Expression of progesterone and oestrogen receptors by early intrauterine equine conceptuses

Progesterone and oestrogen play essential roles in the maintenance of pregnancy in eutherian mammals and are thought to exert their effects on the developing conceptus indirectly, via the endometrium. In some species, early embryos have themselves been shown to express steroid receptors, thereby suggesting that reproductive steroids may also influence embryonic development directly. The aim of this study was to determine whether early intrauterine equine conceptuses express either the classical intracellular progesterone (PR) and oestrogen receptors (ERalpha and ERbeta) or the more recently characterised membrane-bound progesterone receptors (PGRMC1 and mPR). Horse conceptuses recovered on days 7, 10 and 14 after ovulation (n=8 at each stage) were examined for steroid receptor mRNA expression using quantitative rtPCR. Where commercial antibodies were available (PR, ERbeta), receptor localisation was examined immunohistochemically in day 10, 12, 14, 15 and 16 conceptuses (n=2 at each stage). mRNA for PR, PGRMC1 and mPR was detected at all stages examined, but while PGRMC1 and mPR expression increased during the day 7-14 period, PR expression decreased. ERalpha mRNA was not detected at any stage examined, whereas ERbeta mRNA was detected in all day 14, some day 10 and no day 7 conceptuses. Immunoreactive ERbeta receptors were localised to the trophectoderm of day 14-16 conceptuses; PR were not detected immunohistochemically in conceptus tissue. In summary, this study demonstrates that equine conceptuses express mRNA and, in the case of ERbeta, protein for steroid hormone receptors during the period encompassing rapid conceptus growth, differentiation and maternal pregnancy recognition.     

Cloning of the novel isoform of the estrogen receptor beta cDNA (ERbeta isoform M cDNA) from the human testicular cDNA library

Our recent report has revealed the existence of the progesterone receptor (PR) isoform S, which consists of the novel PR exon S and exons 4-8 of the PR gene in the human testicular cDNA library. More recently, we have cloned the human estrogen receptor alpha (ERalpha) isoform S cDNA from the library. The ERalpha isoform S cDNA also contains the novel ERalpha exon S and exons 4-8 of the ERalpha cDNA. Based on these findings, we assumed that the novel isoform of cDNA like the PR- and ERalpha isoforms might exist in the human ER beta (ERbeta). In order to investigate this possibility, we have screened the human testicular cDNA library using the exons 4-8 corresponding sequence of the human ERbeta cDNA. Consequently, we have cloned a novel isoform of the ERbeta cDNA that consists of a previously unidentified 5'-sequence and the exons 5-8 of the ERbeta gene. We termed this isoform cDNA the "ERbeta isoform M cDNA". The 5'-sequence of the ERbeta isoform M cDNA was confirmed to be derived from a novel exon (termed the "exon M") by analysis of the genomic DNA. Moreover, we have analyzed the molecular size of the ERbeta isoform M encoded by the ERbeta isoform M mRNA by transient expression of the ERbeta isoform M cDNA in the 293T cell. The approximately 28 kDa protein, which was recognized by the anti-rat ERbeta antibody against the carboxyl-terminal region, was synthesized in the cells. Thus, we concluded that the ATG in the exon M could be used as the translation initiation codon. This report revealed for the first time the existence of the ERbeta mRNA isoform that is not caused by the skipping of one or more exons, by the alternative usage of the multiple exon 8s, nor by the alternative utilization of the untranslated 5'-exons located on the upstream region of the exon 1.     

Messenger RNA levels of estrogen receptors alpha and beta and progesterone receptors in the cyclic and inseminated/early pregnant sow uterus

The aim of the present study was to investigate differences in the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus at different stages of the estrous cycle as well as in inseminated sows at estrus and during early pregnancy by use of solution hybridization and in relation to plasma levels of estradiol and progesterone. Uterine samples were collected at different stages of the estrous cycle and after insemination/early pregnancy. In the endometrium, the expression of ERalpha mRNA and PR mRNA was similar for cyclic and early pregnant groups. Both were highest at early diestrus/70 h after ovulation and ERalpha mRNA was lowest at late diestrus/d 19 while PR mRNA was lowest at diestrus and late diestrus/d 11 and d 19. The expression of endometrial ERbeta was constantly low during the estrous cycle but higher expression was found in inseminated/early pregnant sows at estrus and 70 h after ovulation. In the myometrium, high expression of ERalpha mRNA and PR mRNA was observed at proestrus and estrus in cyclic sows and at estrus in newly inseminated sows. Higher expression of myometrial ERbeta mRNA was found in inseminated/early pregnant sows compared with cyclic sows, although significant only at estrus. In conclusion, the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus differed between endometrium and myometrium as well as with stages of the estrous cycle and early pregnancy. In addition to plasma steroid levels, the differences between cyclic and inseminated/early pregnant sows suggest that other factors, e.g. insemination and/or the presence of embryos, influence the expression of these steroid receptor mRNAs in the sow uterus.     

Phytoestrogen-mediated inhibition of proliferation of the human T47D breast cancer cells depends on the ERalpha/ERbeta ratio

This study investigates the importance of the intracellular ratio of the two estrogen receptors ERalpha and ERbeta for the ultimate potential of the phytoestrogens genistein and quercetin to stimulate or inhibit cancer cell proliferation. This is of importance because (i) ERbeta has been postulated to play a role in modulating ERalpha-mediated cell proliferation, (ii) genistein and quercetin may be agonists for both receptor types and (iii) the ratio of ERalpha to ERbeta is known to vary between tissues. Using human osteosarcoma (U2OS) ERalpha or ERbeta reporter cells it was shown that compared to estradiol (E2), genistein and quercetin have not only a relatively greater preference for ERbeta but also a higher maximal potential for activating ERbeta-mediated gene expression. Using the human T47D breast cancer cell line with tetracycline-dependent ERbeta expression (T47D-ERbeta), the effect of a varying intracellular ERalpha/ERbeta ratio on E2- or pythoestrogen-induced cell proliferation was characterised. E2-induced proliferation of cells in which ERbeta expression was inhibited was similar to that of the T47D wild type cells, whereas this E2-induced cell proliferation was no longer observed when ERbeta expression was increased. With increased expression of ERbeta the phytoestrogen-induced cell proliferation was also reduced. These results point at the importance of the cellular ERalpha/ERbeta ratio for the ultimate effect of (phyto)estrogens on cell proliferation.     

Estradiol receptor-alpha expression in hypothalamic and limbic regions of ewes is influenced by physiological state and maternal experience

The influence of estrus, pregnancy, parturition, and maternal experience on the expression of estrogen receptor-alpha (ERalpha) was investigated in hypothalamic and limbic regions of the sheep brain, using immunocytochemistry. Four days before parturition, previous maternal experience was associated with a higher density of ERalpha-labeled neurons in the paraventricular and supraoptic nuclei, the medial preoptic area, and the medial amygdala, but not in the mediobasal hypothalamus. Furthermore, an interaction was found between physiological state and experience in the peripartum period as the effect of experience existing 4 days prepartum was not found at parturition, when densities were lowest both in primiparous and in multiparous ewes. An additional effect of physiological state was also observed between parturition and estrus, densities being significantly lower at parturition than at estrus in the SON, PVN, and MPOA, but not in the medial amygdala. These results indicate that in sheep ERalpha expression is influenced by previous physiological and/or maternal experience at specific times of the reproductive cycle. They are also congruent with the higher ability of multiparous than nulliparous ewes to show maternal behavior several days prepartum.     

Quantitative analysis of estrogen receptor mRNA in human endometrium throughout the menstrual cycle using a real-time reverse transcription-polymerase chain reaction assay

We conducted a quantitative analysis of ERalpha and ERbeta mRNA expression in normal human endometrium throughout the menstrual cycle in regular menstruating premenopausal women, taking advantage of this real-time PCR assay. Endometrial dating was determined from the histology of the endometrium and classified into: proliferative endometrium and secretory endometrium. Both ERalpha and ERbeta mRNA expression were detected in all endometrial samples at both proliferative and secretion phase. However ERalpha mRNA expression level was higher than that of ERbeta specially during proliferative phase. These results suggest that estrogenic effects occur predominantly through ERalpha than ERbeta.     

Cellular turnover in the rat uterine cervix and its relationship to estrogen and progesterone receptor dynamics

Histoarchitectural changes of the uterine cervix allow its successful adaptation to different physiological conditions. In this study, we evaluated cell turnover in each cellular compartment of the uterine cervix in association with steroid hormone receptor expression in order to establish the range of physiological changes. Proliferation, apoptosis, and progesterone receptor (PR) and estrogen receptor alpha (ERalpha) expression were evaluated in cycling, pregnant, and postpartum rats. In estrus and diestrus II, ERalpha and PR expression exhibited variations according to the region evaluated. Proliferation and apoptosis showed a reciprocal pattern, the epithelium being the region with higher cell turnover. High apoptotic index (AI) in estrus was associated with the lowest ERalpha and the highest PR scores. During pregnancy, proliferation of the epithelium was the predominant event and AI was low. On Postpartum Day 1 (PPD1), proliferation decreased while apoptosis increased. As described for the estrous cycle, during pregnancy and PPD1, AI and ERalpha were negatively correlated. In the fibroblastic stroma, low proliferation was observed throughout pregnancy; however, there was a net increase in cell number because very few cells underwent apoptosis. No difference in ERalpha was observed in fibroblastic cells during pregnancy and postpartum; however, a great decrease of this receptor in the epithelial compartment was observed after delivery. Unlike cervical epithelium, PR was highly expressed in stromal cells. At term, a dramatic increase in epithelial PR was observed. While epithelial PR remained high on PPD1, a decrease was observed in muscle stroma. These results show that, in all stages studied, 1) ERalpha and PR have different patterns of expression with differential responses to signals that modulate proliferation and/or apoptosis depending on the cellular compartment, and 2) even though the epithelium is the region with the highest cell turnover, the fibroblastic and muscle stroma are active regions that have their own patterns of behavior.     

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.     

Behavioral and physiological changes in early-weaned multiparous and primiparous beef cows

Early weaning is used to advance postpartum ovulation in beef cows, but triggers a stress response, affecting cows' and calves' welfare. Our aim was to compare the response to early weaning in multiparous and primiparous cows. Seventy-one days after birth, calves from 14 primiparous and 11 multiparous cows were weaned. Since birth, cow's body weight (BW), body condition (BC), milk yield and calves' BW were recorded. Basophil/neutrophil relationship, total protein and albumin concentrations were measured before and after weaning. The distance between each cow and its calf before weaning, as well as main behaviors of the cows before and after weaning were recorded using 10 min scan sampling. BW and BC were greater in multiparous than in primiparous cows (P < 0.0001 for both). There was an interaction between parity (P = 0.004) and gender of the calf (P = 0.007): both BW and BC were greater in multiparous than primiparous cows, but multiparous cows with male calves had lower BW and BC than those with females (P < 0.001 for both variables). Milk yield was greater in multiparous than in primiparous cows (P = 0.02), and there was an interaction, with greatest milk yield in multiparous cows that calved males (P = 0.02). Total protein blood concentration increased with time (P < 0.0001), similarly in both groups. Albumin concentration profiles were similar despite parity, and decreased since 10 days after weaning (P < 0.0001). Basophil/neutrophil ratio was not affected by parity, and increased the day of weaning, remaining high until day 10 (P < 0.05). Frequency of standing decreased during the 2 days after weaning, with a greater decrease in multiparous than in primiparous cows (P < 0.0001). Pacing increased after weaning (P = 0.001), but increased significantly more in multiparous cows (P = 0.006), with a significant time after weaning by parity interaction (P = 0.01) reflected in greater frequencies in multiparous cows in the 2 days. Walking increased with time (P < 0.0001), and was more frequent in primiparous cows (P = 0.008). Ruminating, which was less frequent in multiparous cows (P = 0.014), decreased and remained low during the 3 days following weaning (P < 0.0001), but there was a significant time by parity interaction (P < 0.0001). The frequency of vocalizations increased dramatically after weaning (P < 0.0001), and was greater in multiparous cows (P = 0.006), and decreased more slowly during the 2nd and 3rd days after weaning (P = 0.005) in these animals. In conclusion, behavioral changes induced by weaning were greater in multiparous than primiparous cows, indicating a more intense response to cow-calf separation in the multiparous animals.