mediate some effects of progesterone on the follicular development. Superficial stroma cells showing high expression might have a similar influence on primordial and primary follicles. In general, luteal cells had a lower expression than follicle cells, which may be explained by the down-regulatory effect of locally produced progesterone. The lower expression in luteal cells during pregnancy can be due to the longer life span of this corpus luteum and concomitant degeneration of its PR. The high and rather constant expression of PR in cells of the surface epithelium remains to be elucidated.

Cell-specific distribution of oestrogen receptor-alpha in the bovine ovary.

In this study, the expression of estrogen receptor alpha (ERalpha) in the bovine ovary is described. ERalpha was visualized by immunohistochemistry on paraffin sections of ovaries obtained from 11 non-pregnant and 2 pregnant animals. In general, ERalpha 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 ERalpha was obvious. Intermediate to high ERalpha expression was present in thecal cells and in cells of the superficial and deep stroma, tunica albuginea and surface epithelium. Furthermore, the expression of ERalpha 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 ERalpha expression in pregnant cows was in general, lower than in non-pregnant cows with similar plasma progesterone levels. The relatively high expression of ERalpha 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 ERalpha in the bovine ovary together with the cycle-dependent variations in ERalpha expression remain to be elucidated.

Changes in sex hormone receptors during administration of progesterone to prevent estrus in the bitch.

The development of lesions and the changes in sex hormone receptors were studied in the uteri of bitches under progesterone treatment. Twelve weeks after the onset of treatment, there was atrophy of the endometrium and increased thickness of the myometrium, without cystic dilatation of endometrial glands. This was accompanied by a dramatic reduction in estrogen-alpha and progesterone receptors in all cell types of the uterine wall. By 24 weeks after the onset of treatment, however, the endometrium was thickened due to the development of cysts of endometrial glands, while the myometrium thickness had returned to normal. The estrogen-alpha and progesterone receptors in most cell types of the uterine wall were again within the normal range. These results clarify and reconcile some apparent contradictions in the literature. They show that sex hormone receptors in most cell types of the uterine wall, especially endometrial gland cells and stromal cells, escape progestin (down) regulation after prolonged exogenous administration of progesterone.

Cell-specific distribution of progesterone receptors in the bovine ovary.

This study describes the localization of progesterone receptors (PR) in the bovine ovary. Ovaries were obtained from 11 non-pregnant and two pregnant cows. Progesterone receptors were visualized by immunohistochemistry on paraffin sections. Nuclear staining for PR was observed in cells of the follicles, corpora lutea, theca layers, surface epithelium, tunica albuginea, and in superficial and deep stroma cells. No staining was noticed in apoptotic bodies of atretic follicles. Expression of PR in follicle cells indicates an intrafollicular role of progesterone. The higher expression in thecal cells compared with follicle cells indicates that thecal cells mediate some effects of progesterone on the follicular development. Superficial stroma cells showing high expression might have a similar influence on primordial and primary follicles. In general, luteal cells had a lower expression than follicle cells, which may be explained by the down-regulatory effect of locally produced progesterone. The lower expression in luteal cells during pregnancy can be due to the longer life span of this corpus luteum and concomitant degeneration of its PR. The high and rather constant expression of PR in cells of the surface epithelium remains to be elucidated.

Estrogen-alpha and progesterone receptor expression in cystic endometrial hyperplasia and pyometra in the bitch.

Estrogen-alpha receptor (ER) and progesterone receptor (PR) were examined immunohistochemically in uteri of normal bitches, in uteri of bitches with cystic endometrial hyperplasia-mucometra (CEH-M) and in uteri of bitches with endometritis-pyometra (E-P), under exogenous progesterone treatment. In the CEH-M group, the ER- and PR-scores of all uterine cell types were higher than the ER- and PR-scores of normal uteri, although these differences were not always statistically significant. The ER-scores of E-P group were significantly lower than the ER-scores of the normal uteri and CEH-M group. The PR-scores of the E-P group tended to be higher than the PR-scores of the normal uteri, except for the surface epithelium, although these differences were not statistically significant. Exogenous progesterone treated bitches with CEH-M or E-P showed reduced ER- and PR-scores in the different uterine cell types, compared with the corresponding nontreated CEH-M or E-P group. The differences in ER and PR expression between CEH-M and E-P suggest different factors in the pathogenesis of both entities. Although, these changes in ER and PR expression do not seem to be directly involved in the pathogenesis of CEH-M and E-P. It is suggested that for CEH-M and progestin induced CEH-M a hormone dependent pathway is responsible. For P, the trigger may be bacterial infection.

[Cystic ovarian disease in dairy cattle: aetiology, pathogenesis, and risk factors]. / Cysteuze ovariële follikels bij melkvee: etiologie, pathogenese en risicofactoren.

Cystic ovarian disease (COD) is an important ovarian dysfunction in dairy cattle, especially during the early postpartum period. The endocrinology and the symptoms of this disorder vary widely due to the many different forms of cysts that exist. For these reasons, there is currently no clear and unambiguous definition of COD. When ovulation does not occur, a follicle may evolve into an ovarian cyst. Folliculogenesis and ovulation are regulated by the hypothalamic-pituitary-gonadal axis. A dysfunction can occur at different levels of this neuroendocrine system, causing COD. The primary factor is thought to be a deficient luteinizing hormone surge prior to ovulation. What causes this alteration is not yet known. Many factors increase the incidence of COD and are involved in the very complex pathogenesis of this disease.

Immunolocalization of androgen receptors in the canine ovary and their relation to sex steroid hormone concentrations.

Androgens play an essential role as autocrine or paracrine agents in ovarian follicular growth, maturation and luteinization. The aim of this study was to describe the normal cellular distribution of androgen receptors in the canine ovary at different stages of the oestrous cycle. Samples of both ovaries were obtained from 34 dogs, including six pregnant animals and three that had just produced litters. Presence of androgen receptors was visualized by immunohistochemistry on paraffin wax sections using a polyclonal antibody. Nuclear staining for androgen receptors was observed in the surface epithelium, cortical tubules, rete ovarii, follicle cells, thecal cells, luteal cells, granulosa cell cords and ovarian stroma, indicating that androgens have important roles in ovarian function in bitches. In theca interna cells of tertiary follicles, androgen production seems to be more important than androgen receptivity, as immunostaining for androgen receptors in these cells was weak compared with that in other ovarian stromal cells. In primordial and primary follicles, the immunostaining for androgen receptors was rather weak, indicating that androgens are of minor importance in early preantral follicles. In follicle cells of larger preantral and antral follicles, the immunostaining for androgen receptors increased with the stage of the follicle. Corpora lutea expressed less immunostaining, which was not correlated with serum progesterone concentrations, although local actions of progesterone on androgen receptors in corpora lutea cannot be excluded. In general, few correlations were found between immunostaining for androgen receptors and serum sex steroid concentrations, indicating that other factors regulate androgen receptors in the canine ovary.

Immunolocalization of progesterone receptors in the canine ovary and their relation to sex steroid hormone concentrations.

The aim of the present study was to describe the normal cellular distribution of progesterone receptors in the canine ovary at different stages of the oestrous cycle. Samples of both ovaries were obtained from 75 healthy adult bitches of various breeds and ages, including five pregnant bitches and three bitches that had just delivered. The presence of progesterone receptors was visualized by immunohistochemistry on paraffin wax sections using a monoclonal antibody. Nuclear staining for progesterone receptors was observed in the surface epithelium, cortical tubules, rete ovarii, follicle cells, thecal cells, luteal cells, granulosa cell cords and ovarian stroma. The staining intensity for progesterone receptors in the follicle cells increased with the stage of follicle development, indicating an intrafollicular role of progesterone in the mechanism of ovulation and luteinization. The stronger staining intensities for progesterone receptors in thecal cells compared with follicle cells may be explained by the fact that thecal cells mediate some effects of steroid hormones on the follicle cells in secondary and tertiary follicles. Little correlation was found between the expression of progesterone receptors in follicle cells and oestradiol, progesterone or testosterone concentrations. This finding indicates a different regulating mechanism for progesterone receptors in canine ovarian follicles compared with other tissues of the genital tract. During pregnancy all groups of ovarian cells had lower staining intensity scores than during the oestrous cycle, although the sex steroid hormone concentrations in pregnant bitches were similar to those in non-pregnant bitches during the luteal phase of the oestrous cycle. The lower expression of progesterone receptors during pregnancy may be due to higher tissue concentrations of progesterone that are not reflected in the serum because of haemodilution and increased metabolism and clearance during pregnancy.

Cystic endometrial hyperplasia-pyometra complex in the bitch: should the two entities be disconnected?

The uteri of 26 clinically healthy bitches and 42 bitches with a clinical suspicion of pyometra were examined histologically using a computerized image analysis system. Histologic lesions were characterised mainly by thickening or atrophy of the endometrium and by varying degrees of cystic changes of the glands. These lesions were observed in most of the clinically healthy bitches as well as in all of the clinically ill animals. In most of the ill bitches a variable degree of inflammation also was found. Some bitches with clinical signs indicative for pyometra had no inflammatory reaction in the uterus. These bitches were misdiagnosed as suffering from pyometra, confirming the difficulty of diagnosing pyometra by simple clinical examination. Determination of sex hormone serum levels revealed that all dogs in both groups were either in metestrus or in anestrus. Based on the results of this study the cystic endometrial hyperplasia-pyometra complex can be divided in two entities: a cystic endometrial hyperplasia-mucometra complex and an endometritis-pyometra complex. Both entities bear many similarities with each other, except for the inflammatory reaction in the endometritis-pyometra complex. It is concluded from this study that the latter complex probably does not necessarily follow the former, but that both can arise de novo.

Risk factors for post partum ovarian dysfunction in high producing dairy cows in Belgium: a field study.

An epidemiological study of risk factors for postpartal ovarian disturbances was carried out on 334 high-yielding dairy cows in 6 well-managed Belgian herds. Ovarian activity was closely monitored using progesterone profiles, based on twice weekly RIA-analysis for progesterone in milk fat, starting at 10 d after calving and continuing until the confirmation of a new pregnancy. Attention was focused on abnormal cyclicity during the preservice, postpartum period; cows were divided into 6 different categories. Three of these categories (normal profile, delayed cyclicity, and prolonged luteal phase) were of major importance and were analyzed using a multiple variable logistic regression model. Season of calving (stable vs pasture, odds ratio (OR)=5.7), an extended length of the previous dry period (> 77 vs < or = 63 d, OR=2.9), problem calvings (OR=3.6), abnormal vaginal discharge (OR=4.5), health problems during the first month of lactation (clinical disease, OR=5.4; ketosis, OR=11.3), and clinical parameters illustrating the appearance of a severe negative energy balance significantly increased the risk for delayed cyclicity before service. Parity (> or = 4 vs 1, OR=2.5), problem calvings (OR=2.9), occurrence of puerperal disturbances (OR ranged from 3.5 to 11.0), health problems during the first month of lactation (OR=3.1), and an early resumption of ovarian cyclicity after calving (< 19 d vs > 32 d, OR=2.8) increased the risk for prolonged luteal cycles before service.

Immunohistochemical detection of progesterone receptors in the canine uterus and their relation to sex steroid hormone levels.

The aim of this immunohistochemical study is to describe the normal distribution of progesterone receptors in the various cell types of the canine uterine horns, body and cervix. The results can be used for research on uterine and endocrinological pathology, since the impact of progesterone on different uterine cell types is partly determined by the receptor availability. Nuclear staining for progesterone receptors was observed in epithelial cells of the surface epithelium, glandular ducts and basal glands of the endometrium, in endometrial stroma cells and in myometrial smooth muscle cells. This staining was positively correlated with the estradiol-17 beta:progesterone ratio, and reflects the positive effect of estradiol-17 beta and the negative influence of progesterone on the receptors. Staining scores were high during proestrus and decreased through estrus to early metestrus. In late metestrus, staining scores of the stromal and smooth muscle cells increased again. In anestrus, high scores of the surface-epithelial cells contrasted with minimal scores of the basal glands. This finding suggests a different hormonal regulation of the progesterone receptor expression in both epithelial cell groups. The higher staining intensities for progesterone receptors in stromal cells compared with epithelial cells might be explained by the fact that stromal cells mediate some effects of steroid hormones on the epithelial cells in the genital tract. Therefore, the role of stromal cells in regulation of the cyclic endometrial changes and in pathologic changes of uterine tissue should not be underestimated.

Insulin resistance: the link between metabolic disorders and cystic ovarian disease in high yielding dairy cows?

A preliminary investigation was performed to examine whether insulin resistance is a factor in the pathogenesis of cystic ovarian disease (COD) in high-yielding dairy cows. In total 30 cows, of which 15 were diagnosed as suffering from COD based on the anamnesis and clinical examination, and the other 15 served as matched controls, were subjected to an intravenous glucose tolerance test (IVGTT). The aim of the study was to investigate whether insulin activity was altered in COD cows. Differences in glucose clearance between the COD cows and their controls were analyzed comparing the fractional turnover rate (k), the glucose half-time (T1/2), and the area under the curve (AUC) 60 and 120 min after infusion. Differences in insulin response were analyzed comparing the insulin increment, the insulin peak concentration, and the AUC 60 and 120 min after glucose infusion. Although insulin resistance, attended by a secondary hyperinsulinemia, is stated to directly contribute to the ovarian abnormalities that characterize the polycystic ovary syndrome (PCOS) in human medicine, this was not observed in COD cows. On the contrary, COD cows appeared to have a low insulin response following an intravenous glucose load as compared with their matched controls. This was illustrated by significantly lower insulin increments (P = 0.04) and lower insulin peak concentrations (P = 0.04). As COD cows had a significantly lower insulin response to a standard glucose load, it was concluded that insulin could be a factor in the pathogenesis of COD in dairy cows.

Immunohistochemical detection of estrogen receptors in the canine uterus and their relation to sex steroid hormone levels.

Cyclic changes in estrogen receptor expression in the uterine tissue of 60 female dogs were evaluated, using an immunohistochemical technique on formalin-fixed paraffin-embedded sections. The expression of estrogen receptors in the uterine horns, body and cervix was quantified by means of an immunohistochemical score. A negative correlation was found between staining scores in the uterine horns and serum progesterone levels. Generally, staining scores in the uterine horns were highest during proestrus, declined during estrus and were lowest during early metestrus. During anestrus high staining scores for estrogen receptors were observed, indicating sensitivity for estrogens in a sexual quiescence stage. Compared with the uterine horns, high staining scores were found in the uterine body and cervix during estrus and metestrus. No positive staining for estrogen receptors was detected in 1 pregnant uterus. Fluctuations in estrogen receptors were more pronounced in endometrial stroma cells than in epithelial cells of the uterine horns. The importance of stromal cells in the sexual cyclicity of the canine uterus should not be underestimated when studying uterine endocrinology and pathology.