Interferon-tau upregulates prolactin receptor mRNA in the ovine endometrium during the peri-implantation period

Our objective was to determine the effect of ovine interferon-tau (IFN-tau) on prolactin receptor (PRL-R) gene expression in the ovine endometrium. IFN-tau is an embryonic cytokine which, via its paracrine anti-luteolytic activity, plays a critical role in maternal recognition of pregnancy in ruminants. Using ribonuclease protection assay procedures, we compared endometrial PRL-R mRNA levels in ewes that were intrauterine injected with either 2 mg bovine serum albumin or 2 mg recombinant ovine IFN-tau on day 10 of the oestrous cycle (day 0 = day of oestrus). IFN treatment significantly increased the abundance of both the long and short forms of PRL-R mRNA in the ovine uterus, but had no effect on the long:short form ratio. In situ hybridization experiments revealed that the increase in abundance of PRL-R mRNA in the uterus was localized to the glandular compartment of the endometrium. In pregnant ewes, a similar increase in PRL-R mRNA abundance was found to occur in ovine endometrium on days 14-15 post conception. Collectively, these data provided strong evidence that IFN-tau modulates the level of lactogenic hormone receptor mRNA in the ovine uterus. Whether the effect of IFN-tau on PRL-R expression is mediated directly or influenced, at least in part, by progesterone remains to be elucidated.     

Identification of genes regulated by interleukin-1beta in human endometrial stromal cells

Interleukin-1beta (IL-1b) is an important immune regulatory factor that in human endometrium plays a role in both menstruation and implantation in the event of pregnancy. It promotes inflammatory-like processes and also stimulates tissue remodelling. We present a cDNA microarray study documenting the major effects of IL-1beta on gene expression in stromal cells from human endometrium. Endometrial stromal cells from five normal healthy women at the mid secretory phase were cultured with or without IL-1beta at 50 and 500 pg/ml for 48 h. cDNA microarrays were used to compare the levels of gene expression in total RNA isolated from cells stimulated with IL-1beta. These cDNA arrays were produced containing 15 164 sequence-verified clones, which included genes known to be important in angiogenesis, immune modulators, apoptosis, cell signalling, extra-cellular matrix (ECM) remodelling and cell cycle regulation. Genes which were regulated by IL-1beta were identified by analysis of the microarray data using the Significance Analysis of Microarrays software package. Upregulated (n = 23) and downregulated (n = 6) different genes were observed, which changed at least 3-fold, at a false discovery rate of less than 2% (P < 0.02). Our results have identified genes regulated by IL-1beta, which are involved in leukocyte recruitment, ECM remodelling and other cellular functions. Changes in three genes, IL-8, colony-stimulating factor 2 and aldoketo reductase family 1 member 1, which were upregulated by IL-1beta, were verified using real-time PCR. Novel functions regulated by IL-1beta in endometrium, including genes involved in free radical protection, and fatty acid metabolism were also identified. These results also provide new insights into the role of IL-1beta in disorders of the endometrium, especially in implantation-related infertility and endometriosis, in which this cytokine plays a major role.     

Localization of mRNA for vascular endothelial growth factor (VEGF), angiopoietins and their receptors during the peri-implantation period and early pregnancy in marmosets (Callithrix jacchus)

Implantation of a blastocyst into a receptive endometrium is a prerequisite for successful pregnancy. Angiogenesis is a key event in this process but the mechanisms by which localized changes in vascular permeability and angiogenesis occur have yet to be elucidated. Vascular endothelial growth factor (VEGF) and its receptors VEGFR-1 and VEGFR-2 have been implicated as key players in vascular remodelling and placentation. Angiopoietins also appear to have a significant role in regulation of blood vessel growth, maturation and regression. The aim of this study was to describe the molecular regulation of angiogenesis in the first month of pregnancy in marmosets and to address the putative physiological roles for these factors. Uteri were studied at weeks 2, 3 and 4 of pregnancy and compared with late secretory non-pregnant endometrium. Implantation in marmosets occurs at day 11 of pregnancy; hence, these time points were chosen so that the peri-implantation period and very early pregnancy could be studied. mRNAs for VEGF, VEGFR-1 and VEGFR-2, angiopoietin 1, angiopoietin 2 and their receptor Tie-2 were localized and quantified by in situ hybridization. Endothelial cells were identified by CD31 immunocytochemistry. VEGF mRNA was present in all compartments except endothelial cells, and its expression generally increased throughout pregnancy except in upper zone glandular epithelium and luminal epithelium, where a decrease in expression was observed. VEGF receptor mRNAs were found in endothelial cells of the upper zones immediately surrounding glandular epithelium. Angiopoietin 1 mRNA was localized to glandular epithelium of the upper and lower zones throughout pregnancy, and increased in stroma at week 4. Expression of angiopoietin 2 mRNA was localized exclusively to endothelial cells of large luminal vessels and was higher in endometrium from marmosets at week 4 of pregnancy than in endometrium from all other stages. These data provide comprehensive evidence that VEGFR-1 and -2, and angiopoietin 1, angiopoietin 2 and Tie-2 interactions may be involved in the preparation of endometrium for implantation, remodelling of the maternal vasculature and trophoblast invasion during the peri-implantation period in this primate species.     

Temporal and spatial associations of oestrogen receptor alpha and progesterone receptor in the endometrium of cyclic and early pregnant mares

Uterine function is primarily controlled by the combined actions of oestrogen and progesterone working through their cognate nuclear receptors. The mechanism of establishment of pregnancy in the mare is of interest because it involves prolonged pre-attachment and conceptus migration phases, and both invasive and non-invasive placental cell types, and as such has been an important comparative model. This study characterised regulation of oestrogen (ER) and progesterone (PR) receptors in the endometrium of the mare during the oestrous cycle and early pregnancy. Endometrial tissues collected during the oestrous cycle and early pregnancy were analysed for steady-state levels of ER and PR mRNA and protein. Steady-state levels of ER and PR mRNA were highest on days 0, 17 and 20 in cyclic mares and lowest on days 11 and 14. A day-by-status interaction was detected, indicating that day 17 and day 20 pregnant mares exhibited low levels of ER and PR compared with the corresponding days of the oestrous cycle. In situ hybridisation analyses showed receptor mRNA localisation primarily in the luminal epithelium (LE), glandular epithelium (GE) and stroma around oestrus. During dioestrus and early pregnancy, receptors were not detected in the LE, and were lower in the stroma and deeper GE. Changes in hybridisation intensity in these cell types were consistent with changes in mRNA levels detected by slot-blot hybridisation. ER and PR proteins were detected in the nuclei of LE, GE and stromal cells. Consistent with results from in situ hybridisation, levels of ER and PR immunoreactivity were higher around oestrus, declined to low levels during dioestrus and remained low during early pregnancy. Results described here for temporal and spatial changes in steroid receptor gene expression in mares show the greatest similarities with those described for cattle and sheep.     

Mast cell degranulation in rat uterine cervix during pregnancy correlates with expression of vascular endothelial growth factor mRNA and angiogenesis

Vascular growth of the uterine cervix during pregnancy is associated with mast cell (MC) degranulation. To better understand the mechanism underlying this process, uterine cervices of intact pregnant rats were dissected and endothelial cell proliferation was measured by a bromodeoxyuridine incorporation technique. Total vascular endothelial growth factor (VEGF) mRNA expression and the relative abundance of VEGF splice variants (120, 164, and 188) were determined by RT-PCR. VEGF protein expression was evaluated by immunohistochemistry. To investigate the role of MCs on cervical angiogenesis, a second set of pregnant animals were treated with an MC stabilizer (disodium cromoglycate) to inhibit MC degranulation. Furthermore, 17beta-estradiol (E(2)) serum levels were established by RIA. In intact pregnant rats, VEGF mRNA expression was positively correlated with endothelial cell proliferation and circulating E(2) levels. All selected splice variants of VEGF gene were detected and their relative abundance did not show any change throughout pregnancy. Animals treated with disodium cromoglycate showed a decrease in endothelial cell proliferation and in VEGF mRNA expression compared with controls. Relative abundance of VEGF mRNA splice variants and E(2) serum levels showed no differences between these experimental groups. These results show a time-dependent correlation between VEGF mRNA expression and E(2) serum levels in the uterine cervix of intact pregnant rats, while MC stabilizer-treated animals reduced the VEGF expression without modifying E(2) serum levels. We suggest that cervical angiogenesis during pregnancy could be regulated by a mechanism which involves endogenous E(2) and chemical mediators stored in MC granules via a VEGF-dependent pathway.     

Microarray assessment of the influence of the conceptus on gene expression in the mouse uterus during decidualization

During pregnancy in several species including humans and rodents, the endometrium undergoes decidualization. This process of differentiation from endometrial to decidual tissue occurs only after the onset of implantation in mice. It can also be artificially induced causing the formation of deciduomal tissue. The purpose of this study was to compare the gene expression profile of the developing decidua in pregnant mice with the deciduoma formed after artificial induction in an effort to identify conceptus-influenced changes in uterine gene expression during decidualization. We induced decidualization artificially by transferring blastocyst-sized ConA-coated agarose beads into the uterus on day 2.5 of pseudopregnancy. Recently published work has found this model to be more 'physiological' than other methods. Total RNA was isolated from blastocyst and bead-induced 'implantation' sites of the uteri of day 7.5 pregnant (decidua) and pseudopregnant (deciduoma) mice respectively. This RNA was then used for microarray analysis using Mouse Illumina BeadArray chips. This analysis revealed potential differential mRNA levels of only 45 genes between the decidua and bead-induced deciduoma tissues. We confirmed the differential mRNA levels of 31 of these genes using quantitative RT-PCR. Finally, the level and localization of some of the mRNAs for select genes (Aldh3a1, Bcmo1, Guca2b, and Inhbb) identified by our microarray analysis were examined in more detail. This study provides the identity of a small set of genes whose expression in the uterus during decidualization may be influenced by molecular signals from the conceptus.     

Modulation of peroxisome proliferator-activated receptor delta and gamma transcripts in swine endometrial tissue during early gestation

Recent evidence points to a role for peroxisome proliferator-activated receptors (PPARs) delta and gamma in embryo implantation and survival. In this study, we report the porcine PPARdelta complete coding sequence and mRNA abundance of PPARdelta, PPARgamma1 and gamma2, angiopoietin-like protein 4 (ANGPTL4) and adipocyte determination and differentiation-dependent factor 1 (ADD1) genes in the pregnant sow endometrium. Real-time PCR analysis was used to study the effect of parity (Yorkshire-Landrace multiparous (YL) and nulliparous (YLn)), site of endometrial tissue sampling (between and at embryo attachment sites) in crossbred DurocxYorkshire-Landrace (DYL) sows and stages of pregnancy (non-pregnant, day 15 and day 25 after mating) in Meishan-Landrace (ML) on mRNA levels. Parity effects were observed for PPARdelta, ANGPTL4, and ADD1, with higher mRNA levels in YL than YLn sows. In DYL sows, lower mRNA levels were present at attachment sites compared to between attachment sites for PPARdelta, PPARgamma1, and ANGPTL4. Finally, day 15 pregnant ML sows had lower PPARdelta mRNA levels compared to day 15 cycling ML sows. A significant increase of PPARgamma1 mRNA levels was found on day 25 pregnant ML and DYL sows relative to day 15 ML or DYL pregnant sows. PPARdelta and gamma immunostaining was detected in endometrial tissue of day 15 cycling sows, day 15 and 25 pregnant sows and epithelial cells of day 25 embryos. Collectively, our results suggest a role for PPARdelta, PPARgamma1, and ANGPTL4, but not PPARgamma2, during the peri-implantation period in pregnant sows.     

The expression of transforming growth factor beta in pregnant rat myometrium is hormone and stretch dependent

From a quiescent state in early pregnancy to a highly contractile state in labor, the myometrium displays tremendous growth and remodeling. We hypothesize that the transforming growth factor beta (TGFbeta) system is involved in the differentiation of pregnant myometrium throughout gestation and labor. Furthermore, we propose that during pregnancy the mechanical and hormonal stimuli play a role in regulating myometrial TGFbetas. The expression of TGFbeta1-3 mRNAs and proteins was examined by real-time PCR, Western immunoblot, and localized with immunohistochemistry in the rat uterus throughout pregnancy and labor. Tgfbeta1-3 genes were expressed differentially in pregnant myometrium. Tgfbeta2 gene was not affected by pregnancy, whereas the Tgfbeta1 gene showed a threefold increase during the second half of gestation. In contrast, we observed a dramatic bimodal change in Tgfbeta3 gene expression throughout pregnancy. Tgfbeta3 mRNA levels first transiently increased at mid-gestation (11-fold on day 14) and later at term (45-fold at labor, day 23). Protein expression levels paralleled the changes in mRNA. Treatment of pregnant rats with the progesterone (P4) receptor antagonist RU486 induced premature labor on day 19 and increased Tgfbeta3 mRNA, whereas artificial maintenance of elevated P4 levels at late gestation (days 20-23) caused a significant decrease in the expression of Tgfbeta3 gene. In addition, Tgfbeta3 was up-regulated specifically in the gravid horn of unilaterally pregnant rats subjected to a passive biological stretch imposed by the growing fetuses, but not in the empty horn. Collectively, these data indicate that the TGFbeta family contributes in the regulation of myometrial activation at term integrating mechanical and endocrine signals for successful labor contraction.     

Hot topic: Pregnancy-induced expression of interferon-stimulated genes in the cervical and vaginal mucosal membranes

In ruminants, IFN-tau (IFNT) is a pregnancy recognition signal secreted by the embryonic trophectoderm before implantation, and it induces the expression of IFN-stimulated genes (ISG) in the uterine endometrium and blood leukocytes. The expression of ISG in blood leukocytes could indicate the presence of a viable conceptus before return of the next estrus; however, expression levels have high variation for confirming pregnancy. We hypothesized that the secreted IFNT in the uterus would affect ISG expression in cervical and vaginal tissues because they are directly adjacent to the uterus. To prove the hypothesis, we investigated the expression of 3 ISG (ISG15, MX1, and MX2) in cervical and vaginal mucosal membranes collected from pregnant (n = 12) and nonpregnant (n = 11) lactating Holstein cows at 17 to 18 d after artificial insemination. Mucosal membrane samples of the cervical canal near the external os (cervix) and deep vaginal wall surrounding the external os (vagina) were collected separately by simply scraping with a curette on d 17 or 18 of pregnancy (d 1 = ovulation), at which time IFNT secretion into the maternal uterus is maximal. After pregnancy diagnosis on d 30 and 60, separately collected samples confirmed as pregnant and nonpregnant were used for evaluation of the expression of IFN-stimulated protein 15 kDa (ISG15) and myxovirus-resistance protein 1 and 2 (MX1, MX2) with quantitative real-time PCR. The collected mucosal membrane samples from cervix contained mostly cell clots showing membrane structure and a low content of blood cells. The expression levels of all 3 genes were significantly increased in pregnant cows compared with nonpregnant cows in both cervical and vaginal samples. These results suggest that increased expression of ISG in the cervix and vagina is a pregnancy-associated phenomenon and is highly affected by IFNT secreted from the conceptus through the uterus.     

Nitric oxide in blastocyst implantation in the rhesus monkey

Successful blastocyst implantation depends on the interaction between cells of maternal endometrium and conceptus, as well as adequate blood supply to the site of blastocyst implantation. Nitric oxide (NO) generally plays a significant role in the local regulation of vascular physiology in a variety of mammalian tissue systems, however, its role in blastocyst implantation and placentation in the primate is not known. The aim of the present study was to examine: (i) NADH-diaphorase activity and expression of three isoforms of nitric oxide synthase (NOS), namely endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) in pre-implantation stage monkey embryos, morula (n = 4) and blastocyst (n = 10), as well as, in different compartments of conceptus and maternal endometrium at primary implantation sites during lacunar (n = 6) and villous (n = 9) stages of placentation in the rhesus monkey, and (ii) the potential anti-nidatory effect of vaginal administration of NOS inhibitor during the peri-implantation period of conception cycles in rhesus monkeys. Pre-implantation stage blastocysts exhibited marked NADPH-diaphorase activity along with immunopositive iNOS mainly in the inner cell mass. During the lacunar stage, marked eNOS expression was observed in cytotrophoblast cells lining the embryonic cavity. However, cytotrophoblast cells lining villi, forming columns, and constituting anchoring villi expressed all the three isoforms of NOS in villous placenta stage tissue. During the lacunar stage, eNOS and iNOS protein expressions were observed in epithelial and decidual cells of endometrium. As gestation advanced, mRNAs for all three isoforms of NOS were observed to increase in epithelial and decidual cells, however, with no marked change in protein expression. Vaginal administration of a NOS inhibitor (N(G)-nitro-l-arginine methyl ester, L-NAME, 4, 6, and 8 mg/kg body weight or aminoguanidine, AG, 4 mg/kg body weight) during days 6 to 12 after ovulation resulted in pregnancy failure in a higher number of animals (L-NAME: 8 confirmed pregnancies in 25 animals; AG: 2 confirmed pregnancies in 8 animals) compared with control animals (5 pregnancies in 7 animals). It appears that NO may play an important role in the establishment of pregnancy in the rhesus monkey.     

Angiogenesis and microvascular development in the marmoset (Callithrix jacchus) endometrium during early pregnancy

The aim of the study was to describe and quantify the changes in the maternal vasculature and angiogenesis during early pregnancy in the marmoset endometrium using bromodeoxyuridine (BrdU) to identify proliferating cells, CD31 to label endothelial cells and dual staining to identify proliferating endothelial cells. Non-pregnant animals from mid- and late secretory stages were studied and compared with pregnant animals at weeks 2, 3 and 4 of pregnancy. Qualitative and morphometric analyses of angiogenesis and vascular area were performed. The results show that pregnancy is associated with increasing angiogenesis in the upper zone of the endometrium, becoming significantly increased at 3 weeks. This is associated with an increase in the vessel area and diameter in this zone. These results provide the platform from which to design studies in which specific angiogenic factors can be targeted in vivo during early pregnancy in order to determine their role in regulating these vascular changes.