Christopher R. Murphy

Plasma membrane transformation: a common response of uterine epithelial cells during the peri‐implantation period

The plasma membrane of uterine epithelial cells and its changes during early pregnancy are reviewed. The review first examines morphological alterations in rats and mice and laboratory rodents and finds that similar changes in membrane organization accompany the peri‐implantation period: long, thin, regular microvilli are gradually converted into irregular, flattened projections. It is also found however, that in many other species related plasma membrane alterations are seen during early pregnancy. Molecular alterations in the membrane are also examined and although the evidence is so far limited, striking similarities are noted across species. The review also examines some new morphological studies on the alterations in the plasma membrane of uterine epithelial cells during early pregnancy and concludes that a process of plasma membrane transformation may be a common response across species.

Evolution of viviparity: what can Australian lizards tell us?

Historically, Australia has been important in the study of, and the development of hypotheses aimed at understanding, the evolution of viviparity in amniote vertebrates. Part of the importance of Australia in the field results from a rich fauna of skinks, including one of the broadest ranges of diversity of placental structures within one geographic region. During the last decade, we have focussed our studies on one lineage, the Eugongylus group of skinks of the subfamily Lygosominae because it contains oviparous species and some that exhibit complex placentae. Our specific objective has been to attempt to understand the fundamental steps required when viviparity, and ultimately complex placentae, evolve from oviparous ancestors. We have taken a three-prong approach: (1) detailed study of the morphology and ontogeny of the placentae of key species at the light microscope level; (2) study of changes in the uterus associated with pregnancy, or the plasma membrane transformation; and (3) measures of the net exchange of nutrients across the placenta or eggshell of key species. In turn, we have found that: (1) details of the morphology and ontogeny of placentae are more complex that originally envisaged, and that the early conclusions about a sequence in the evolution of complex placentae was naïve; (2) a plasma membrane transformation occurs in viviparous, but not oviparous lizards, and thus may be a fundamental feature of the evolution of viviparity in amniotes; and (3) species with more complex chorioallantoic placentae tend to transport more nutrients across the placenta during pregnancy than those with simpler chorioallantoic placentae but, because the correlation is not tight, the importance of the omphaloplacenta in transporting nutrients may have been overlooked. Also, the composition of yolk of highly matrotrophic species is broadly similar, but not identical, to the yolk of oviparous species. Some of the interpretation of our data within the context of our specific objective is not yet possible, pending the publication of a robust phylogeny of Eugongylus group skinks. Once such a phylogeny is available, we are in a position to propose specific hypotheses about the evolution of viviparity that can be tested using another lineage of amniotes, possibly Mabuya group skinks.

Endometrial cell death during early pregnancy in the rat.

In a study of early pregnancy in the rat, a high proportion of morphologically apoptotic, TUNEL and P2X7 positive cells were found to be present in the luminal epithelium and stroma prior to implantation. At the time of implantation on Day 6, apoptosis as measured by these indicators was reduced up to 4-fold in the non-implantation uterine epithelium but was markedly increased adjacent to the implanting blastocyst. It is proposed that apoptotic cell death is an important regulatory factor involved in uterine remodelling prior to and during implantation.

Integrin β3 in rat blastocysts and epithelial cells is essential for implantation in vitro: studies with Ishikawa cells and small interfering RNA transfection

BACKGROUND Integrins are involved in the process of embryo-endometrium interaction during implantation. We investigated the localization of integrin β3 in the rat blastocyst and Ishikawa cells using an in vitro co-culture model of implantation. METHODS Zona pellucida-free rat blastocysts were co-cultured with the Ishikawa cells (endometrial adenocarcinoma cell line) to observe the attachment between the embryo and endometrium. Immunofluorescence staining was used to investigate the localization of integrin β3 in rat embryos at different stages of development (each n= 3 embryos) and at the embryo/endometrium interface, observed by confocal microscopy. The Ishikawa cells were transfected with integrin β3 small interfering RNA (siRNA) for 48 h and then co-cultured with Day 5 rat blastocysts to observe the effect on attachment. RESULTS Integrin β3 staining in the rat embryos increased at the blastocyst stage being highly concentrated in the cytoplasm of trophoblast cells (n= 9 embryos). Integrin β3 was localized on the apical surface of the Ishikawa cells (n= 3 experiments). However, integrin β3 relocated to the apical membrane of trophoblast cells in response to attachment to Ishikawa cells (n= 6 embryos). Moreover, when Ishikawa cells were transfected with integrin β3 siRNA, blastocyst attachment was significantly reduced compared with those transfected with control siRNA (16.7 versus 92.3%, respectively, P< 0.05). CONCLUSIONS Integrin β3, localized apically in the blastocyst and the Ishikawa cells, is important during initial attachment of the blastocyst to endometrial cells. This study provides further evidence of the importance of integrins during implantation and may aid in elucidating the molecular mechanism of implantation failure and infertility in women.

Aquaporins are upregulated in glandular epithelium at the time of implantation in the rat

Regulation of luminal fluid is essential for blastocyst implantation. While it has been known for quite some time that there is a reduction in the amount of luminal fluid at the time of implantation, the mechanisms regulating this process are only just emerging. Previous studies have shown an upregulation of aquaporin (AQP) 5 channels in luminal epithelial cells at the time of implantation providing a mechanism for fluid reabsorption across the surface epithelium. However to date the contribution of fluid reabsorption by glandular epithelial cells has not been established. This study using reverse transcriptase polymerase chain reaction demonstrates the presence of several AQP isoforms in the rat uterus at the time of implantation while immunofluorescence data demonstrates an apical distribution of AQPs5 and 9 in the glandular epithelium at the time of implantation. The presence of AQPs5 and 9 in the apical plasma membrane of the glandular epithelium seen in this study provides a mechanism for transcellular fluid transport across these glandular epithelial cells similar to that seen in luminal epithelial cells. The reabsorption of glandular fluid via AQP channels may also regulate luminal fluid volume and be involved in the reduction in luminal fluid seen at the time of implantation.

Alterations in tight junction molecules of uterine epithelial cells during early pregnancy in the rat.

Distribution patterns of the tight junction associated proteins ZO-1, claudin-1 and occludin were investigated in rat uterine epithelial cells during early pregnancy. Light microscopy and immunohistochemical labelling were used to detect these proteins on days 1, 3, 6 and 7 of pregnancy. Intense staining of claudin-1 at the apical region of the lateral plasma membrane accompanied diffuse staining throughout the cytoplasm. ZO-1 was also localised in the apical region, but ZO-1 was not present in the lower two thirds of the lateral plasma membrane or in the cytoplasm. Occludin was present only on days 6 and 7 of pregnancy. Labelling was also localised in the apical region of the lateral plasma membrane where tight junctions are known to be present. Our results show that ZO-1, claudin-1 and occludin are present in the apical region of uterine epithelial cells, and appear to play a role in the very dynamic tight-junctional network of uterine epithelial cells during early pregnancy. In particular, occludin appears only during uterine receptivity for implantation.

Distributional changes of purinergic receptor subtypes (P2X 1-7) in uterine epithelial cells during early pregnancy.

Expression of each of the purinergic receptor subtypes (P2X1–7) was studied by immunohistochemical localization in the apical, lateral and basal plasma membranes of rat uterine epithelial cells during early pregnancy to the time of implantation on Day 6. Labelling for each P2X subtype was seen in the apical, lateral and basal compartments on Days 1 and 3, except for P2X2 which was only observed in the basement membrane. The P2X5 signal was similar in temporal and spatial expression to the other subtypes, but with a greatly reduced intensity. At the time of implantation on Day 6, this pattern altered dramatically. Apical expression markedly increased for most subtypes while the lateral and basal signals were markedly reduced. The exceptions to this pattern were P2X2, which displayed both a strong basal and apical label, and P2X4 which became de-expressed in all areas. We propose that the changing spatial and temporal expression of the P2X receptors is a significant factor in the regulation of events during early pregnancy. They are expressed in the same location as remodelling, apoptosis, and protein activation events prior to implantation on Day 6. These observations suggest an up-regulation of calcium-mediated events, including cytoskeletal alterations, a decrease in luminal pH and transmembrane molecule activation.

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

Summary— Actin filaments were identified in the epithelial cells of rat uterus following detergent extraction and decoration of microfilaments (MF) with myosin subfragment 1 (SI). 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.

β1 and β3 integrins disassemble from basal focal adhesions and β3 integrin is later localised to the apical plasma membrane of rat uterine luminal epithelial cells at the time of implantation

The present study investigated the expression of integrin subunits that are known to be associated with focal adhesions, namely β(1) and β(3) integrins in rat uterine luminal epithelial cells during early pregnancy. The β(1) and β(3) integrins were concentrated along the basal cell surface and were colocalised and structurally interacted with talin, a principal focal adhesion protein, on Day 1 of pregnancy. At the time of implantation, β(1) and β(3) integrins disassembled from the site of focal adhesions, facilitating the removal of uterine luminal epithelial cells for embryo invasion. Also at this time, β(3) integrin markedly increased along the apical membrane, suggesting a role in embryo attachment. This distributional change in β(1) and β(3) integrins seen at the time of implantation was predominantly under the influence of progesterone. Taken together, β(1) and β(3) integrin disassembly from focal adhesions and the increase in β(3) integrin apically are key components of hormonally regulated endometrial receptivity.

Pan-cadherin concentrates apically in uterine epithelial cells during uterine closure in the rat.

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.