Tissue- and hormone- dependent progesterone receptor distribution in the rat uterus

BACKGROUND: Estradiol (E2) and progesterone (P) are well known regulators of progesterone receptor (PR) expression in the rat uterus. However, it is not known which receptor subtypes are involved. Little knowledge exist about possible differences in PR regulation through ERalpha or ERbeta, and whether the PR subtypes are differently regulated depending on ER type bound. Thus, in the present study PR immunostaining has been examined in uteri of ovariectomized (ovx) rats after different treatments of estrogen and P, in comparison with that in immature, cycling, and pregnant animals. METHODS: The uteri were collected from 1) ovx rats treated with E2 and/or P; 2) immature rats, intact cycling rats and animals pregnant day 8 and 18; 3) ovx rats treated with E2 or an estrogen receptor (ER)alpha agonist or an ERbeta agonist. Two antibodies were used, one detecting PRA+B and another one specific for PRB. Real-time PCR was used to determine mRNA levels for PRAB and PRB in experiment 3. RESULTS: In stroma and myometrium faint staining was detected in ovx controls (OvxC), whereas E2 treatment resulted in strong staining. In contrast to this, in luminal epithelium (LE) the staining was strong in the OvxC group, whereas E2 treatment during the last 24 hrs before sacrifice caused a decrease. Similar to OvxC the LE of the immature animals was strongly stained. In the pregnant rats LE was negative, well in agreement with the results seen after E2 treatment. In the pregnant animals the stroma and decidua was strongly stained for PRAB, but only faint for PRB, indicating that PRA is the most expressed isoform in this state. The increase in stromal and myometrial immunostaining after E2 treatment was also found after treatment with the ERalpha agonist PPT. The ERbeta agonist DPN caused a decrease of the PR mRNA levels, which was also found for PRAB and PRB immunostaining in the GE. CONCLUSION: Stromal and myometrial PRAB levels are increased via ERalpha, as shown by treatment with E2 and the ERalpha agonist PPT, while the levels in LE are decreased. The uterine stroma of pregnant rats strongly expressed PRAB, but very little PRB, which is different to E2 treated ovx animals where both PRAB and PRB are strongly expressed. The ERbeta agonist DPN decreased the mRNA levels of PRAB and PRB, as well as the PRAB protein level in GE. These results suggest that ERbeta signals mainly down-regulate PR levels in the epithelial cells. ERalpha, on the other hand, up-regulates PR levels in the stroma and myometrium while it decreased them in LE. Thus, the effects from E2 and PPT on the mRNA levels, as determined by PCR, could be annihilated since they are increased and decreased depending on cell type. The distribution and amount of PR isoforms strongly depend on the hormonal milieu and cell type within the rat uterus

Impaired leukocyte influx in cervix of postterm women not responding to prostaglandin priming

<p>Abstract</p> <p>Background</p> <p>Prolonged pregnancies are associated with increased rate of maternal and fetal complications. Post term women could be divided into at least two subgroups, one where parturition is possible to induce by prostaglandins and one where it is not. Our aim was to study parameters in cervical biopsies in women with spontaneous delivery at term (controls) and compare to those that are successfully induced post term (responders), and those that are not induced (non-responders), by local prostaglandin treatment.</p> <p>Methods</p> <p>Stromal parameters examined in this study were the accumulation of leukocytes (CD45, CD68), mRNAs and/or proteins for the extracellular matrix degrading enzymes (matrix metalloproteinase (MMP)-2, MMP-8 and MMP-9), their inhibitors (tissue inhibitor of MMP (TIMP)-1 and TIMP-2), interleukin-8 (IL-8), the platelet activating factor-receptor (PAF-R), syndecan-1 and estrogen binding receptors (estrogen receptor (ER)α, ERβ and G-coupled protein receptor (GPR) 30) as well as the proliferation marker Ki-67.</p> <p>Results</p> <p>The influx of leukocytes as assessed by CD45 was strongest in the responders, thereafter in the controls and significantly lower in the non-responders. IL-8, PAF-R and MMP-9, all predominantly expressed in leukocytes, showed significantly reduced immunostaining in the group of non-responders, while ERα and GPR30 were more abundant in the non-responders, as compared to the controls.</p> <p>Conclusion</p> <p>The impaired leukocyte influx, as reflected by the reduced number of CD45 positive cells as well as decreased immunostaining of IL-8, PAF-R and MMP-9 in the non-responders, could be one explanation of the failed ripening of the cervix in post term women. If the decreased leukocyte influx is a primary explanation to absent ripening or secondary, as a result of other factors, is yet to be established.</p

An immunohistochemical study on the regulation of estrogen receptor α\alpha by estradiol in the endometrium of the immature ewe

The effects of estradiol-17$\beta$ (E2) on the expression of estrogen receptor $\alpha$ (ER$\alpha$) in stromal and epithelial cells of endometrium in prepubertal lambs were investigated. Twenty three-month-old lambs were treated or not treated with one, two or three i.m. injections of E2 (1 $\mu$g$\cdot$kg$^{-1}$) in corn oil at intervals of 24 h. Lambs were slaughtered 12 or 24 h after the last injection. An immunohistochemical technique was used to visualize ER$\alpha$ immunostaining which was then analyzed quantitatively by a computer imaging analysis system. Seven endometrial compartments defined by cell type and location were analyzed separately. Positive staining of ER$\alpha$ was seen in the nuclei of stromal and epithelial cells. Glandular epithelium located next to the myometrium was stained more intensely than that next to the luminal epithelium and this phenomenon was maintained during treatment. Significantly less immunostaining was found in stromal cells 12 and 24 h after the first injection compared to the control group. A similar pattern was found in the glandular epithelium, although the decrease was more pronounced and the restoration of ER$\alpha$ was faster. This study shows that E2 treatment down regulates ER$\alpha$ in the endometrium temporarily in both stromal and epithelial cells, but the characteristics of this effect seems to be cell type specific.Étude immuno-cytologique de la régulation du récepteur aux œstrogènes ER$\alpha$ par l'œstradiol dans l'endomètre de la brebis impubère. Les effets de l'œstradiol-17$\beta$ (E2) ont été étudiés sur l'expression du récepteur aux œstrogènes ERa dans les cellules du stroma et de l'épithélium de l'endomètre chez la brebis impubère. Vingt agnelles âgées de 3 mois ont reçu ou non une, deux ou trois administrations intramusculaires de E2 (1 $\mu$g$\cdot$kg$^{-1}$ en solution huileuse) à intervalle de 24 h. Elles ont été sacrifiées 12 ou 24 h après la dernière injection. ER$\alpha$ a été mis en évidence par immuno-cytologie et sa quantification a été faite à l'aide d'un système d'analyse d'images. Sept compartiments de l'endomètre, définis par leur type cellulaire et leur localisation, ont été analysés séparément. Un marquage positif a été constaté dans les noyaux des cellules du stroma et de l'épithélium. L'épithélium glandulaire situé prés du myomètre est marqué plus intensément que celui qui borde la lumière utérine et ce phénomène est encore observé à la suite des traitements. Un marquage significativement plus faible est constaté dans les cellules du stroma 12 et 24 h après la première injection par comparaison aux témoins. Une réponse identique est vue dans l'épithélium glandulaire bien que la diminution de marquage ait été plus prononcée et la restauration de ER$\alpha$ plus rapide. Cette étude montre que E2 régule négativement les récepteurs ER$\alpha$ de l'endomètre dans les cellules du stroma et de l'épithélium et cet effet semble être spécifique du type cellulaire

A biphasic action of estradiol on estrogen and progesterone receptor expression in the lamb uterus

Regulation of the uterine expression of estrogen and progesterone receptors was studied in 20 three-month-old lambs that were not treated or treated with estradiol-17$\beta$. Determinations of receptors were performed by binding assays in the nuclear and cytosolic fractions, receptor mRNAs by solution hybridization, and estrogen receptor protein by an enzyme-immunoassay. Estradiol treatment decreased the receptor binding capacity of both receptors and the levels of immunoreactive estrogen receptor 12 h after injection in the absence of decreased receptor mRNAs, suggesting that the initial decrease is due to degradation of the proteins or that mRNAs are translated into new receptor proteins at a reduced rate. The mRNA levels increased after estradiol treatment suggesting that the replenishment phase consists of synthesis of new receptors rather than recycling of inactivated receptors.Action biphasique de l'œstradiol sur l'expression des récepteurs aux œstrogènes et à la progestérone dans l'utérus de l'agnelle. La régulation de l'expression des récepteurs aux œstrogènes et à la progestérone au niveau de l'utérus a été étudiée chez des agnelles immatures âgées de 3 mois ($n=20$), traitées et non traitées avec œstradiol-17$\beta$. Les déterminations des récepteurs ont été faites par des essais de liaison dans les fractions nucléaires et cytosoliques, les ARNm des récepteurs par la méthode d'hybridation en solution et la protéine du récepteur aux œstrogènes par un essai immuno-enzymatique. Le traitement à l'œstradiol réduit la capacité de liaison des deux récepteurs et les niveaux des récepteurs aux œstrogènes immunoréactifs 12 h après l'injection en absence d'une diminution des ARNm des récepteurs, suggèrent que la réduction initiale est due à la dégradation des protéines ou à une traduction à taux réduit des ARNm dans des nouveaux récepteurs. Les niveaux augmentés des ARNm après le traitement à l'œstradiol suggèrent que la phase de remplissage consiste en la synthèse des nouveaux récepteurs plutôt qu'au recyclage des récepteurs inactivés

Factors involved in the inflammatory events of cervical ripening in humans

<p>Abstract</p> <p>Background</p> <p>Cervical ripening is an inflammatory reaction. The glucocorticoid receptor (GR) mediates glucocorticoid anti-inflammatory reactions, whereas nuclear factor (NF)kappaB is a key pro-inflammatory transcription factor. Prostaglandins as well as platelet activating factor (PAF) are inflammatory mediators. Inducible nitric oxide synthase (iNOS) regulates the level of nitric oxide (NO) in response to various inflammatory stimuli. We hypothesize that a changed biological response to glucocorticoids could be a mechanism regulating the inflammatory events resulting in cervical ripening.</p> <p>Methods</p> <p>We monitored GR and NFkappaB, prostaglandin synthases cyclooxygenase (COX)-1 and -2, iNOS, as well as the PAF-receptor (PAF-R) in the uterine cervix from term pregnant women (with unripe cervices) before the onset of labor (TP), immediately after parturition (PP), as compared to non-pregnant (NP), using immunohistochemistry and RT-PCR.</p> <p>Results</p> <p>The GR protein was detected by immunohistochemistry in the nuclei of stroma and squamous epithelium (SQ). Stromal GR staining was increased in TP as compared to the NP group and decreased again after parturition. GR staining in SQ was decreased after parturition as compared to term. NFkappaB was present in SQ and glandular epithelium (GE), stroma and vascular endothelium. Increased nuclear NFkappaB staining was observed postpartum as compared to term pregnancy in stroma and GE. Stromal immunostaining for COX-1 as well as COX-2 was increased in the TP and PP groups as compared to the NP, and GE displayed an intensely increased COX-2 immunostaining at term and postpartum. Stromal PAF-R immunostaining was highest at term, while it was greatly increased in GE postpartum.</p> <p>No difference in the immunostaining for iNOS was found between the groups. RT-PCR showed a predominance of GRalpha to GRbeta mRNA in cervical tissue. The COX-2 mRNA level was increased in the PP group as compared to the TP group.</p> <p>Conclusions</p> <p>There is a decrease in GR levels in human cervix at parturition. Concomitantly there is an increase of factors such as NFkappaB, PAF-R, COX-1 and COX-2, suggesting that they may participate in the sequence of events leading to the final cervical ripening.</p