Secretory endometrium highly expresses urocortin messenger RNA and peptide: possible role in the decidualization process

BACKGROUND: Urocortin (UCN) gene expression and synthesis have been reported in epithelial and stromal cells of the human endometrium. In this study we evaluated (i) UCN messenger RNA (mRNA) expression and peptide production in uterine specimens collected throughout the endometrial cycle, (ii) UCN secretion after decidualization of cultured human endometrial stromal cells (HESCs) and (iii) the effect of UCN on endometrial decidualization. METHODS: HESCs were isolated from samples of human endometrium collected from healthy patients with normal menstrual cycle and cultured in presence of cAMP, 17-beta-estradiol (E(2)) + medroxyprogesterone acetate (MPA) and UCN. UCN levels were measured in endometrial extracts by an enzyme immunoassay, and changes of endometrial UCN mRNA expression were measured by RT-PCR analysis. RESULTS: UCN peptide concentrations and mRNA expression were highest in the secretory phase of the menstrual cycle (P < 0.001, late secretory versus early and late proliferative phase) and higher in the late than the early secretory phase (P < 0.01). After decidualization of HESC with cAMP or E(2) + MPA, UCN levels rose in parallel with prolactin concentrations by days 6 (P < 0.01, for all). Finally, the addition of UCN to HESCs, with or without E(2) + MPA, induced the release of prolactin. CONCLUSIONS: The evidence that (i) UCN is highly expressed in the secretory phase of the endometrial cycle; (ii) cAMP and E(2) + MPA modulate secretion of UCN and (iii) UCN induces HESCs decidualization together suggest a possible role for UCN in endometrial physiology.     

Insulin-like growth factor binding protein-1 induces decidualization of human endometrial stromal cells via alpha5beta1 integrin

Progesterone is known to induce decidualization of human endometrial stromal cells in vitro. Decidualized stromal cells produce insulin-like growth factor binding protein-1 (IGFBP-1) as well as prolactin (PRL). In this study, we tested the possibility that IGFBP-1 directly stimulates endometrial stromal cell decidualization. Endometrial stromal cells were obtained from normal menstruating patients with uterine myoma at hysterectomy. Stromal cells were cultured for up to 4 weeks with estradiol (E(2)) and/or medroxy progesterone acetate (MPA) in the presence or the absence of IGFBP-1 and, LR(3)-IGF-I (an IGF-I analogue) that binds to the IGF-I receptor but has reduced affinity for IGFBPs. Decidualization of endometrial stromal cells was evaluated by morphological changes and PRL release into culture media. The binding of IGFBP-1 to endometrial cells was analysed using a biosensor. MPA and E(2) induced decidualization of stromal cells, while LR(3)-IGF-I inhibited decidualization by MPA and E(2) as well as PRL and IGFBP-1 secretion into medium. IGFBP-1 induced decidualization of stromal cells in the absence of MPA and E(2) in the medium. IGFBP-1-induced decidualization was not inhibited by the addition of LR(3)IGF-1 but was inhibited by the addition of an RGD peptide, however, the RGD peptide had no effect on decidualization when added alone. The binding analysis showed that IGFBP-1 bound to the surface of endometrial stromal cells and an anti-alpha5beta1 integrin antibody inhibited its binding. These results suggest that IGFBP-1 produced by endometrium can mediate progesterone-induced decidualization possibly by interacting with alpha5beta1 integrin on the surface of endometrial stromal cells.     

Interleukin 11 advances progesterone-induced decidualization of human endometrial stromal cells

Differentiation of endometrial stromal cells into decidual cells is crucial for embryo implantation and placentation. Interleukin (IL)-11 signalling is essential for adequate decidualization in the mouse uterus. We examined the role of IL-11 during progesterone-induced decidualization of human endometrial stromal cells over a 10-12 day period, using prolactin (PRL) production as a decidual marker. These cells produced biologically active IL-11 and expressed IL-11, IL-11Ralpha and PRL mRNA during decidualization. Neutralization of endogenous IL-11 with an anti-human (hu)IL-11 antibody (AB) reduced production of PRL from day 8 and insulin-like growth factor binding protein (IGFBP)-1, another marker of decidualization, from day 10 of culture. Following AB washout, PRL and IGFBP-1 secretion increased. Addition of recombinant (r)huIL-11 (10 or 100 ng/ml) to endometrial stromal cells increased secretion of PRL from day 4 and IGFBP-1 from day 6 compared with progesterone alone. Morphological signs of differentiation accompanied biochemical differentiation in the progesterone-treated cells and were further induced by exogenous rhuIL-11. Our observations demonstrate that human endometrial stromal cells produce biologically active IL-11, which promotes progesterone-induced decidualization. These results suggest that IL-11 has both paracrine and autocrine actions on human endometrial stromal cells and plays an important role in preparing the human endometrium for implantation.     

Blastocyst invasion and the stromal response in primates

One of the most remarkable processes associated with the establishment of pregnancy in the primate is the process of decidualization. This transformation of a stromal fibroblast to a fully differentiated decidual cell is required for implantation and embryo survival in early pregnancy. Although the morphological and biochemical characteristics of the primate decidual cell have been extensively studied, the precise cellular, biochemical and molecular signals required for this transformation have yet to be elucidated. During decidualization, stromal cells first proliferate and then differentiate. Based on our extensive in-vivo and ongoing in-vitro studies, we have suggested that the process of decidualization in the baboon can be divided into two distinct phases. The initial proliferative phase is characterized by the expression of the cytoskeletal protein alpha smooth muscle actin (alphaSMA) in the stromal fibroblasts and is independent of the presence of the conceptus. The second phase of differentiation is characterized by the expression of insulin-like growth factor binding protein-1 (IGFBP-1) and the down-regulation of alphaSMA in the decidualized stromal fibroblast. The expression of IGFBP-1 is dependent on the presence of the conceptus in vivo and is regulated by hormones and cAMP in vitro. We have postulated that, during the initial phase of stromal cell differentiation, alphaSMA expression is regulated by the interaction between stromal cell integrins with the secreted extracellular matrix proteins (ECM). In response to pregnancy a trophoblast 'factor', mediated by cAMP signal transduction, induces IGFBP-1 expression in decidualizing stromal fibroblasts. This induction of IGFBP-1 is associated with the disappearance of alphaSMA and de-novo protein synthesis. Our comparative studies suggest that the process of decidualization in the human and baboon involve similar mechanisms. However, the metabolic pathways required for decidualization in the two species appear to differ in their degree of sensitivity to external stimuli. This review focuses on the cellular events that may potentially regulate decidualization in the primate and its role in regulating trophoblast migration.     

Ghrelin: structure and function

Small synthetic molecules called growth hormone secretagogues (GHSs) stimulate the release of growth hormone (GH) from the pituitary. They act through the GHS-R, a G protein-coupled receptor whose ligand has only been discovered recently. Using a reverse pharmacology paradigm with a stable cell line expressing GHS-R, we purified an endogenous ligand for GHS-R from rat stomach and named it "ghrelin," after a word root ("ghre") in Proto-Indo-European languages meaning "grow." Ghrelin is a peptide hormone in which the third amino acid, usually a serine but in some species a threonine, is modified by a fatty acid; this modification is essential for ghrelin's activity. The discovery of ghrelin indicates that the release of GH from the pituitary might be regulated not only by hypothalamic GH-releasing hormone, but also by ghrelin derived from the stomach. In addition, ghrelin stimulates appetite by acting on the hypothalamic arcuate nucleus, a region known to control food intake. Ghrelin is orexigenic; it is secreted from the stomach and circulates in the bloodstream under fasting conditions, indicating that it transmits a hunger signal from the periphery to the central nervous system. Taking into account all these activities, ghrelin plays important roles for maintaining GH release and energy homeostasis in vertebrates.     

Ghrelin increases intracellular Ca(2+) concentration in the various hormone-producing cell types of the rat pituitary gland

Ghrelin, isolated from the stomach as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), has potent growth hormone release ability in vivo and in vitro. Although GHS-R is abundantly expressed in the pituitary gland, there is no direct evidence of a relationship between hormone-producing cells and functional GHS-R in the pituitary gland. The aim of this study was to determine which anterior pituitary cells respond to ghrelin stimulation in male rats. We performed Fura-2 Ca(2+) imaging analysis using isolated pituitary cells, and performed immunocytochemistry to identify the type of pituitary hormone-producing cells. In Fura-2 Ca(2+) imaging analysis, ghrelin administration increased the intracellular Ca(2+) concentration in approximately 50% of total isolated anterior pituitary cells, and 20% of these cells strongly responded to ghrelin. Immunocytochemical analysis revealed that 82.9±1.3% of cells that responded to ghrelin stimulation were GH-immunopositive. On the other hand, PRL-, LH-, and ACTH-immunopositive cells constituted 2.0±0.3%, 12.6±0.3%, and 2.5±0.8% of ghrelin-responding pituitary cells, respectively. TSH-immunopositive cells did not respond to ghrelin treatment. These results suggest that ghrelin directly acts not only on somatotrophs, but also on mammotrophs, gonadotrophs, and corticotrophs in the rat pituitary gland.     

Symposium: reproduction in baboons. Secretory proteins of the baboon (Papio anubis) endometrium: regulation during the menstrual cycle and early pregnancy

The biological function of uterine endometrial secretory proteinsin the primate remain to be elucidated. In general, during theluteal phase and under progesterone dominance, the glandularepithelial cells synthesize and secrete a number of proteins.Of these, placental protein 14 (PP₁₄; now referred to as glycodelin)and insulin-like growth factor binding protein-1 (IGFBP-1) arethe best characterized. Although induced by progesterone, theirsynthesis increases exponentially during pregnancy. In the baboon,glycodelin is immunolocalized to the mid functionalis and basalglands between days 10 and 12 post-ovulation. In response toeither exogenous or blastocyst-secreted chorionic gonadotrophin,glandular synthesis increases markedly and remains elevatedup to days 18-25 of pregnancy. The decrease in glycodelin inthe endometrium is associated with glandular regression duringthe first third of pregnancy. In contrast, IGFBP-1 is only observedin the deep basal glands during the luteal phase. Followingthe establishment of pregnancy, IGFBP-1 synthesis switches fromglandular to stromal and is correlated with the process of decidualization.IGFBP-1 synthesis continues to increase throughout gestation.We propose that glycodelin may have immunosuppressive propertiesand that IGFBP-1 may regulate trophoblast migration within theuterine endometrium.     

The effect of mifepristone on the expression of insulin-like growth factor binding protein-1, prolactin and progesterone receptor mRNA and protein during the implantation phase in human endometrium

Insulin-like growth factor binding protein-1 (IGFBP-1) and prolactin are recognized as crucial signals for the initiation and maintainance of decidualization. The purpose of the study was to investigate the effect of mifepristone on the expression of IGFBP-1, prolactin and progesterone receptors (PR) during the implantation phase in human endometrium. Eight fertile women were studied during control and treatment cycles. Treatment with 200 mg of mifepristone was administered on day LH +2. Endometrial samples were collected on day LH +6 to +8. Expression of IGFBP-1, prolactin and PR was identified using immunohistochemistry, and mRNA levels were determined with RT-PCR. In control specimens, IGFBP-1 and prolactin were localized to the cytoplasm of the endometrial glandular and to a lesser extent in stromal cells. In the same samples, PR immunoreactivity was detected in the nucleus of the endometrial stromal cells, and was absent from the glandular cells. After mifepristone treatment, there was a significant increase in the immunostaining and mRNA expression for IGFBP-1 and PR. Prolactin expression increased only slightly after treatment. These results support the view that administration of mifepristone in the early luteal phase does not simply retard endometrial development. Our findings provide further insight into the regulation of IGFBP-1 and prolactin by PR in the human endometrium in vivo.     

Retinoic acid suppresses in-vitro decidualization of human endometrial stromal cells

All-trans retinoic acid (RA) has potent effects on cell differentiationand gene expression. Previous studies have demonstrated thathuman endometrial stromal cells express mRNA for retinoic acidreceptors (RARs) and cellular RA-binding protein-ll (CRABP-II).We examined whether RA regulates stromal cell differentiation(decidualization), a critical process in preparation of theuterus for blastocyst implantation. Decidualization was inducedby incubating cultured stromal cells with medroxyprogesteroneacetate (MPA) and oestradiol. Decidualization was defined bythe induction of prolactin, insulin-like growth factor bindingprotein-1 (IGFBP-1), appearance of a differentiated phenotypeand changes in fibronectin expression. RA treatment significantly(P<0.05) suppressed prolactin and IGFBP-1 production associatedwith stromal cells decidualization. The formation of differentiatedcells was inhibited by RA, and consistent with maintenance ofthe undifferentiated phenotype, fibronectin mRNA content was-3.5-times greater than in the absence of RA. Upon inductionof decidualization, the expression of mRNA for the major RAreceptor sub-types (RAR-     

Differential localization and expression of urokinase plasminogen activator (uPA), its receptor (uPAR), and its inhibitor (PAI-1) mRNA and protein in endometrial tissue during the menstrual cycle

Normal endometrium is a highly dynamic tissue, which responds to ovarian steroids with cyclic proliferation, differentiation (secretion), and degradation (menstruation). The urokinase plasminogen activator (uPA)-dependent proteolytic cascade as well as ligand activation of the uPA receptor (uPAR) is critically involved in physiological as well as pathophysiological aspects of tissue expansion and remodelling. Cyclic variation and distribution of uPA, uPAR and plasminogen activator inhibitor 1 (PAI-1) mRNA were examined by in situ hybridization, real-time PCR and northern blot in normal endometrium. Their corresponding proteins were localized with immunohistochemistry. uPA mRNA is exclusively expressed by stromal cells, whereas uPA protein is present in both epithelial and stromal cells. Immunostaining for uPA protein is reduced or undetectable at midcycle, thus coinciding with peak concentration of uPA in the uterine fluid. uPAR mRNA is expressed by epithelial cells in the proliferative phase and by stromal cells in the secretory phase. However, epithelial cells stain for uPAR protein throughout the cycle, suggesting that uPAR may detach from stromal cells and then bind to epithelial cells in the secretory phase. PAI-1 mRNA is located in vessel walls. The late secretory phase has greatly increased expression of all three mRNA and their proteins, mainly in pre-decidual cells in the superficial stroma. Discordant localization of the mRNA and proteins suggest that uPA is produced by stromal cells, released and bound to epithelial cells in both the proliferative and secretory phases, whereas uPAR is released from the stroma and bound to epithelial cells in the secretory phase. Also, the present data together with earlier reports suggest that uPA is released from the epithelial cells to the uterine fluid.     

Histamine uptake by human endometrial cells expressing the organic cation transporter EMT and the vesicular monoamine transporter-2

Cellular reuptake of monoamines, which is mediated by cell membrane transporters, is followed by accumulation in vesicles by vesicular monoamine transporters (VMAT). The aim of this study was to demonstrate the presence of functional monoamine transporters with high affinity for histamine in human endometrial tissue, since histamine has been implicated as a paracrine signal during endometrial decidualization and embryo implantation. In situ hybridization with (35)S-labelled cRNA probes was used for detection of the organic cationic transporter-2 (OCT-2), the extraneuronal monoamine transporter (EMT), and VMAT-2 in cryosections of normal human endometrial tissue. To identify functional transporters for histamine in endometrial cells, we incubated primary cultures of stromal cells and cultures of attached glands with (3)H-labelled histamine. Cultures were pretreated with either corticosterone, a specific inhibitor of EMT, or reserpine, a specific inhibitor of VMAT-2. EMT mRNA was localized in the stroma with peak expression in the secretory phase, whereas OCT-2 mRNA was expressed by few cells in the stroma throughout the cycle. VMAT-2 mRNA was localized in the stroma during the proliferative phase and in the epithelium during the secretory phase. Thus, EMT and VMAT-2, which both have high affinity for histamine, are strongly expressed in endometrial cells. Both corticosterone and reserpine significantly reduced the uptake of (3)H-histamine in stromal cells during the proliferative as well as the secretory phase. This indicates the presence of functional EMT and VMAT-2 transporter proteins throughout the cycle, even though their periods of maximal mRNA expression were limited. The results of uptake experiments with glandular epithelial cells confirmed not only the presence of functional VMAT-2 transporter protein in the secretory phase but also the absence of a histamine-specific plasma membrane transporter throughout the cycle. Thus, endometrial tissue contains both plasma membrane and vesicular membrane monoamine transporters with high affinity for histamine. They can potentially influence the reproductive process by the uptake of extracellular histamine and subsequent release on demand.     

Ghrelin对糖尿病大鼠下丘脑弓状核胃牵张敏感神经元放电活动及胃运动的调控

 目的：观察链脲佐菌素(STZ)所致糖尿病（DM）大鼠下丘脑弓状核（Arc）胃牵张（GD）敏感神经元放电活动及胃运动改变,探讨ghrelin对DM大鼠下丘脑Arc GD敏感神经元放电活动和胃运动的影响。方法：采用STZ腹腔注射诱导DM大鼠模型;通过细胞外记录神经元单位放电和在体胃运动方法,观察ghrelin及其受体阻断剂［D-Lys3］-GHRP-6对DM大鼠下丘脑Arc GD敏感神经元自发放电活动和胃运动的影响;应用real-time PCR和荧光免疫组化方法,探讨DM大鼠Arc内ghrelin受体（GHS-R1a）mRNA及其免疫阳性物的表达。结果：在正常大鼠Arc记录到的98个GD敏感神经元中,64.3%为GD兴奋性（GD-E）神经元,35.7%为GD抑制性（GD-I）神经元。在63个GD-E神经元中,Arc微量注射ghrelin可使其中73.0%神经元兴奋,其放电频率与生理盐水组比较显著增加（P<0.05）;而在35个GD-I神经元中,Arc微量注射ghrelin可抑制其中60.0%神经元,放电频率显著降低（P<0.01）;ghrelin改变GD神经元放电效应可被ghrelin 受体阻断剂［D-Lys3］-GHRP-6阻断（P<0.05）;在DM大鼠,Arc记录到的66个GD敏感神经元中有56.1%为GD-E神经元,43.9%为GD-I神经元。Arc注射ghrelin可兴奋其中35.1%GD-E神经元,放电频率与生理盐水比较显著增加（P<0.05）;而在29个GD-I神经元中,ghrelin可抑制其中21个神经元（72.4%）,放电频率显著降低（P<0.01）。与正常大鼠比较,DM大鼠Arc GD敏感神经元中的GD-E和GD-I比例无显著改变（P>0.05）,但ghrelin使GD-E神经元兴奋的比率明显降低（P<0.05）,放电频率平均增加率也显著下降（P<0.05）;但ghrelin使GD-I 神经元抑制比率和放电频率平均减少率均无显著改变（P>0.05）。在体胃运动研究结果显示,Arc微量注射ghrelin,可显著促进正常和DM大鼠胃运动,且呈显著量效关系（P<0.05,P<0.01）,但ghrelin对正常大鼠的促胃运动作用显著强于其对DM大鼠的作用（P<0.05）,［D-Lys3］-GHRP-6可完全阻断ghrelin该作用。Real-time PCR研究结果显示,DM大鼠下丘脑Arc GHS-R1a mRNA表达较正常大鼠明显减少（P<0.05）;免疫荧光研究进一步证实DM大鼠下丘脑Arc GHS-R1a 免疫阳性物表达较正常大鼠明显减少（P<0.05）。结论：下丘脑Arc ghrelin参与DM大鼠GD敏感神经元自发放电活动,并参与胃运动的调控,该效应可能是通过作用于ghrelin受体而实现的。     

Different mechanisms for the induction of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone in human endometrial stromal cells

BACKGROUND: The present study was undertaken to investigate the cAMP-dependent regulation of copper-zinc superoxide dismutase (Cu,Zn-SOD) and manganese SOD (Mn-SOD) by ovarian steroids in human endometrial stromal cells (ESC). METHODS and RESULTS: To examine the effect of cAMP on SOD expression, ESC were incubated with dibutyryl-cAMP (db-cAMP, 0.5 mmol/l), forskolin (25 micromol/l), or estradiol (E(2), 10(-8) mol/l) + medroxyprogesterone acetate (MPA, 10(-6) mol/l), for 18 days. E(2) + MPA significantly increased Cu,Zn-SOD activity and mRNA concentrations, whereas db-cAMP and forskolin had no effect. On the other hand, Mn-SOD activity and mRNA concentration were significantly increased by all of these treatments. Insulin-like growth factor-binding protein-1, a marker of decidualization, was clearly induced by db-cAMP, forskolin or E(2) + MPA, accompanied by morphological changes characteristic of decidualization. To study whether the increase in Mn-SOD by db-cAMP or E(2) + MPA was mediated by cAMP-dependent protein kinase A (PKA), ESC were incubated with protein kinase inhibitor (PKI) (10 microg/ml), an inhibitor of PKA, in the presence of db-cAMP or E(2) + MPA. The increase in Mn-SOD activity following db-cAMP or E(2) + MPA was completely inhibited by PKI. CONCLUSIONS: In the process of decidualization, E(2) + MPA increases Mn-SOD expression via a cAMP-dependent pathway. Cu,Zn-SOD is also up-regulated by E(2) + MPA, but via a different pathway from that involving cAMP.     

Expression of activin receptors, follistatin and betaglycan by human endometrial stromal cells; consistent with a role for activins during decidualization

Decidualization of the human endometrium is critical for implantation, but the mechanisms involved are largely unknown. Activin subunits are expressed in endometrium during decidualization. From its known actions in cell differentiation and tissue remodelling, we hypothesized that activin A is involved in the paracrine regulation of decidualization. We examined the expression of activin receptors (ActRs) by semi-quantitative and real-time RT-PCR. mRNA for all ActR subtypes (Ia, Ib, IIa and IIb) was detected in endometrium, with maximal expression in the early secretory phase and in early pregnancy. ActR protein was localized exclusively to stromal and endothelial cells. This expression pattern was confirmed by in-situ hybridization. Activin bioavailability is locally regulated by its binding protein, follistatin, and also by the antagonist, inhibin. Inhibin competition for ActRII binding is enhanced by the binding protein, betaglycan. Follistatin and betaglycan were also detected in the endometrium, localized to stromal and epithelial cells. This co-expression of activin subunits, receptors and binding proteins indicates that stromal cells are capable of responding to activin, and that there is tight local regulation of activin action within the endometrium. As activin production is up-regulated in decidual cells, this provides further evidence for an involvement of activins during stromal cell decidualization.