Closure of the uterine lumen and the plasma membrane transformation do not require blastocyst implantation

Ultrastructural changes in the plasma membrane of uterine epithelial cells in the pseudopregnant rat were examined to determine if these changes resemble those found during normal pregnancy and also to examine if the well-known membrane alterations of early pregnancy are intrinsic to uterine epithelial cells. Changes in the surface contours of uterine epithelial cells from the afternoon of day 6 to the morning of day 9 of pseudopregnancy were similar to those present after attachment in normal pregnancy although somewhat delayed. The presence of short, irregular microvilli was seen from as early as day 7 of pseudopregnancy, with regular microvilli returning to the epithelial surface by days 8-9 of pseudopregnancy but to a slightly lesser extent as compared to normal pregnancy. Furthermore, observations made on the afternoon of day 6 to the morning of day 7 of pseudopregnancy showed that the uterine lumen was closed down and that complete membrane flattening between opposing uterine epithelial cells was seen all along the uterus in the absence of a blastocyst. These observations establish that the "plasma membrane transformation" does not depend on blastocyst implantation.     

Cytoskeletal control of the apical surface transformation of rat uterine epithelium

Summary— During early pregnancy, in the lead up to blastocyst implantation, the apical cell surface of luminal epithelial cells of the rat uterus undergo a dramatic shape transformation. This study aims to investigate the role of the cytoskeleton in this apical transformation by considering the effects of the drugs cytochalasin D and colchicine on the uterine luminal cell surface. The results are determined using transmission and scanning electron microscopy. In vivo exposure to cytochalasin D during oestrus, as well as on day 1 of pregnancy, did not affect the long, regular surface microvilli. This drug, however, did disrupt the terminal web within the apical cytoplasm of these cells. Disruption of microfilament (MF) polymerization by cytochalasin D on day 4 of pregnancy induced a cell surface transformation, resulting in the appearance of numerous irregular projections normally present during blastocyst implantation on day 6 of pregnancy. Colchicine did not alter the uterine microvilli of oestrus or day 1 pregnant tissue. Unlike the effect of cytochalasin D, colchicine-induced microtubule (MT) disruption on day 4 of pregnancy did not increase irregular projections and hence this treatment did not result in the cell surface appearance associated with blastocyst implantation. These results indicate that the disruption of MF, rather than MT, contributes to the transformation of the uterine luminal cell surface during the lead up to blastocyst attachment.     

Junctional adhesion molecule 2 mediates the interaction between hatched blastocyst and luminal epithelium: induction by progesterone and LIF

Background

Junctional adhesion molecule 2 (Jam2) is a member of the JAM superfamily. JAMs are localized at intercellular contacts and participated in the assembly and maintenance of junctions, and control of cell permeability. Because Jam2 is highly expressed in the luminal epithelium on day 4 of pregnancy, this study was to determine whether Jam2 plays a role in uterine receptivity and blastocyst attachment in mouse uterus.

Methodology/Principal Findings

Jam2 is highly expressed in the uterine luminal epithelium on days 3 and 4 of pregnancy. Progesterone induces Jam2 expression in ovariectomized mice, which is blocked by progesterone antagonist RU486. Jam2 expression on day 4 of pregnancy is also inhibited by RU486 treatment. Leukemia inhibitory factor (LIF) up-regulates Jam2 protein in isolated luminal epithelium from day 4 uterus, which is blocked by S3I-201, a cell-permeable inhibitor for Stat3 phosphorylation. Under adhesion assay, recombinant Jam2 protein increases the rate of blastocyst adhesion. Both soluble recombinant Jam2 and Jam3 can reverse this process.

Conclusion

Jam2 is highly expressed in the luminal epithelium of receptive uterus and up-regulated by progesterone and LIF via tyrosine phosphorylation of Stat3. Jam2 may play a role in the interaction between hatched blastocyst and receptive uterus.     

ICAM-2 and lipid rafts disappear from the basal plasma membrane of uterine epithelial cells during early pregnancy in rats

Adhesion molecules are redistributed in rat uterine epithelial cells (UECs) during early pregnancy for endometrial receptivity and implantation. Intercellular adhesion molecule-2 (ICAM-2) is located as an oligomer on the basal plasma membrane of non-receptive UECs on day 1 of pregnancy and colocalizes with the lipid raft marker flotillin-2. At the time of implantation in rats and in ovariectomized rats primed with progesterone, ICAM-2 disappears from the basal plasma membrane and lipid rafts redistribute to the apical membrane. The loss of ICAM-2 might render UECs less adherent to the underlying basal lamina and more prone to apoptosis. Flotillin-2 in the apical plasma membrane at the time of implantation might provide an anchoring point for several adhesion molecules that are known to localize to this region at this time. We suggest that flotillin-2 is involved with adhesion between UECs and the implanting blastocyst, whereas ICAM-2 is associated with the ability for UECs to be removed at the time of implantation.     

Alkaline phosphatase distribution in the plasma membrane of uterine epithelial cells is markedly altered during early pregnancy in the rat

Ultrastructural and light microscopic cytochemical methods were used to study the distribution and changes in distribution of alkaline phospatase in the apical plasma membrane of rat uterine epithelial cells during different stages of early pregnancy up to the time of attachment of the blastocyst. Reaction product generated by alkaline phosphatase (AP) was located along the apical plasma membrane at each stage investigated. However, a very different organization of reaction product was observed depending on the time during early pregnancy with a continuous pattern appearing all along the microvilli on day 1. This pattern was subsequently converted into a clumped and highly ‘patchy’ appearance around the time of blastocyst attachment by day 6 of pregnancy. This change in pattern and distribution was only seen on the luminal epithelial cells with glandular epithelial cells and blood vessels displaying an unchanging distribution.     

Altered fetal pituitary-adrenal function in the ovine fetus treated with RU486 and meloxicam, an inhibitor of prostaglandin synthase-II

Term and preterm labor are associated with increased fetal hypothalamic-pituitary-adrenal (HPA) activation and synthesis of prostaglandins (PGs) generated through the increased expression of prostaglandin H synthase-II (PGHS-II) in the placenta. Inhibition of PGHS-II has been advocated as a means of producing uterine tocolysis, but the effects of such treatment on fetal endocrine functions have not been thoroughly examined. Because PGE(2) is known to activate the fetal HPA axis, we hypothesized that administration of meloxicam, a PGHS-II inhibitor, to sheep in induced labor would suppress fetal HPA function. Chronically catheterized pregnant ewes were treated with RU486, a progesterone receptor antagonist, to produce active labor, and then treated with either high-maintenance-dose meloxicam, graded-maintenance-dose meloxicam, or a saline infusion. Maternal uterine contraction frequency increased 24 h after the RU486 injection and the animals were in active labor by 48 +/- 4 h. RU486 injection led to increased concentrations of PGE(2), ACTH, and cortisol in the fetal circulation, and increased concentrations of 13,14 dihydro 15-ketoprostaglandin F(2 alpha) (PGFM) in the maternal circulation. Uterine activity was inhibited within 12 h of beginning meloxicam infusion at both infusion regimes. During meloxicam infusion there were significant decreases in fetal plasma PGE(2), ACTH, and cortisol concentrations, and PGFM concentrations in maternal plasma. In control animals, frequency of uterine contractions, maternal plasma PGFM, fetal plasma PGE(2), ACTH, and cortisol concentrations increased after RU486 administration, and continued to rise during saline infusion until delivery occurred. We conclude that RU486-provoked labor in sheep is associated with activation of fetal HPA function, and that this is attenuated during meloxicam treatment to a level considered compatible with pregnancy maintenance.     

CD43 is relocated from the basal to the apical plasma membrane of rat uterine epithelial cells by progesterone

Adhesion molecules play an important part in preparing uterine epithelial cells for receptivity to the implanting embryo, and their rearrangement is crucial in allowing successful implantation. CD43 is an adhesion molecule which has previously been suggested to take part in implantation in mice. Indirect immunofluorescence microscopy localising CD43 was performed on uterine tissue during early pregnancy, and tissue obtained from ovariectomised rats administered with ovarian hormones. Western blotting was performed during early pregnancy on isolated epithelial cells and ovariectomised rats for comparison of the amount of CD43. Immunofluorescence microscopy showed CD43 was situated basally in uterine luminal epithelial cells on day 1 of pregnancy and during oestrogen administration, corresponding to a 95-kDa band of CD43 seen in western blotting. At the time of implantation, and during progesterone or progesterone plus oestrogen combined treatment, CD43 is apical in uterine luminal epithelial cells, resulting in an 85-kDa form of CD43. We suggest that a de-glycosylated form of CD43 moves from basally to apically at the time of implantation, thus facilitating blastocyst attachment to uterine epithelial cells as well as their removal.     

Ovarian hormones regulate expression of the focal adhesion proteins,talin and paxillin,in rat uterine luminal but not glandular epithelial cells

During early pregnancy in the rat, focal adhesions disassemble in uterine luminal epithelial cells at the time of implantation to facilitate their removal so that the implanting blastocyst can invade into the underlying endometrial decidual cells. This study investigated the effect of ovarian hormones on the distribution and protein expression of two focal adhesion proteins, talin and paxillin, in rat uterine luminal and glandular epithelial cells under various hormone regimes. Talin and paxillin showed a major distributional change between different hormone regimes. Talin and paxillin were highly concentrated along the basal cell surface of uterine luminal epithelial cells in response to oestrogen treatment. However, this prominent staining of talin and paxillin was absent and also a corresponding reduction of paxillin expression was demonstrated in response to progesterone alone or progesterone in combination with oestrogen, which is also observed at the time of implantation. In contrast, the distribution of talin and paxillin in uterine glandular epithelial cells was localised on the basal cell surface and remained unchanged in all hormone regimes. Thus, not all focal adhesions are hormonally dependent in the rat uterus; however, the dynamics of focal adhesion in uterine luminal epithelial cells is tightly regulated by ovarian hormones. In particular, focal adhesion disassembly in uterine luminal epithelial cells, a key component to establish successful implantation, is predominantly under the influence of progesterone.     

RU 486 completely inhibits the action of progesterone on cell proliferation in the mouse uterus

The antiprogestagen RU 486 completely inhibited the progesterone-induced switch in cell proliferation from the luminal and glandular epithelia to the stroma in response to oestradiol-17 beta. It also inhibited the progesterone-induced differentiation of the uterine epithelium. Since the proliferative switch of the uterus and the differentiation of the epithelium are prerequisites for implantation, these inhibitory actions may, in part, explain the ability of RU 486 to prevent implantation. Furthermore, it also suggests that the proliferative response to oestradiol in the presence of progesterone may be a sensitive assay for compounds with anti-progestational activity.     

Reorganization of the apical cytoskeleton of uterine epithelial cells during early pregnancy in the rat: a study with myosin subfragment 1.

Actin filaments were identified in the epithelial cells of rat uterus following detergent extraction and decoration of microfilaments (MF) with myosin subfragment 1 (S1). MF connections with cytoplasmic organelles and the apical plasma membrane are also described. Transmission electron microscopy revealed that the regular microvilli of non-pregnant, oestrous animals contain several decorated MF with rootlets descending into a densely filamentous terminal web. Following mating, the actin cytoskeleton was examined on days 1, 3 and 6 of pregnancy. In this period, the irregular projections that replace MV assumed an underlying, dense network of decorated MF, whilst smoother surfaces displayed few cytoplasmic filaments. At the time of blastocyst implantation, a structured terminal web was no longer present. Structural details were revealed concerning the contents of large, bleb-like projections found on the apical surface.     

A genomic approach to identify novel progesterone receptor regulated pathways in the uterus during implantation

The cellular actions of steroid hormone progesterone (P) are mediated via its nuclear receptors, which regulate the expression of specific target genes. The identity of gene networks that are regulated by the P receptors (PRs) in the uterus at various stages of the reproductive cycle and pregnancy, however, remain largely unknown. In this study, we have used oligonucleotide microarrays to identify mRNAs whose expression in the pregnant mouse uterus is modulated by RU486, a well-characterized PR antagonist, which is also an effective inhibitor of implantation. We found that, in response to RU486, expression of mRNAs corresponding to 78 known genes was down-regulated at least 2-fold in the preimplantation mouse uterus. The PR regulation of several of these genes was ascertained by administering P to ovariectomized wild-type and PR knockout (PRKO) mice. Detailed spatio-temporal analysis of these genes in the pregnant uterus indicated that their expression in the epithelium and stroma could be correlated with the expression of PR in those cell types. Furthermore, time-course studies suggested that many of these genes are likely primary targets of PR regulation. We also identified 70 known genes that were up-regulated at least 2-fold in the pregnant uterus in response to RU486. Interestingly, initial examination of a number of RU486-inducible genes reveals that their uterine expression is also regulated by estrogen. The identification of several novel PR-regulated gene pathways in the reproductive tract is an important step toward understanding how P regulates the physiological events leading to implantation.     

Viviparous lizard, Eulamprus tympanum, shows changes in the uterine surface epithelium during early pregnancy that are similar to the plasma membrane transformation of mammals

The "plasma membrane transformation" describes a series of ultrastructural, biochemical, and morphological changes that occur in the uterus of many mammals at the time of blastocyst attachment. These changes, regardless of placental type or length of gestation, include alterations to microvillar length and density and the presence or absence of pinopods or uterodomes. Scanning electron microscopy (SEM) was used to 1) document the topographical ultrastructure of the uterus of Eulamprus tympanum, an eastern Australian viviparous skink with a simple chorioallantoic placenta, for the first time; and 2) determine whether changes identified as "plasma membrane transformation" in mammals occur in E. tympanum. Tissues collected over three seasons from nonreproductive subadult females, preovulatory, postovulatory, and early to mid-gestational females were examined. At low magnification the uterine epithelium of subadults displays a distinctive pattern of tissue folding that includes rectangular areas of tissue delineated by deep lateral and transverse folds. At higher magnification, the uterine epithelium surface is composed of two dominant cell types, i.e., those covered by microvilli and ciliated cells. The folding pattern observed in subadults is less evident in vitellogenic females and the cell surfaces appear highly secretory, with bulging cell apices. Tissue from postovulatory lizards has no distinctive folding pattern and cell surfaces are frequently smooth and lack microvilli. Uterine egg chambers lack ciliated cells at the embryonic pole, but display abundant secretory droplets. Thus, the uterus of E. tympanum undergoes a plasma membrane transformation. The scope of this transformation is not fully understood but may be related to the complexity of placental structure and the development of the embryo/fetus at parturition.     

Progesterone, but not luteal estrogen, is required for the establishment of pregnancy in the new world monkey Cebus apella

The aim of this study was to examine the requirement of luteal progesterone or luteal estrogen for the establishment of pregnancy in the Cebus monkey and to test in a primate species the synergism between RU 486 and letrozole (LTZ) found in rodents for inhibiting implantation. Exposure of target tissues to either hormone was suppressed during the mid-luteal phase of mating cycles by subcutaneous administration of the antiprogestin (RU 486), the aromatase inhibitor LTZ or the antiestrogen (ICI 182780) on days 4-7 of the luteal phase. Administration of 0.1 or 0.5 mg/kg of LTZ on days 5-7 of the luteal phase caused a profound drop in the levels of E(2) in all animals, whereas administration of ICI 182780 0.2 mg/kg on days 4-6 of the luteal phase had the opposite effect. The pregnancy rate in vehicle treated cycles of the same females was (58.3%). Treatment with RU 486, 0.8 mg/kg/day on days 5-7 of the luteal phase-induced endometrial bleeding in 3/5 mated females none of which became pregnant, whereas pregnancy was confirmed in one of the two animals that did not bled. Treatment with RU 486, 0.4 mg/kg/day alone or with LTZ on days 5-7 or ICI 182780 alone, on days 4-6 of the luteal phase failed to induce bleeding, allowing the establishment of pregnancy in 50.0-66.6% of the animals in these groups. We conclude that in Cebus monkeys, progesterone but not luteal estradiol is required for the establishment of pregnancy and that RU 486 and LTZ do not exhibit in this species the synergism found in rodents.     

Uterine vascular permeability, blood flow and extracellular fluid space during implantation in rats

Vascular permeability to plasma proteins in uterine implantation and non-implantation sites (i.e. dye sites and non-dye sites) was assessed quantitatively by a method which accounts for steady-state volumes of distribution. Extracellular fluid volume and uterine blood flow were also determined. On both the evening of Day 5 and the morning of Day 6, vascular permeability to 125I-labelled human serum albumin, extracellular fluid volume and blood flow were significantly increased in implantation sites compared to non-implantation sites. Vascular permeability in implantation sites was increased significantly between Days 5 and 6, whereas that in non-implantation sites was unchanged. This increase in vascular permeability between Days 5 and 6 was not accompanied by further increases in extracellular fluid volume and blood flow. This result shows a dissociation between vascular permeability and extracellular fluid volume immediately after the onset of implantation and raises important questions as to whether the rat uterus undergoes a truly oedematous response at implantation as has been generally accepted.     

Regulation of uterine progesterone receptors by the nonsteroidal anti-androgen hydroxyflutamide

We have recently reported that the anti-androgen hydroxyflutamide causes delayed implantation and exhibits antideciduogenic activity in the rat. The present experiments were conducted to examine whether hydroxyflutamide binds to the uterine progesterone receptors and/or alters the progesterone binding sites in the uterus. Cytosol and nuclear fractions from decidualized rat uterus were incubated with [3H]-R5020 without or with increasing concentrations of radioinert R5020, RU486, dihydrotestosterone, or hydroxyflutamide. From the log-dose inhibition curves, the relative binding affinity of both hydroxyflutamide and dihydrotestosterone was less than 0.1% and 2%, compared with R5020 (100%) for displacing [3H]-R5020 bound to uterine cytosol and nuclear fractions, respectively. Injection of estradiol-17 beta (1 microgram/rat) to ovariectomized prepubertal rats induced a 1.85-fold increase in uterine weight by 24 h. Hydroxyflutamide at 2.5 or 5.0 mg did not significantly alter the estrogen-induced increase in uterine weight. Compared to vehicle alone, estrogen induced an approximately 5-fold increase in uterine cytosolic progesterone binding sites. Hydroxyflutamide at both 2.5- and 5.0-mg doses significantly attenuated the estrogen-induced elevation in uterine progesterone binding sites. These studies demonstrate that hydroxyflutamide does not bind with high affinity to progesterone receptors, but suppresses the estrogen-induced elevation in progesterone receptor levels in the uterus.     

Profiling of E-cadherin, beta-catenin and Ca(2+) in embryo-uterine interactions at implantation

Establishment of early pregnancy is promoted by a complex network of signalling molecules that mediate cell-to-cell and cell-to-extracellular matrix communications between the receptive endometrium and the invasive trophectoderm. In this study, we have attempted to evaluate the expression profiles of cadherin and catenin during embryo implantation in the mouse. Western blotting studies along with immunocytochemical analysis revealed that E-cadherin is expressed rather ubiquitously in the uterine epithelial cells, distinct enrichment is observed on the apical membrane in the endometrium of peri-implantation uterus specifically at the implantation sites and not at the inter-implanation sites. beta-Catenin also is upregulated and is specifically restricted to apical membrane of epithelial cells of implantation sites. Progesterone induced expression of E-cadherin and 17beta-estradiol regulated the expression of catenin in implantation-delayed uteri. Interestingly, estradiol imparted negative modulation on cadherin expression when co-administered with progesterone. On the contrary, trophoblast exhibits a striking down regulation of cadherin, catenin and Ca(2+) at peri implanting stage. These observations suggest that the trophoblasts exhibited an invasive phenotype while the endometrial epithelium displayed an adhesive phenotype during the window of implantation. Thus, embryo implantation presents an instance where two interacting surfaces showed mutually complementing interaction phenotypes.     

ICAM1 and fibrinogen-γ are increased in uterine epithelial cells at the time of implantation in rats

Uterine epithelial cells transform into a receptive state to adhere to an implanting blastocyst. Part of this transformation includes the apical concentration of cell adhesion molecules at the time of implantation. This study, for the first time, investigates the expression of ICAM1 and fibrinogen‐γ (FGG) in uterine epithelial cells during normal pregnancy, pseudopregnancy and in hormone‐treated rats. An increase (P < 0.05) in ICAM1 was seen at the apical membrane of uterine epithelial cells at the time of implantation compared with day 1 of pregnancy. ICAM1 was also increased (P < 0.05) on day 6 of pseudopregnancy as well as in ovariectomized rats treated with progesterone plus oestrogen. These results show that ICAM1 up‐regulation at the time of implantation is under the control of progesterone, and is not dependent on cytokine release from the blastocyst or in semen. FGG dimerization increased (P < 0.05) on day 6 of pregnancy compared with day 1, and was not up‐regulated in day 6 pseudopregnant animals, suggesting this increase is dependent on a developing blastocyst. The presence of ICAM1 and FGG in the uterine epithelium at the time of implantation in the rat is similar to that seen in lymphocyte–endothelium adhesion, and we suggest a similar mechanism in embryo–uterine epithelium adhesion is utilized. Mol. Reprod. Dev. 78:318–327, 2011. © 2011 Wiley‐Liss, Inc.