Laura A. Lindsay

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.

CALCIUM TRANSPORT ACROSS THE UTERINE EPITHELIUM OF PREGNANT LIZARDS

ABSTRACT Calcium is a major requirement for developing embryos, but an insufficient amount is present in egg-yolk to sustain embryonic development in most reptiles. Consequently, the eggshell acts as an extra-lectithal source of calcium in oviparous amniote vertebrates, including squamates. A number of anatomical and physiological changes must accompany the evolutionary transitions from oviparity to viviparity, including the reduction or elimination of the eggshell. In our quest to understand the evolution of viviparity, we have used indirect immunofluorescence to characterize Ca2+ATPase pumps in the uterus of a targeted range of species of Eugongylus group skinks and bird species at various stages of the reproductive cycle, including pregnancy. During egg-shelling, apical localisation of Ca2+ATPase pumps occurs in the glandular epithelium of the uterus in the oviparous lizard Lampropholis guichenoti and the King quail Coturnix chinensis, presumably to allow movement of calcium against its electrochemical gradient into the lumen of the glandular duct and ultimately to the uterine lumen and developing embryo. Interestingly, structurally similar shell glands containing Ca2+ATPase pumps occur in the uterine lamina propria (stroma) of viviparous species, although we have so far seen them only in early vitellogenic N. ocellatus and non-pregnant female Pseudemoia spenceri. Our findings suggest that the down regulation of Ca2+ATPase in the shell glands may be an important contributor to the evolution of viviparity through the loss of an eggshell.

Ovarian hormones control the changing expression of claudins and occludin in rat uterine epithelial cells during early pregnancy

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.

Aquaporin-1 Increases in the Rat Myometrium During Early Pregnancy

Immunofluorescence and immunogold techniques were used to determine the presence and distribution of aquaporin-1 (AQP1) within the rat uterus. Uterine tissue from non-pregnant (proestrus) as well as pregnant (days 1, 3, 6 and 7) rats were used. It was found that this water channel was present in the myometrium of the pregnant rat uterus with the intensity of AQP1 immunoreactivity increasing from day 1 to day 6 of pregnancy. In particular, an increase was also observed in mesometrial as compared to antimesometrial myometrium. Immunolocalization at the electron microscope level indicated that AQP1 was localized to the plasma membrane of smooth muscle cells found within the inner circular layer. It is suggested that AQP1 plays a role in stromal oedema, uterine closure and orientation of the blastocyst.

VEGF111: new insights in tissue invasion

Vascular endothelial growth factor is a secreted glycoprotein that acts on endothelial cells to induce developmental and physiological angiogenesis. It has also been implicated in angiogenesis occurring in several pathologies, most notably, cancer. Alternative splicing of VEGF mRNA transcripts results in several isoforms with distinct properties depending on their exon composition. Recently, a new isoform has been identified, VEGF111 with a unique exon composition responsible for its high angiogenic potential. In humans, the only known inducer of VEGF111 is DNA damage but its natural presence in the uterus of the viviparous lizard, Saiphos equalis, suggests other mechanisms of regulation. Most interestingly, the possible relationship between the evolution of viviparity and the associated increased risk in developing cancer may be important in understanding the mechanisms underlying tumor development.

Aging Impairs VEGF-Mediated, Androgen-Dependent Regulation of Angiogenesis

There is a progressive impairment of vascular repair mechanisms with advancing age concomitant with a steady decline in circulating androgen levels in men. Emerging evidence indicates androgens regulate angiogenesis; however, little research has focused on the impact of age upon androgen-mediated regulation of angiogenic mechanisms. Human dermal fibroblasts from young (<30 years) and older (>65 years) men were incubated with DHT, with or without androgen receptor antagonist hydroxyflutamide, or phosphoinositide 3-kinase inhibitor. Fibroblast-conditioned medium was used to stimulate angiogenic functions in human umbilical vein endothelial cells. Nuclear fractionation and fluorescence microscopy were used to study androgen receptor (AR) distribution. Conditioned medium from fibroblasts of young men, but not old men, treated with DHT produced a 3-fold increase in human umbilical vein endothelial cell tubulogenesis and 2-fold increase in migration via increased vascular endothelial growth factor (VEGF) expression and secretion, predominantly of VEGF145. DHT-induced VEGF secretion from fibroblasts of young men was AR-dependent and increased AKT phosphorylation, which was abrogated by phosphoinositide 3-kinase inhibition. By contrast, fibroblasts from older men were unresponsive to DHT and lacked androgen-mediated enhancement in VEGF production. These findings were associated with reduced AR nuclear translocation in old fibroblasts. The failure of DHT-induced paracrine stimulation of angiogenesis in fibroblasts from older men is likely due to defective nuclear translocation of AR. This first demonstration of androgen resistance (or insensitivity) acquired by human fibroblasts with aging suggests that pharmacological testosterone therapy for old men may be less effective in enhancing angiogenesis and facilitating tissue regeneration mechanisms reliant on paracrine release of VEGF.

Ovarian hyperstimulation affects fluid transporters in the uterus: a potential mechanism in uterine receptivity

Controlled ovarian hyperstimulation is commonly used in fertility treatment. Evidence suggests that this could alter the endometrial environment and influence implantation rate. However, the mechanisms underlying this disruption are unknown. A recently developed rat ovarian hyperstimulation (OH) model found alterations in the localisation and expression of several molecules associated with implantation, as well as an increase in luminal fluid at the time of implantation. The present study investigated the effects of OH in rats on the expression of fluid-transporting molecules aquaporin 5 (AQP5) and claudin 4. The expression of these proteins was investigated in uterine luminal epithelial cells of rats undergoing OH and compared with normal pregnancy. There was a significant increase in AQP5 protein in OH rats at the time of implantation, along with a loss of the mesometrial staining gradient, which is thought to contribute to implantation position. At the same time, there was a significant decrease in claudin 4 protein. These results suggest that OH in rats causes a dysregulation in uterine fluid dynamics through modifications to fluid-transporting molecules, resulting in an unfavourable implantation environment for the blastocyst.

Caveolins redistribute in uterine epithelial cells during early pregnancy in the rat: An epithelial polarisation strategy?

Abstract At the time of implantation, uterine luminal epithelial cells undergo a dramatic change in all plasma membrane domains. Changes in the basolateral plasma membrane at the time of implantation include progression from smooth to highly tortuous, as well as a loss of integrin-based focal adhesions. Another aspect of the basolateral plasma membrane that has not been studied in uterine epithelial cells are caveolae, which are omega-shaped invaginations of the plasma membrane known to be involved in endocytosis and contribute to membrane curvature. The current study investigated caveolin, a major protein of caveolae, to explore the possible roles that they play in the remodelling of the basolateral plasma membrane of uterine epithelial cells during early pregnancy in the rat. Morphological caveolae were found at the time of implantation and were significantly increased compared to day 1 of pregnancy. Caveolins 1 and 2 were found to shift to the basolateral plasma membrane of uterine epithelial cells at the time of implantation as well as when treated with progesterone alone, and in combination with oestrogen. A statistically significant increase in the amount of caveolin-1 and a decrease in caveolin-2 protein in uterine epithelial cells was observed at the time of implantation. Caveolin-1 also co-immunoprecipitated with integrin β1 on day 1 of pregnancy, which is a protein that has been reported to be found in integrin-based focal adhesions at the basolateral membrane on day 1 of pregnancy. The localisation and expression of caveolin-1 at the time of implantation is consistent with the presence and increase of morphological caveolae seen at this time. The localisation and expression of caveolins 1 and 2 in luminal uterine epithelium at the time of implantation suggest a role in trafficking proteins and the maintenance of a polarised epithelium.

Ezrin and EBP50 redistribute apically in rat uterine epithelial cells at the time of implantation and in response to cell contact

Uterine epithelial cells (UECs) undergo extensive morphological remodelling in preparation for an implanting blastocyst. This remodelling involves changes in the actin cytoskeleton and surface structures including microvilli. Ezrin and ezrin-radixin-moesin-binding protein-50-kDa (EBP50) link actin filaments to intra-membranous adhesion molecules and are important molecules in polarised epithelia. The current study is the first to describe the colocalisation and molecular association of ezrin and EBP50 in rat UECs by using immunofluorescence microscopy and immunoprecipitation techniques. These proteins have also been localised in relation to uterine epithelial cytoskeletal rearrangement during early pregnancy in the rat and to the effect of apical surface contact between opposing epithelial cells, blastocyst contact and contact with a silicon filament. Immunofluorescence microscopy has revealed that ezrin and EBP50 respond to contact between opposing epithelial cells and increase apically on day 6 of pregnancy. This apical distribution is also observed in UECs in contact with a silicon filament. Ezrin and EBP50 are however absent within the implantation chamber itself, seemingly mimicking the events that take place in leucocyte-endothelium binding. Thus, ezrin and EBP50 occur apically in UECs at the time of implantation in the rat and in response to a substitute blastocyst (filament) suggesting a role for these proteins in the cytoskeletal rearrangements that facilitate uterine receptivity and blastocyst-epithelial adhesion. Their loss within the implantation chamber possibly allows the subsequent invasion of the embryo.

The adherens junction is lost during normal pregnancy but not during ovarian hyperstimulated pregnancy.

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.