Immunohistochemical demonstration of cyclooxygenase-2 (COX-2) and prostaglandin receptors EP2 and FP expression in the bovine intercaruncular uterine wall around term

During parturition, uterine-derived prostaglandins (PG) play an outstanding role regarding the functional elimination of the corpus luteum and the promotion of uterine contraction. The rate-limiting enzyme cyclooxygenase-2 (COX-2), highly regulated in a cell-type and localization specific manner throughout pregnancy, is involved in uterine prostanoid production. Prostaglandins exert their effects via G-protein-coupled receptors. Distribution and cellular localization of these receptors are decisive factors for prostaglandin-mediated actions. Since both COX-2 and PG receptors have only been assessed during pregnancy in the cow, these parameters were localized immunohistochemically near term to evaluate their specific role at parturition. Thus, during two periods, segments of the intercaruncular uterine wall were collected from cows at slaughter being eight and nine months pregnant, from cattle during caesarean section, and after spontaneous calving.

Results reveal that COX-2 was mainly localized in the cytoplasm of surface epithelial cells with a high expression in animals with induced parturition. The enzyme could also be found in lower concentrations within the glandular epithelium without any effect of gestational time or labour. In contrast to relaxant prostaglandin E receptor type 2 (EP2), not showing any change in all tissue layers observed, contractile prostaglandin F₂ receptor (FP) was modulated during the peripartal period revealing a peak expression in animals with induced parturition. FP was localized in surface and glandular epithelial cells as well as in endometrial stroma and myometrial smooth muscle cells.

Our study indicates that labour and induction of parturition may have an effect on amounts of immunohistochemically detectable COX-2 and FP. EP2 remains rather unchanged during the peripartal period. COX-2 and FP thus contribute via changes in amount and distribution to mechanisms associated with parturition.     

Pregnancy effects on distribution of progesterone receptors, oestrogen receptor alpha, glucocorticoid receptors, Ki-67 antigen and apoptosis in the bovine interplacentomal uterine wall and foetal membranes

Until recently, studies dealing with the uterus of the pregnant cow focus primarily on the placentome or on early and late pregnancy. Thus, there is a paucity of information about many aspects of the interplacentomal uterine wall including adherent foetal membranes. Corresponding tissue specimens were collected at the slaughterhouse and in animals undergoing premature caesarean section. Two specimens per month of pregnancy were assessed immunohistochemically for progesterone receptors, oestrogen receptor alpha and glucocorticoid receptors, Ki-67 protein and TUNEL procedure was performed. The latter two methods were employed in three animals each per months 1 and 2, 3 and 4, 7 and 8 and in six animals undergoing caesarean section at days 274 and 275 post insemination or during spontaneous labour. Results indicate that proliferation and apoptosis are of minor importance for tissue homeostasis since both can histochemically be detected only sporadically. Thus, at the sites investigated here, cellular hypertrophy plays an important role for tissue growth during pregnancy. Progesterone receptors, oestrogen receptor alpha and glucocorticoid receptors, however, exhibit cell type and pregnancy stage specific distribution patterns within the tissues assessed. Progesterone receptor immunoreactive scores remained fairly unchanged during pregnancy. Oestrogen receptor alpha scores, however, generally decreased and glucocorticoid receptors increased with ongoing gestation. Progesterone receptors and oestrogen receptor alpha were present in endometrial stroma and in myometrial smooth muscle cells during whole pregnancy. Oestrogen receptor alpha was detectable during whole pregnancy also in uterine glands. Progesterone receptors were, however, present at a very low level at the latter site only during months 1-3 and 6-9. Oestrogen receptor alpha and glucocorticoid receptors may also mediate uterine blood flow since they were present in the tunica media of uterine blood vessels. Results of the present study indicate, that progesterone and its receptor play an important role during whole gestation, mainly for uterine quiescence. Glucocorticoids and their receptors - possibly in cooperation with oestrogens and decreasing amounts of the oestrogen receptor alpha - should trigger processes initiating parturition, such as endometrial prostaglandin production. Further studies - including the periparturient period - should help to understand the exact role of the extraplacental compartment of the uterine wall for the initiation and progress of parturition.     

Regulation of progesterone receptors and decidualization in uterine stroma of the estrogen receptor-alpha knockout mouse

Regulation of progesterone receptor (PR) in uterine stroma (endometrial stroma plus myometrium) by estrogen was investigated in estrogen receptor-alpha (ERalpha) knockout (alphaERKO) mice. 17 beta-Estradiol (E(2)) increased PR levels in uterine stroma of ovariectomized alphaERKO mice, and ICI 182 780 (ICI) inhibited this E(2)-induced PR expression. Estrogen receptor-beta(ER beta) was detected in both uterine epithelium and stroma of wild-type and alphaERKO mice by immunohistochemistry. In organ cultures of alphaERKO uterus, both E(2) and diethylstilbestrol induced stromal PR, and ICI inhibited this induction. These findings suggest that estrogen induces stromal PR via ERbeta in alphaERKO uterus. However, this process is not mediated exclusively by ERbeta+, because in ERbeta knockout mice, which express ERalpha, PR was up-regulated by E(2) in uterine stroma. In both wild-type and alphaERKO mice, progesterone and mechanical traumatization were essential and sufficient to induce decidual cells, even though E(2) and ERalpha were also required for increase in uterine weight. Progesterone receptor was strongly expressed in decidual cells in alphaERKO mice, and ICI did not inhibit decidualization or PR expression. This study suggests that up-regulation of PR in endometrial stroma is mediated through at least three mechanisms: 1) classical estrogen signaling through ERalpha, 2) estrogen signaling through ERbeta, and 3) as a result of mechanical stimulation plus progesterone, which induces stromal cells to differentiate into decidual cells. Each of these pathways can function independently of the others.     

Effect of plane of nutrition on endometrial sex steroid receptor expression in ewes

The effect of plane of nutrition on progesterone receptor (PR) and estrogen receptor alpha (ERalpha) expression in ovine endometrium was investigated. Rasa Aragonesa ewes (n=26) were fed diets to provide either 1.5 (Group C) or 0.5 (Group L) times the daily maintenance requirement and were slaughtered at Days 5 or 10 of the estrous cycle (Day 0=estrus). PR and ERalpha immunoreactivity were analyzed in eight endometrial cell compartments, defined by cell type and location. Group L had less PR immunostaining on Day 5 (P<0.05), which is consistent with lesser endometrial content of progesterone found in such animals. Most cell types of Group C had down regulation of PR at Day 10, but in Group L, this pattern was observed only in three cell compartments. The lesser PR contents found at Day 5 in Group L ewes may explain the lack of inhibition of PR. No effect of treatment or day of the estrous cycle was observed in ERalpha. Results indicate that endometrial PR is affected in a cell type, in specific manner, by plane of nutrition.     

Progesterone receptor,estrogen receptor alpha,and the type II glucocorticoid receptor are coexpressed in the same neurons of the ovine preoptic area and arcuate nucleus: a triple immunolabeling study

The neuroendocrine reproductive and stress axes are known to be closely linked, but the mechanisms underlying these links remain poorly understood. In the ovine brain, GnRH neurons do not contain type II glucocorticoid (GR), progesterone (PR), or alpha estrogen (ERalpha) receptors. We sought to determine whether PR, ERalpha, and GR coexist within the same hypothalamic neurons. A triple immunocytochemical study, involving antisera raised in three different species, was performed on cryostat sections from ovariectomized ewes treated either with estradiol and progesterone or with progesterone alone. All PR-immunoreactive neurons contained ERalpha, and about 95% of ERalpha were PR immunoreactive in the preoptic area and arcuate nucleus. Although the PR with ERalpha colocalization ratio was not affected by the steroid treatments, immunolabeling for PR was weaker in animals that did not receive estradiol. Numerous PR- and ERalpha-immunoreactive cells contain GR. PR+ERalpha+GR-immunoreactive cells represent 70% of PR, 65% of ERalpha, and 72% of GR in the preoptic area and 70% of PR, 66% of ERalpha, and 63% of GR in the arcuate nucleus. These results suggest that estrogen, progesterone, and glucocorticoids may influence the activity of the same neurons to modulate both reproductive and stress axes.     

Uterine oxytocin receptors in cyclic and pregnant cows.

Binding of [3H]oxytocin to uterine subcellular preparations ('oxytocin receptor concentrations') was measured in uterine tissue of heifers and multiparous dairy cows at various stages of the oestrous cycle and during early pregnancy. A method for the assay of ovine uterine oxytocin receptors was optimized for use on bovine tissue. Oxytocin receptor concentrations were increased in cyclic animals around the period of luteolysis and oestrus, rising on Day 15 in endometrium and on Day 17 in myometrium while pregnant animals showed no comparable rise. Receptor concentrations then declined on Day 3 after oestrus in myometrium and on Day 5 in endometrium. Some cyclic animals did not show the expected rise in receptors in the late luteal phase; these animals had abnormally high progesterone concentrations for this stage of the cycle. In animals slaughtered on Day 18 after oestrus and/or insemination which had low oxytocin receptor levels, plasma progesterone concentrations were consistently high; while all animals showing the late luteal phase elevation in receptor values had low progesterone concentrations. Oxytocin receptor and progesterone concentrations were negatively correlated (P less than 0.05). These data support the hypothesis that oxytocin receptor level is a key factor in the process of luteolysis in cattle and that in pregnancy there is suppression of uterine oxytocin receptor at the expected time of luteolysis. We suggest that uterine oxytocin receptor levels are partly controlled by circulating steroid hormones and are suppressed during early pregnancy.     

Expression of prostaglandin transporter in the bovine uterus and fetal membranes during pregnancy

Uteroplacental prostaglandins (PGs) play pivotal roles in the maintenance and termination of pregnancy in mammals. In the present study, we have characterized the expression of prostaglandin transporter (PGT) in placentome caruncles, intercaruncular tissues, fetal membranes, and utero-ovarian plexus during pregnancy in cattle. Pregnant bovine uteri were collected and classified into six groups covering the entire gestational length. In caruncles and intercaruncular tissues, PGT mRNA (also known as SLC02A1) and PGT protein were highly expressed at the late stage of pregnancy compared to the early and mid stages, whereas the level of expression is constant and low in fetal membranes throughout pregnancy. PGT mRNA and PGT protein were expressed at a constant level in the utero-ovarian plexus both ipsilateral and contralateral to corpus luteum throughout the course of pregnancy. Overall, the relative expression of PGT mRNA and PGT protein were higher in caruncles than in intercaruncular tissue and fetal membranes, whereas no differences were detected between intercaruncular tissues and fetal membranes at any stage of gestation. Immunohistochemistry indicated that PGT was preferentially expressed in caruncular epithelial cells of placentomes and endometrial luminal epithelial and myometrial smooth muscle cells of the intercaruncular regions. The level of PGT expression was comparatively higher in maternal components than in fetal components. In conclusion, differential spatiotemporal tissue-specific expression of PGT in uterine and intrauterine tissues suggests a role for this transporter in the exchange of PGs between the maternal and the fetal compartments, as well as for intrauterine metabolism of PGs during pregnancy.     

Effects of dystocia, fetotomy and caesarian sections on the liver enzymes activities and concentrations of some serum biochemical parameters in dairy cattle

The aim of the present study was to determine the level of serum liver enzymes, triglyceride and some metabolites in cows with or without difficulties during parturition. The second goal was to compare between the possible effects of caesarian section and fetotomy on these parameters. A total number of 24 native breed cows at full term were included in this study. Out of them, 8 gave normal parturition, 16 cows were admitted with dystocia. The group of dystocia was subdivided into two groups; fetotomy (n=8) and caesarian (n=8) group. In the caesarian group, 4 cows were with uterine torsion. Five blood samples were collected from each cow: directly pre-partum, during and just after delivery and at, 24, 48 and 72 h post-partum. Serum samples were used for determination of aspartate amino transferase (AST), glutamate dehydrogenase (GLDH), gamma glutamyl transferase (gamma-GT), creatine phosphokinase (CK), glucose, total bilirubin, cholesterol and triglyceride. The results showed that AST, GGT, GLDH and CK activities were significantly increased in the group with caesarian sections due to uterine torsion than the control and fetotomy groups. There were insignificant changes in serum GGT and GLDH activities between control, fetotomy and dystocia group without uterine torsion at pre-partum and at 24 and 48 h post-partum. At 72 h post-partum, there was a significant increase in GLDH activity without significant increase in serum GGT activity. The concentrations of cholesterol and triglycerides did not differ in cows with dystocia compared to normal cows. In conclusion, cattle subjected to caesarian section and especially those with uterine torsion are associated with hepatic dysfunction. On the other hand, fetotomy has no effect on hepatocellular damage. The type of parturition has no effect on the bilirubin, cholesterol and triglyceride concentrations just before parturition to the 3rd day post-partum. It is recommended to supply cows with liver supportive therapy after caesarian section with uterine torsion.     

Endometrial mRNA expression of oestrogen receptor alpha, progesterone receptor and insulin-like growth factor-I (IGF-I) throughout the bovine oestrous cycle

This study characterized endometrial expression of mRNAs of oestrogen and progesterone receptors (ER, PR) and insulin-like growth factor-I (IGF-I) during the oestrous cycle. Seven Holstein heifers that showed standing oestrus on the same day (day 0) were selected and blood samples for oestradiol (E2) and progesterone (P4) determinations by RIA were taken daily until day 23. Endometrial samples were taken by transcervical biopsies on days 0, 5, 12 and 19 for mRNA determination by solution hybridization. The highest endometrial mRNA levels of ERalpha and PR were observed at oestrus and a decline was observed already at day 5, which then decreased progressively at the end of the luteal phase. IGF-I mRNA levels were higher at day 0 and 5 than at day 12. At day 19, mRNA levels of ERalpha, PR and IGF-I were the lowest in heifers that were at the end of their luteal phase (n=4), but were high again in heifers which P4 levels were basal (n=3). The temporal changes in mRNA endometrial expression of ERalpha, PR and IGF-I and their relation to the changes in steroid concentrations during the bovine oestrus cycle are described.     

Dienogest is a selective progesterone receptor agonist in transactivation analysis with potent oral endometrial activity due to its efficient pharmacokinetic profile

Dienogest was introduced as an oral progestin. Yet its strong oral potency on endometrial activity is not clearly explained. To circumvent this situation, steroid hormone receptor profiling using transactivation assay and endometrial activity test in rabbits were carried out with determination of plasma drug concentration. Agonistic/antagonistic activity on human progesterone receptor (PR), androgen receptor (AR), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), estrogen receptor alpha (ERalpha), or estrogen receptor beta (ERbeta) were determined. Dienogest activate PR (EC50=3.4 or 10.5 nmol/l) with antagonistic activity on AR (EC50=420.6 or 775.0 nmol/l) but not agonistic nor antagonistic action on GR, MR (3000 nmol/l). Dienogest activate neither ERalpha nor ERbeta (3000 nmol/l). Progesterone activated PR with antagonistic activity on AR and on MR. Dydrogesterone showed a similar profile to progesterone. Norethisterone activated PR, AR, and ERalpha. Medroxyprogesterone acetate activated PR, AR, and GR. Danazol activated PR and AR. Collectively, dienogest has a good specificity to PR compared with the other drugs. By oral treatment, dienogest showed the strongest endometrial activity (ED50=0.0042 mg/kg) in McPhail test among other progestins (ED50 values for MPA, DYG, NES were 0.074, 1.9, >0.05 mg/kg, respectively). Dienogest showed higher plasma concentrations than those of the other progestins with higher doses. The estimated plasma concentration of dienogest at ED50 (3.66 nmol/l) was close to its EC50 value to activate PR. Thus, the stronger oral activity of dienogest could be explained simply by its in vitro potency on PR and its oral pharmacokinetic profile.     

Secretion of angiogenic activity by placental tissues of cows at several stages of gestation

Samples of maternal and fetal placental tissues were obtained from cows on Days 100 (N = 4), 150 (N = 5), 200 (N = 6) and 250 (N = 6) of gestation and incubated for 24 h. Conditioned media from caruncular explants were mitogenic for bovine aortic endothelial cells (BAEC) on all days of gestation. Media from intercaruncular endometrium were stimulatory for proliferation of BAEC on Day 100 but inhibitory on Days 150, 200 and 250. Media from cotyledonary and intercotyledonary tissues inhibited proliferation of BAEC on all days. Caruncular-conditioned media stimulated migration of BAEC on Days 150, 200 and 250. Cotyledonary-conditioned media inhibited migration of BAEC on all days. Effects of media from intercaruncular and intercotyledonary tissues on migration of BAEC varied with stage of gestation. Angiogenic activity of media from caruncular (all stages) and intercaruncular (Day 100) tissues appeared to have an Mr greater than 100,000. In cows, therefore, the maternal placentome (caruncle) appears to be the primary source of placental angiogenic activity throughout gestation. The fetal placentome (cotyledon) secretes activity which inhibits two major components of angiogenesis (proliferation and migration of endothelial cells) throughout gestation. Intercaruncular and intercotyledonary tissues may modulate placental angiogenesis throughout gestation. Placental vascular development in the cow is therefore probably controlled by an interaction between stimulatory and inhibitory factors produced by the placenta itself.     

Nuclear estrogen and progesterone receptors in the oviduct of heifers under natural and superovulated estrous cycles

Oviducts from 22 crossbred heifers were examined for the presence of nuclear estrogen (ERalpha) and progesterone (PR) receptors at different phases (estrus, metaestrus and diestrus) of naturally occurring estrous cycles and estrous cycles during which superovulation was induced. Receptors were detected by immunohistochemistry in the epithelial cells, connective tissue and muscular layer of oviductal infundibulum, ampulla, ampullary/isthmic transition and isthmus. Epithelial ERalpha was found along the entire oviduct regardless of the cycle phase and of the circulating concentrations of 17beta-estradiol (E(2)) and progesterone (P(4)). Epithelial PR was found mainly at the ampullary-isthmic transition and isthmus and more intensely at the estrus phase but their amount was not correlated with P(4) concentrations. ERalpha in the connective tissue was more abundant at the infundibulum and ampulla, regardless of the phase of the estrous cycle and of E(2) and P(4) circulating concentrations. PR in the connective tissue was found mostly at the ampulla, regardless of the estrous cycle phase but no correlations were found between amount and P(4) concentrations. ERalpha staining intensity in the muscular layer was similar at all phases of the estrous cycle and at all anatomical segments of the oviducts. However, PR staining was more intense at the isthmus during the metaestrus phase and it was negatively correlated with P(4) concentrations. In general, data from the present research suggest that P(4) exerts an inhibitory role upon ERalpha and PR. Also, no differences were found between animals subjected or not to superovulation.     

The activation function-1 domain of estrogen receptor alpha in uterine stromal cells is required for mouse but not human uterine epithelial response to estrogen

The activation function-1 (AF-1) domain of the estrogen receptor alpha (ERalpha) in stromal cells has been shown to be required for epithelial responses to estrogen in the mouse uterus. To investigate the role of the stroma in estrogenic responses of human uterine epithelium (hUtE), human/mouse chimeric uteri composed of human epithelium and mouse stroma were prepared as tissue recombinants (TR) that were grown in vivo under the renal capsule of female nude mouse hosts. In association with mouse uterine stroma (mUtS), hUtE formed normal glands surrounded by mouse endometrial stroma and the human epithelium influenced the differentiation of stroma into myometrium, such that a histologically normal appearing uterine tissue was formed. The hUtE showed a similar proliferative response and increase in progesterone receptors (PR) in response to 17beta-estradiol (E2) in association with either human or mUtS, as TRs. However, under identical endocrine and micro-environmental conditions, hUtE required 5-7 days exposure to E2 rather than 1 day, as shown for mouse uterine epithelium, to obtain a maximal proliferative response. Moreover, this extended length of E2 exposure inhibited mouse epithelial proliferation in the presence of mouse stroma. In addition, unlike the mouse epithelium, which does not proliferate or show regulation of PR expression in response to E2 in association with uterine stroma derived from mice that are null for the AF-1 domain of ERalpha, hUtE proliferates and PR are up-regulated in response to E2 in association genetically identical ERalpha knock-out mouse stromal cells. These results clearly demonstrate fundamental differences between mouse and human uterine epithelia with respect to the mechanisms that regulate estrogen-induced proliferation and expression of PR. Moreover, we show that genetically engineered mouse models could potentially aid in dissecting molecular pathways of stromal epithelial interactions in the human uterus.     

Uterine estrogen receptor alpha and progesterone receptor during the follicular and luteal phase in llamas

Estrogen receptor-alpha (ERalpha) and progesterone receptor (PR) were characterized in different endometrial cell types as luminal and glandular epithelium and stroma during the follicular (FP) and the luteal phase (LP) in llamas. Animals were examined daily by transrectal ultrasonography for the determination of the presence of an ovulatory follicle and ovulation was immediately induced by a GnRH injection (Day 0). Endometrial samples were obtained by transcervical biopsies from the left uterine horn on Day 0 (FP) and 9 days after the GnRH injection (Day 9, LP). Blood samples were collected on these days for estradiol 17beta and progesterone determination by RIA. An immunohistochemical technique was used to visualize ERalpha and PR immunostaining which was then analyzed by two independent observers. Total positive area and average staining for ERalpha were affected by the phase of the ovarian activity: in the three cell types there was more positive area and intense staining during the FP than during the LP. Similar findings were observed for PR, more positive stained areas were found during the FP than during the LP in the epithelia. In addition, the three cell types had more intense staining during the FP than during the LP. An effect of the cell type for ERalpha and PR was observed; epithelia (luminal and glandular) had more positive stained areas and greater intensity than stromal cells. In conclusion, the results of the present study suggest that in llamas, like in other ruminants, estradiol has a stimulatory effect while progesterone downregulates the ERalpha and PR and that the receptor is cell type specific.     

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.     

Concentration of oxytocin receptors in the placenta and fetal membranes of cows during pregnancy and labour.

The concentrations of oxytocin receptors were measured in intercaruncular and caruncular endometrium, fetal cotyledons, chorioallantois and amnion during pregnancy and parturition in cows. Tissues were obtained on days 20 (endometrium only), 50, 100, 150, 200, 225, 250, 275, at term (days 280-284), during labour and within 24 h after calving. Receptor concentrations in intercaruncular endometrium were low on day 20 of pregnancy, 39 +/- 11 fmol mg-1 protein. By day 50, receptor concentrations had increased more than tenfold to 572 +/- 52 fmol and rose steadily until day 250 and then levelled off at about 4500 fmol mg-1. Shortly before parturition, on day 282 +/- 1, a further rise to 7300 +/- 1418 fmol mg-1 was observed, these concentrations were maintained throughout labour. By contrast, caruncular endometrial receptor concentrations remained low until term, mean 145 +/- 15 fmol mg-1, and then rose to 720 +/- 163 fmol mg-1 during labour (cervix 17 cm--fully dilated). Fetal cotyledons and membranes had very low oxytocin receptor concentrations during most of pregnancy, on average only 20 fmol mg-1 protein. At term and during labour, receptor concentrations were significantly increased in both tissues. Mean concentrations during labour were 163 +/- 36 fmol mg-1 for cotyledons, 270 +/- 61 fmol mg-1 for chorioallantois and 311 +/- 121 fmol mg-1 for amnion.(ABSTRACT TRUNCATED AT 250 WORDS)