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During early pregnancy in the rat, the luminal uterine epithelial cells (UECs) must transform to a receptive state to permit blastocyst attachment and implantation. The implantation process involves penetration of the epithelial barrier, so it is expected that the transformation of UECs includes alterations in the lateral junctional complex. Previous studies have demonstrated a deepening of the tight junction (zonula occludens) and a reduction in the number of desmosomes (macula adherens) in UECs at the time of implantation. However, the adherens junction (zonula adherens), which is primarily responsible for cell-cell adhesion, has been little studied during early pregnancy. This study investigated the adherens junction in rat UECs during the early stages of normal pregnancy and ovarian hyperstimulated (OH) pregnancy using transmission electron microscopy. The adherens junction is present in UECs at the time of fertilisation, but is lost at the time of blastocyst implantation during normal pregnancy. Interestingly, at the time of implantation after OH, adherens junctions are retained and may impede blastocyst penetration of the epithelium. The adherens junction anchors the actin-based terminal web, which is known to be disrupted in UECs during early pregnancy. However, artificial disruption of the terminal web, using cytochalasin D, did not cause removal of the adherens junction in UECs. This study revealed that adherens junction disassembly occurs during early pregnancy, but that this process does not occur during OH pregnancy. Such disassembly does not appear to depend on the disruption of the terminal web.

Summary Regulation of the uterine luminal environment is important for successful attachment and implantation of the blastocyst. The contents and volume of luminal fluid are regulated in part by the tight junctions. Using immunofluorescence microscopy, protein and RNA analysis, the cellular distributions of tight junction components claudins and occludin were observed during early pregnancy and under various hormonal regimens. Results indicate that occludin and claudin-4 distribution changed during early pregnancy and in response to ovarian hormones. At the time of implantation and in response to progesterone administration to ovariectomised rats, occludin and claudin-4 showed increased immunolabelling in luminal epithelium. Interestingly, occludin protein detection in uterine luminal epithelial cells at the time of implantation was statistically significantly decreased at the time of implantation compared to day 1 of pregnancy. This suggests that a cytoplasmic pool of occludin is present at day 1 of pregnancy and is redistributed to the tight junctions at the time of implantation. The presence of occludin and claudin-4 in the tight junctions at the time of implantation and in response to progesterone suggests that the paracellular pathway is impermeable to water and Na + at this time, and that the transport of such substances takes place via the transcellular pathway.

The aim of this study was to investigate the presence of aquaporins 1, 4 and 5 in uterine epithelial cells during the early stages of pregnancy in the rat. Immunofluorescent and immunogold techniques showed that there was a shift of aquaporin 5 to the apical surface of uterine epithelial cells in the mesometrial pole of the uterus at the time of implantation. This study also revealed the absence of aquaporin 4 in the rat uterus during early pregnancy. Aquaporin 1 was observed in stromal blood vessels but there was no change in uterine epithelial cells during early pregnancy. These results suggest that aquaporin 5 plays an important role in the removal of uterine luminal fluid at the time of implantation in the rat and may contribute to the antimesometrial positioning of the implanting blastocyst.

Immunohistochemical staining of 5 cytoskeletal proteins (actin, alpha-actinin, gelsolin, plectin and plakoglobin) was used to investigate changes in distribution patterns of these proteins after the period of uterine receptivity for blastocyst implantation in the rat. Actin was found throughout the cytoplasm but it was concentrated along the apical plasma membrane on day 1 of pregnancy, decreased by day 6 and then increased again at day 9. Alpha-actinin and gelsolin were localized in distinctive bands along the apical plasma membrane at day 6 of pregnancy but became diffusely distributed at day 9. Plectin was localized along the apical and basal plasma membranes at day 6 but in higher amounts apically and at day 9, it was concentrated in apical and basal zones in the cells. Plakoglobin was found along the lateral and basal membranes with increased intensity along the apical third of the lateral plasma membrane from day 6 to day 9 of pregnancy. These results show that all 5 cytoskeletal proteins redistributed after the period of uterine receptivity: some exhibited a similar pattern of labelling to that found during the prereceptive state, whereas others only partially returned to the pre-receptive state. This change in distribution patterns may reflect differences in the epithelial barrier function before and after the period of receptivity.

Summary Using immunohistochemical methods at both light and electron microscopical levels, we have studied epidermal growth factor in the uterine epithelium during early pregnancy and up to the time of blastocyst implantation. We report that the distribution of this growth factor changed markedly over the period of study and that it was gradually de-expressed across the entire uterine epithelium as pregnancy advanced towards the time of implantation. At the electron microscopical level, immunogold labelling clearly showed label associated with both lateral and basal plasma membranes very early in pregnancy but not by the time of implantation. We suggest that the de-expression of this molecule may indicate its role in the removal of mechanisms which inhibit uterine receptivity for implantation.

The extensive actin cytoskeletal remodelling of the uterine stroma during early pregnancy involves changes in actin-binding proteins. This study provides the first detailed localisation of the actin-binding protein, moesin, in rat uterine endometrium during this period. Western blot analysis and immunofluorescence microscopy showed that the amount of moesin in the uterus peaked at the time of implantation, corresponding to the presence of intensely immunolabelling decidual cells. Furthermore, moesin increased in active membrane/cytoskeleton bound protein at the time of implantation, concomitantly decreasing in cytosolic protein. The increase of moesin at decidualisation corresponds with the appearance of alpha smooth muscle actin (alphaSMA) suggesting that decidual cells have contractile abilities that may aid in containing an invasive trophoblast. The results of this study suggest that moesin is important in developing a specialised cytoskeleton and increased adhesiveness of decidual cells, possibly functioning to bridge adhesion molecules to the underlying cytoskeleton.

Summary Non-specific acid phosphatase activity was studied in the rat uterine luminal and glandular epithelium at oestrous, late dioestrous, and 6th d of pregnancy using the substrates β-glycerophosphate and p -nitrophenyl phosphate, at both pH = 5.0 and pH = 6.0. At late dioestrous and 6th d of pregnancy, a significantly larger lysosomal population in the luminal and glandular epithelium was operating at pH = 5.0 rather than pH = 6.0. At oestrous, the epithelial lysosomes showed no preference for an operational pH = 5.0 or 6.0. Uniquely, the luminal epithelial lysosomes showed a significant preference for the hydrolysis of p-nitrophenyl phosphate over β-glycerophosphate at 6th d of pregnancy, compared to no substrate preference at oestrous and late dioestrous. The activity of 5-Nucleotidase was also estimated semi-quantitatively at oestrous, late dioestrous, and 6th d of pregnancy in the luminal and glandular epithelium. 5-Nucleotidase activity was greatest in the luminal and glandular epithelium at 6th d of pregnancy. These findings are considered with respect to the possible mechanisms involved in implementing the changes and the relevance to blastocyst implantation.

Summary In this retrospective and quantitated study on banked tissue we found that, compared to normal uterine epithelial cells, growth hormone (GH) is increased 3.4-fold in endometriosis and 3.8-fold in endometrial adenocarcinoma. Similarly, interleukin-6 (IL-6) is increased 2.4-fold in endometriosis and 4.4-fold in endometrial adenocarcinoma. These proteins appear to be involved in the progression of both these conditions. GH is particularly interesting in this context since it is known to not only promote cellular proliferation but also reduces cell–cell adhesion, thus allowing individual cells to break away from their parent architecture. Our results suggest that both IL-6 and GH may play a role in the progression of both endometriosis and endometrial carcinoma.

Summary Uterine epithelium undergoes dramatic changes during early pregnancy in preparation for implantation. We have studied distribution patterns of the desmosomal marker, desmoglein 1&2, in rat uterine epithelial cells during early pregnancy as well as in hormonally stimulated ovariectomised animals. On day 1 of pregnancy as well as in oestradiol treated rats, desmoglein 1&2 staining was localized along the entire length of the lateral plasma membrane. By day 3 and on subsequent days of pregnancy as well as in ovariectomised animals treated with progesterone alone or in combination with oestradiol, desmoglein 1&2 staining was concentrated at the apical portion of the lateral plasma membrane. We suggest that the reorganisation of these desmosomal cadherins is an important component of uterine epithelial receptivity and this relocation is under the control of the ovarian hormone progesterone.

The present study further elucidates the complex effects of a commonly-prescribed fertility drug upon a target organ in an animal model. In the human condition, its effects are rarely observed without the influence of endogenous ovarian hormones. The aim of the study was to investigate how the administration of a single dose of clomiphene citrate (CC) given prior to an implantation-priming sequence of ovarian hormones would affect the expression of surface oligosaccharides and membrane architecture of uterine epithelium. Ovariectomized rats were given a single dose of either 0.25 mg or 1.25 mg of CC prior to a hormone-priming regime of progesterone (P4) for 3 days with a single additional administration of oestradiol 17beta (E2) on day 3. Animals were killed 24 h after final treatment. Uterine tissue was labelled with the lectin Phytolacca americana conjugated with avidin, subsequently labelled with biotinylated ferritin and prepared for transmission electron microscopy. Results indicate that CC, when administered prior to the implantation hormone-priming regime, is able to act as a super oestrogen and upregulates expression of oligosaccharides on the plasma membrane surface and increases the density and length of microvilli on the surface of the cells when compared with other treatment regimes. Understanding of the effects of CC at the uterine level at the time of implantation enables manipulation of uterine receptivity to control fertility and to improve the outcome of assisted reproductive procedures.

The presence and distribution pattern of Muc-1 was determined immunohistochemically in rat uterine epithelial cells and localised ultrastructurally on the plasma membrane of uterine epithelial cells. Rat uterine tissue was examined on days 1, 3 and 6 of pregnancy and it is shown to be expressed in uterine luminal epithelial cells on day 1 of pregnancy with decreasing expression towards the time of implantation on day 6. It was demonstrated ultrastructurally that Muc-1 is expressed at the external surface of the plasma membrane of uterine epithelial cells thus confirming that it is well positioned to act as an anti-adhesive molecule during the nonreceptive stage of pregnancy. Ultrastructural localisation also revealed that Muc-1 was not present on the outer surface of the plasma membrane during the receptive phase.

In the mature rat ventral prostate, epithelial proliferation is accompanied by significant upregulation of tyrosine kinase A, alpha1B and muscarinic acetylcholine M2 receptors as well as the synaptic vesicle-associated membrane proteins synaptobrevin and SV2 as compared with immature prostate tissue. The adrenergic receptors beta1, alpha2A and alpha1 were also up-regulated and translocated in mature rat prostate tissue. Expression of the Schwann cell/axonal marker S100 remained unchanged. These results are suggestive of a marked increase in metabolic activity, calcium influx and autonomic receptor expression in the aging prostate. These changes were not accompanied by an increase in the number of axons.

We used a single administration of clomiphene citrate (CC), a synthetic oestrogen that is prescribed for infertility treatment, in combination with either a single administration of oestradiol 17beta (E2) or progesterone (P4) to assess the combined effects of these hormones on the uterine surface. The aim of these experiments was to investigate how CC in combination with these hormones affected both expression of oligosaccharides on the uterine surface and membrane architecture further elucidating CC's agonistic/antagonistic properties. Ovariectomized sexually mature rats were given combinations of E2 and CC (E2 + CC) or P4 and CC (P4 + CC) or P4 and E2 (P4 + E2) and were killed 24 h later. Uterine tissue was labelled with the lectin Phytolacca americana conjugated with avidin and subsequently labelled with biotinylated ferritin and prepared for transmission electron microscopy. Results of the administration of these hormone combinations indicate that CC, when administered in conjunction with E2, had the ability to downregulate expression of oligosaccharides on the membrane surface caused by E2. When administered with P4, CC had the ability to upregulate the effects of P4. Thus, when combined with E2, CC has an antagonistic effect but when combined with P4, CC has an agonistic effect.

Summary We have studied histochemically the effects of clomiphene citrate on the expression of oligosacchrides on the apical plasma membrane of uterine epithelial cells using the lectin Phytolacca americana . Ovariectomized sexually mature rats were given a single injection of either clomiphene in two concentrations or estradiol 17p or progesterone and were killed 24 hr later. Uterine tissue was labeled with Phytolacca americana conjugated with avidin and subsequently labeled with biotinalyted ferritin and prepared for transmission electron microscopy. Our results indicate that clomiphene and to a lesser degree progesterone significantly increased lectin binding. However, the increase was not as large as that observed with a single dose of estrodiol 17p. When the proportion of lectin positivity in relation to total membrane length was analyzed, treatment with clomiphene and progesterone did not have significantly different effects. Low dose clomiphene did not have a significant effect as compared with controls. Our data show that clomiphene has a dose-dependent adverse effect on lectin binding as compared with ovarian hormones. We suggest that these effects contribute to low pregnancy rates with clomiphene use.

The ubiquitous cadherin probe, pan-cadherin was used to study changes in the distribution of cadherins in the plasma membrane of rat uterine epithelial cells during early pregnancy when the uterine lumen closes down. A major reorganisation of cadherin expression was observed to occur in uterine epithelial cells between days 1 and 6 of pregnancy with a clear shift in molecular distribution from a basal location on day 1 to a distinctly and almost exclusively apical distribution by day 6 of pregnancy when the blastocyst is attached. We suggest these results indicate participation of this molecule in closure of the uterine lumen, a phenomenon well-known to occur in the rat uterus during early pregnancy and in which apical surfaces of opposing uterine epithelial cells cohere.

We have studied the arrangement of microfilaments in uterine microvilli during early pregnancy, using transmission electron microscopy and immunofluorescence microscopy. Observations indicate that changes in actin microfilament organization occur in association with the alterations to the uterine cell surface which precede blastocyst implantation. We consider these findings with reference to the possible mechanisms involved.

Cationic Ferritin and Ruthenium Red were used to label rat uterine luminal epithelial cells at estrus and 6th d of pregnancy. Labelling was heavy at estrus and light on 6th d. Trypsin and pronase reduced labelling at estrus and restored labelling at 6th d suggesting that negatively charged carbohydrates may become masked at the time of implantation.

Summary Cytochemistry using biotinylated lectins and an avidin-ferritin label has been combined with freeze-fracture to study the relationship between surface carbohydrates and intramembranous particles in uterine epithelial cells. We find no structural relationship between the 2 classes of membrane components and consider the significance of this finding.