Maternal Uterine Tissues Research Articles

Down-regulation of uterine LIF expression induced by the hormonal level disorder causes embryo implantation loss after mice exposed to carbon disulfide at peri-implantation

Carbon disulfide (CS2) exposure can cause embryo implantation loss but the mechanism remains unclear. Earlier study revealed that the 4th day of gestation (GD4) and GD5 were the most sensitive exposure time on which the number of implanted embryos decreased obviously in mice. Leukemia inhibitory factor (LIF) in maternal uterine tissue is involved in embryo implantation, which is produced by endometrium and Th2 cells that participate in cellular adhesion of maternal–fetal interface. We herein investigated the effect of CS2 on the expression of LIF in uterine tissue and its regulatory mechanism in Kunming mice. Exposure was on GD3, GD4, GD5 and GD6, respectively, single administration (631.4 mg/kg), and the indexes were arranged in time series after exposure. The results showed that LIF gene breakage was captured at the 18th hour after exposure by Comet-FISH and the protein and mRNA of LIF in uterine tissue were down-regulated after exposure through the peri-implantation period. In addition, sex steroid hormones, progesterone (P4) and oestrogen (E2) were detected since they can stimulate synthesis of LIF from endometrial cells. Results showed that P4 and E2 in serum were down-regulated at all the endpoints of CS2 exposure groups. These findings suggested that the down-regulated LIF induced by the decreased P4 and E2 after mice exposure to CS2 might be important reasons for implantation disorders.

LIMK1 Regulates Human Trophoblast Invasion/Differentiation and Is Down-Regulated in Preeclampsia

Successful human pregnancy requires extensive invasion of maternal uterine tissues by the placenta. Invasive extravillous trophoblasts derived from cytotrophoblast progenitors remodel maternal arterioles to promote blood flow to the placenta. In the pregnancy complication preeclampsia, extravillous trophoblasts invasion and vessel remodeling are frequently impaired, likely contributing to fetal underperfusion and maternal hypertension. We recently demonstrated in mouse trophoblast stem cells that hypoxia-inducible factor-2 (HIF-2)-dependent Lim domain kinase 1 (LIMK1) expression regulates invasive trophoblast differentiation by modulating the trophoblast cytoskeleton. Interestingly, in humans, LIMK1 activity promotes tumor cell invasion by modulating actin and microtubule integrity, as well as by modulating matrix metalloprotease processing. Here, we tested whether HIF-2α and LIMK1 expression patterns suggested similar roles in the human placenta. We found that LIMK1 immunoreactivity mirrored HIF-2α in the human placenta in utero and that LIMK1 activity regulated human cytotrophoblast cytoskeletal integrity, matrix metallopeptidase-9 secretion, invasion, and differentiation in vitro. Importantly, we also found that LIMK1 levels are frequently diminished in the preeclampsia setting in vivo. Our results therefore validate the use of mouse trophoblast stem cells as a discovery platform for human placentation disorders and suggest that LIMK1 activity helps promote human placental development in utero.

Maternal cardiac metabolism in pregnancy

Pregnancy causes dramatic physiological changes in the expectant mother. The placenta, mostly foetal in origin, invades maternal uterine tissue early in pregnancy and unleashes a barrage of hormones and other factors. This foetal 'invasion' profoundly reprogrammes maternal physiology, affecting nearly every organ, including the heart and its metabolism. We briefly review here maternal systemic metabolic changes during pregnancy and cardiac metabolism in general. We then discuss changes in cardiac haemodynamic during pregnancy and review what is known about maternal cardiac metabolism during pregnancy. Lastly, we discuss cardiac diseases during pregnancy, including peripartum cardiomyopathy, and the potential contribution of aberrant cardiac metabolism to disease aetiology.

Differential expression of the metastasis suppressor KAI1 in decidual cells and trophoblast giant cells at the feto-maternal interface

Invasion of trophoblasts into maternal uterine tissue is essential for establishing mature feto-maternal circulation. The trophoblast invasion associated with placentation is similar to tumor invasion. In this study, we investigated the role of KAI1, an anti-metastasis factor, at the maternal-fetal interface during placentation. Mouse embryos were obtained from gestational days 5.5 (E5.5) to E13.5. Immunohistochemical analysis revealed that KAI1 was expressed on decidual cells around the track made when a fertilized ovum invaded the endometrium, at days E5.5 and E7.5, and on trophoblast giant cells, along the central maternal artery of the placenta at E9.5. KAI1 in trophoblast giant cells was increased at E11.5, and then decreased at E13.5. Furthermore, KAI1 was upregulated during the forskolinmediated trophoblastic differentiation of BeWo cells. Collectively, these results indicate that KAI1 is differentially expressed in decidual cells and trophoblasts at the maternal-fetal interface, suggesting that KAI1 prevents trophoblast invasion during placentation. [BMB Reports 2013; 46(10): 507-512]

External Mechanical Cues Trigger the Establishment of the Anterior-Posterior Axis in Early Mouse Embryos

Mouse anterior-posterior axis polarization is preceded by formation of the distal visceral endoderm (DVE) by unknown mechanisms. Here, we show by in vitro culturing of embryos immediately after implantation in microfabricated cavities that the external mechanical cues exerted on the embryo are crucial for DVE formation, as well as the elongated egg cylinder shape, without affecting embryo-intrinsic transcriptional programs except those involving DVE-specific genes. This implies that these developmental events immediately after implantation are not simply embryo-autonomous processes but require extrinsic factors from maternal tissues. Moreover, the mechanical forces induce a breach of the basement membrane barrier at the distal portion locally, and thereby the transmigrated epiblast cells emerge as the DVE cells. Thus, we propose that external mechanical forces exerted by the interaction between embryo and maternal uterine tissues directly control the location of DVE formation at the distal tip and consequently establish the mammalian primary body axis.

AKT Isoforms 1 and 3 Regulate Basal and Epidermal Growth Factor-Stimulated SGHPL-5 Trophoblast Cell Migration in Humans1

Protein kinase B/AKT is critically involved in murine placental development and migration of human placental trophoblasts into maternal uterine tissue. However, localization of the three AKT isoforms within human placenta and their roles in extravillous trophoblasts have not been elucidated. Therefore, we analyzed the expression pattern and function of AKT1, AKT2, and AKT3 in migratory human trophoblasts using SGHPL-5 cell pools stably expressing small-hairpin microRNA (shRNAmir) against AKT1, AKT2, or AKT3 as a model. Western blot analyses using isoform-specific antibodies revealed ubiquitous expression of AKT1, AKT2, and AKT3 in primary villous and extravillous trophoblasts and the trophoblastic cell lines JEG-3, HTR-8/SVneo, and SGHPL-5. Immunofluorescence of first-trimester placentae localized AKT2 and AKT3 to the cytoplasm and nucleus, respectively, in all subtypes of cytotrophoblasts, whereas AKT1 was detected in both cellular compartments. A similar distribution of AKT isoforms was detectable in SGHPL-5 cells. Gene silencing using shRNAmir decreased protein expression of AKT1, AKT2, and AKT3 to 16%, 8%, and 11%, respectively, in SGHPL-5 cells. Compared with shRNAmir controls, proliferation and camptothecin-induced apoptosis were not affected in the different AKT knockdown cells. However, basal and epidermal growth factor (EGF)-induced trophoblast migration was significantly reduced in AKT1 and AKT3 gene-silenced cells, whereas downregulation of AKT2 was not effective. Accordingly, a decrease in EGF-stimulated phosphorylation of AKT (Ser473 and Thr308) and its downstream target mTORC1 (Ser2448) was noticed in AKT1 and AKT3 shRNAmir cell pools. In summary, the results suggest that the AKT isoforms 1 and 3 promote basal as well as EGF-induced trophoblast migration.

HLA class I expression in the human placenta

Placental trophoblast cells of the semi-allogenic human conceptus invade deeply into maternal uterine tissue. From a classical immunoiogic point of view this invasion and the following growth and development of the fetus in the uterus have to be tolerated by a pregnant woman's immune system. Among the various possible protective mechanisms that may be involved, the unique expression pattern of HLA class I molecules seems to be relevant. Besides many other differences between placentation and organ transplantation, this extraordinary HLA class I expression on trophoblast explains why pregnancy should not be considered an immunologic paradox but rather a fascinating example of a very special challenge for the female immune system.

IFPA Award in Placentology Lecture: Molecular regulation of human trophoblast invasion

Invasion of extravillous trophoblast cell types into maternal uterine tissues is essential for successful human placental development and progression of pregnancy. Whereas endovascular trophoblasts migrate into the maternal spiral arteries, interstitial trophoblasts invade the decidual stroma, colonize the vessels from outside and communicate with diverse uterine cell types such as decidual stromal cells, macrophages and uterine NK cells. For example, interstitial trophoblasts expressing polymorphic human leukocyte antigen-C interact with uterine NK cells through binding to their killer immunoglobulin-like receptors which likely plays a role in trophoblast invasion and reproductive success of pregnancy. Both extravillous trophoblast subtypes are critically involved in the vascular transformation of the spiral arteries into dilated conduits ensuring appropriate blood flow into the intervillous space. Failures in this remodeling process are thought to be associated with severe forms of fetal growth restriction, preeclampsia and other pregnancy complications warranting studies on the molecular regulation of extravillous trophoblast differentiation. Moreover, interstitial trophoblast-derived hormones may regulate diverse biological functions in the decidua. In particular, human chorionic gonadotrophin has been shown to promote angiogenesis and to suppress apoptosis of endometrial stromal cells. In return, decidual cells produce a plethora of soluble factors controlling trophoblast invasion in a time- and distance-dependent manner. However, the underlying mechanisms have not been fully elucidated. Here, we will summarize autocrine as well as paracrine factors regulating invasion of extravillous trophoblasts and discuss critical signaling cascades involved. In addition, we will focus on key regulatory transcription factors controlling cell column proliferation and differentiation of the human extravillous trophoblast.

A syncytin-like endogenous retrovirus envelope gene of the guinea pig specifically expressed in the placenta junctional zone and conserved in Caviomorpha

Syncytins are genes of retroviral origin that have been co-opted by mammalian hosts for a function in placentation. Two such genes have already been identified in simians, as well as two distinct, unrelated ones in Muridae and a fifth in the rabbit.Here we searched for similar genes in the guinea pig, which belongs to the Caviomorpha lineage within the Hystricognathi suborder of rodents and displays a placental structural organization with several characteristic features comparable to those of the human organ, including deep trophoblast invasion of maternal tissues.An in silico search for envelope (env) genes with full coding capacity identified a candidate gene that showed specific expression in the placenta, as revealed by RT-qPCR using RNAs from a large panel of tissues. This gene belongs to an endogenous retroviral element present at a single-copy in the guinea pig genome, still displaying a retroviral organization – with a degenerate gag and pol, but an intact env gene. In situ hybridization of guinea pig placenta sections demonstrated specific expression at the level of the invasive trophoblast-containing junctional zone, as observed in humans for syncytin-1 and consistent with a role in invasion of the maternal uterine tissues. The identified gene displays a conserved open reading frame in the Caviomorpha, consistent with an entry date >30 million years, and sequence analyses showed purifying selection of the gene. Conclusively, despite the absence of a demonstrated fusogenic activity, it is likely that the identified env gene – that we named syncytin-like env-Cav1 – exerts a physiological function possibly related to trophoblast invasion, in the course of caviomorph placentation.

Endocrine Activity of Extraembryonic Membranes Extends beyond Placental Amniotes

BackgroundDuring development, all amniotes (mammals, reptiles, and birds) form extraembryonic membranes, which regulate gas and water exchange, remove metabolic wastes, provide shock absorption, and transfer maternally derived nutrients. In viviparous (live-bearing) amniotes, both extraembryonic membranes and maternal uterine tissues contribute to the placenta, an endocrine organ that synthesizes, transports, and metabolizes hormones essential for development. Historically, endocrine properties of the placenta have been viewed as an innovation of placental amniotes. However, an endocrine role of extraembryonic membranes has not been investigated in oviparous (egg-laying) amniotes despite similarities in their basic structure, function, and shared evolutionary ancestry. In this study, we ask whether the oviparous chorioallantoic membrane (CAM) of chicken (Gallus gallus) has the capability to synthesize and receive signaling of progesterone, a major placental steroid hormone.Methodology/Principal FindingsWe quantified mRNA expression of key steroidogenic enzymes involved in progesterone synthesis and found that 3β-hydroxysteroid dehydrogenase, which converts pregnenolone to progesterone exhibited a 464 fold increase in the CAM from day 8 to day 18 of embryonic development (F5, 68 = 89.282, p<0.0001). To further investigate progesterone synthesis, we performed explant culture and found that the CAM synthesizes progesterone in vitro in the presence of a steroid precursor. Finally, we quantified mRNA expression and performed protein immunolocalization of the progesterone receptor in the CAM.Conclusions/SignificanceCollectively, our data indicate that the chick CAM is steroidogenic and has the capability to both synthesize progesterone and receive progesterone signaling. These findings represent a paradigm shift in evolutionary reproductive biology by suggesting that endocrine activity of extraembryonic membranes is not a novel characteristic of placental amniotes. Rather, we hypothesize that these membranes may share an additional unifying characteristic, steroidogenesis, across amniotes at large.

Expression of Genes in the Canine Pre‐implantation Uterus and Embryo: Implications for an Active Role of the Embryo Before and During Invasion

The aim of the present study was to assess genes expressed in maternal uterine tissue and pre-implantation embryos which are presumably involved in maternal recognition and establishment of canine pregnancy. For this purpose, 10 pregnant bitches were ovariohysterectomized between days 10 and 12 after mating. Four non-pregnant bitches served as controls. Early pregnancy was verified by flushing the uterine horns with PBS solution. The collected embryos (n = 60) were stored deep-frozen (-80 degrees C). Uterine tissue was excised, snaps frozen in liquid nitrogen and homogenized using TRI Reagent. All embryos from one litter were thawed together and also homogenized in TRI Reagent. RT-PCR was performed to prove mRNA expression of progesterone receptor, key enzymes of the prostaglandin synthesis pathway, selected growth factors, cytokines, immune cell receptors, major histocompatibility complex (MHC) and matrix-metalloproteinases (MMP). Only pregnant uteri revealed the presence of mRNA for interferon (IFN)-gamma, IL-4 and CD-8, which resembles the milieu in humans and other mammalians. Similarly, in day 10 embryos, mRNA for transforming growth factor-beta, insulin-like growth factor-1,-2, hepatocyte growth factor, leukaemia inhibitor factor, tumour necrosis factor-alpha, interleukin-1beta,-6,-8, cyclooxygenase-2, CD4(+) cells, and MMP-2 and -9 were detected, but not MHC-I or -II. We therefore suppose that the canine embryo, like its human counterpart, actively initiates measures to prevent attacks from the maternal immune system to prepare its own adhesion, nidation, growth and further development.

Valproate, thalidomide and ethyl alcohol alter the migration of HTR-8/SVneo cells

BackgroundValproate, thalidomide and alcohol (ethanol) exposure during the first trimester of pregnancy is known to cause several developmental disorders. All these teratogens are known to pass the placental barrier and interfere directly with the normal development of the fetus. However, these teratogens also alter the formation and function of the placenta itself which may in turn affect the proper nourishment and development of the fetus. Optimum development of the placenta requires adequate invasion of trophoblast into the maternal uterine tissues. Changes in the migratory behavior of trophoblast by maternal exposure to these teratogens during placentogenesis may therefore alter the structure and function of the placenta.MethodsIn the present study, the effects of sodium valproate, thalidomide and alcohol on the migration of human first trimester trophoblast cell line (HTR-8/SVneo) were examined in vitro. Cells were cultured in the wells of 48-well culture plates as mono or multilayers. Circular patches of cells were removed from the center of the wells by suction, and the migration of cells into the wound was studied using microscopy. Effects of low and high concentrations of valproate, thalidomide and alcohol were examined on the healing of wounds and on the migration rate of cells by determining the wound areas at 0, 3, 6, 12, 24 and 48 h. Effects of drugs and alcohol on the proliferation and the expression levels of integrin subunits beta1 and alpha5 in cells were examined.ResultsThe migration rates of trophoblast differed between wounds created in mono and multilayers of cells. Exposure to teratogens altered the migration of trophoblast into mono and multilayer wounds. The effects of valproate, thalidomide and alcohol on the proliferation of cells during the rapid migratory phase were mild. Drug exposure caused significant changes in the expression levels of beta1 and alpha5 integrin subunits.ConclusionResults suggest that exposure to valproate, thalidomide or alcohol during the first trimester of pregnancy may change the ultrastructure of the placenta by altering the migration of trophoblast cells and this effect may be mediated by drug- or alcohol-induced changes in the expression levels of beta1 and alpha5 integrin subunits.

Elevated expression of N-acetylglucosaminyltransferase V in first trimester human placenta

In early pregnancy, placental trophoblast cells rapidly grow and invade into maternal uterine tissue. N-Acetylglucosaminyltransferase V (GnT-V) and its product, β1-6-GlcNAc branching glycan, are known to correlate with tumor invasion and metastasis. Since the placentation process resembles invasion of cancer cells, we examined the expression of β1-6-GlcNAc branching glycan and GnT-V in human placenta. Placentas derived from the first trimester contained a larger amount of β1-6-GlcNAc branching glycan, detected by leukoagglutinating phytohemagglutinin lectin blotting, than those at term. Immunohistochemical study revealed that β1-6-GlcNAc branching glycans and GnT-V protein were localized in the trophoblast layer. Both protein expression and the enzyme activity of GnT-V in first trimester placentas were higher than those at term. These results suggest that GnT-V would contribute to placentation in the early phase of pregnancy, possibly regulating the process of invasion of trophoblast cells.

Extravillous trophoblast apoptosis – A workshop report

During normal pregnancy, extravillous trophoblast cells invade maternal uterine tissues. The interstitial trophoblast penetrates decidual tissues reaching the inner third of the myometrium. A subset of the interstitial trophoblast, the intramural/endovascular trophoblast transforms uterine spiral arteries into large-bore conduits to enable the adequate supply of nutrients and oxygen to the placenta and thus the fetus. Control of invasion is still a mystery and therefore, in this workshop report already existing concepts as well as new models are discussed. Maternal cells such as macrophages and endothelial cells have a clear impact on trophoblast invasion and apoptosis. However, the trophoblast cells need to be susceptible to undergo apoptosis. Thus, an intrinsic program within the trophoblast needs to be activated before induction from the outside can be successful. Quantification of apoptosis further clarified that apoptosis of interstitial trophoblast is not the ultimate means to lead to pathologically shallow invasion. On the other hand, apoptosis of intramural/endovascular trophoblast seems to be highly relevant for a correct transformation of spiral arteries.

Role of 14‐bp deletion in the HLA‐G gene in the maintenance of pregnancy

Differential expression of human leukocyte antigens (HLAs) on trophoblast has been the focus of many studies, specially on extravillous cytotrophoblast cells, which migrates into the maternal uterine tissues. These invading cells do not express classical major histocompatibility complex class I (-A and -B) and class II molecules, along with low expression of HLA-C. HLA-G is the predominantly expressed antigen along with HLA-E. Hence, it is believed that expressed antigens may be involved in materno-fetal tolerance. In the present study, we have studied 14-bp deletion polymorphism in the exon-8 of the non-classical HLA-G antigen. There was no difference in the frequency of deletion/insertion polymorphism in fertile normal women and recurrent spontaneous abortion (RSA) women. However, the number of heterozygotes (-14b/+14b) were increased in RSA women. The probable mechanism for the increase of heterozygotes in recurrent fetal loss is discussed in light of soluble HLA-G.

Pre-eclampsia and Maternal Anaemia Display Reduced Apoptosis and Opposite Invasive Phenotypes of Extravillous Trophoblast

During pregnancy extravillous trophoblast invades maternal uterine tissues and remodels spiral arteries. Maternal anaemia and early onset pre-eclampsia are associated with perturbed trophoblast biology. We systematically compared numerical density, invasive depth and apoptosis rates of extravillous trophoblast in uterine tissues taken from hysterectomies following Caesarean section after normal pregnancies (n=4) or pregnancies complicated by pre-eclampsia (n=5) or anaemia (n=6). Full thickness sections of the placental bed were studied by immunohistochemistry using anti-active caspase 3, anti-cytokeratin 7, anti-lamin B, M30, Mib-1, anti-PARP, and by the TUNEL assay. In normal pregnancy extravillous trophoblast invaded 2.04±0.19mm (mean±sem ) from the endometrial–myometrial border into the myometrium; in pre-eclampsia 0.67±0.14mm (P< 0.01), and in anaemia 3.84±0.21mm (P< 0.001). The endometrial trophoblast density in normal pregnancy was 2.44±0.37 cells per 60000μm3, in pre-eclampsia was 1.04±0.15 (P< 0.01), and in anaemia was 3.10±0.32. The rate of apoptotic extravillous trophoblast (M30-positive) in the endometrium in normal pregnancy was 7.17±1.46 per cent, in pre-eclampsia 4.4±0.71, and in anaemia 2.1±0.42 (P< 0.01). Maternal anaemia leads to general tissue hypoxia throughout gestation. Increased invasive depth could be explained by hypoxia-stimulated mitosis and decreased apoptosis of extravillous trophoblast. Reduced trophoblast invasion in pre-eclampsia cannot be explained by higher rates of apoptosis.

The Type 2 Iodothyronine Deiodinase Is Expressed in the Rat Uterus and Induced During Pregnancy*

Thyroid hormones are of considerable importance for vertebrate reproductive function and during development. To further assess the role of these compounds in this capacity, we examined the expression pattern of the type 2 iodothyronine deiodinase (D2), which converts T(4) to the more active hormone T(3), in the rat uterus in both the nonpregnant and the pregnant state. D2 activity was identified as the predominant, if not only, 5'-deiodinase in the nonpregnant rat uterus. The expression of D2 messenger RNA was located by in situ hybridization to the endometrial stromal cells, where the signal was particularly enriched in the region adjacent to the epithelial cells of the uterine lumen. During pregnancy, D2 activity increased, peaking on day 17 of gestation (embryonic day 17). At that time, uterine D2 activity exceeded that in the placenta, as well as that in the fetal tissues. In the earlier stages of pregnancy before placental formation (e.g. embryonic days 10-11), D2 messenger RNA in the rat uterus was located outside the decidual tissue, which was observed, as in previous studies, to highly express the inactivating type 3 deiodinase. In summary, the rat uterus, particularly during pregnancy, seems to be a site of active thyroid hormone metabolism, presumably designed to maintain the optimal thyroid hormone environment for both the fetus and the maternal uterine tissue.

Glucocorticoid Regulation of Human and Ovine Parturition: The Relationship Between Fetal Hypothalamic-Pituitary-Adrenal Axis Activation and Intrauterine Prostaglandin Production

Birth in many animal species and in humans is associated with activation of hypothalamic-pituitary-adrenal function in the fetus and the increased influence of glucocorticoids on trophoblast cells of the placenta and fetal membranes. We suggest that in ovine pregnancy glucocorticoids directly increase fetal placental prostaglandin production, and indirectly increase prostaglandin production by maternal uterine tissues through the stimulation of placental estradiol synthesis. The events of ovine parturition are compared with those of human parturition. In the latter, we suggest similar direct effects of glucocorticoids on prostaglandin synthesis and metabolism in fetal membranes and similar indirect effects mediated by glucocorticoid-stimulated increases in intrauterine corticotropin-releasing hormone expression.

Screening for genes involved in tissue invasion based on placenta formation and cancer cell lines with low and high metastatic potential

During the formation of placenta, trophoblast cells vigorously invade maternal uterine tissues, sharing many features with the invasion of cancers. We applied RNA differential display to placenta tissues from 8.5 to 17.5 days post-coitus (dpc) ICR mice, and isolated 188 cDNA fragments expressed differentially. Among the 25 known cDNA fragments thus far analyzed, six cDNAs have been reported to be relevant to tumor invasion and/or metastasis. Furthermore, 11 of 20 unknown cDNAs isolated showed differential expression between the pairs of cancer cell lines with low and high metastatic potential, indicating potential usefulness of the present two-step approach.

Localization of prostaglandin synthase type-1 (PGHS-1) mRNA and prostaglandin synthase type-2 (PGHS-2) mRNA in ovine myometrium and endometrium throughout gestation.

Increased prostaglandin production by tissues in the sheep uterus and placenta are thought to be important for the onset of parturition. In the sheep placenta, this is most likely due to increased expression of prostaglandin synthase type-2 (PGHS-2) rather than prostaglandin synthase type-1 (PGHS-1). However, there is no information concerning expression of PGHS isoenzymes in maternal uterine tissues during pregnancy. Therefore, the purpose of the present study was to examine the expression of PGHS-1 and PGHS-2 in the sheep myometrium and endometrium during late gestation using in situ hybridization and immunohistochemistry. Using (35)S-labelled oligonucleotide probes, which give specific hybridization signals in other tissues, we localized PGHS-2 mRNA to endometrial epithelium, and apparently to other cells in both endometrium and myometrium. This artefactual signal was still present with 100-fold excess unlabelled oligonucleotide probe and with sense probes, but was resolved with the use of (33)P-oligonucleotides. Using (33)P-labelled oligonucleotide probes we could not detect either PGHS-1 or PGHS-2 mRNA in myometrium, and found expression only of PGHS-2 mRNA in endometrium. PGHS-2 mRNA localized to the endometrial epithelium and was undetectable in glandular epithelium. The level of PGHS-2 expression rose significantly between days 80 and 85 of pregnancy and term, and this corresponded to the appearance of immunoreactive PGHS-2 protein, measured by immunohistochemistry, in the endometrial epithelium. Therefore we conclude that (33)P-labelled probes are preferred for detection of mRNAs encoding PGHS-2 in ovine uterine tissues. Expression of PGHS-2 mRNA is greater than that of PGHS-1, increases during gestation, and predominates in the endometrial epithelium, consistent with the site of PGHS-2 protein localization.