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.     

Expression of cellCAM-105 in the apical surface of rat uterine epithelium is controlled by ovarian steroid hormones

Affinity-purified antibodies to cellCAM-105, an adhesive cell surface glycoprotein, were used in immunohistochemical investigations of rat uteri at various functional stages: (i) the oestrous, pro-oestrous, metoestrous, and dioestrous stages of the oestrous cycle, (ii) Days 1-8 of normal pregnancy, (iii) delayed implantation, (iv) 18 h after oestrogen reactivation from delay of implantation, and (v) juvenile rats, and normal ovariectomized adults, respectively, before and after experimental injection of progesterone and/or oestrogen. CellCAM-105 was present in the apical zones of the luminal and glandular epithelium cells in a stage-specific and hormone-dependent manner. The results indicate that: (1) steroid hormones are essential for the expression of cellCAM-105 in the uterine epithelial cells; (2) progesterone induces cellCAM-105 expression in the glandular epithelium, and oestrogen induces cellCAM-105 expression in the luminal epithelium; (3) progesterone induces down-regulation of cellCAM-105 from the surface of the uterine luminal epithelium of juvenile rats; (4) cellCAM-105 is absent in the luminal epithelial cells but present in the glandular epithelial cells of the rat uterus at the time of blastocyst implantation.     

Calpain 2 activity increases at the time of implantation in rat uterine luminal epithelial cells and administration of calpain inhibitor significantly reduces implantation sites

The present study investigated the role of calpain 2 in rat uterine luminal epithelial cells during early pregnancy. Calpain 2 is an intracellular calcium-dependent proteolytic enzyme which cleaves numerous focal adhesion proteins. Calpain 2 was concentrated along the basal cell surface of uterine luminal epithelial cells at the predicted site of focal adhesions on day 1 of pregnancy and remained unchanged at the time of implantation as observed by immunofluorescence microscopy. However, Western blotting analysis showed a marked increase in the active form and a significant decrease in the latent form of calpain 2 at the time of implantation. The increase in calpain 2 activity coincides with the disassembly of focal adhesion proteins, talin, paxillin, integrin β1 and β3 from the site of focal adhesions. Intraperitoneal injection of calpain inhibitor, calpain inhibitor l (ALLN), significantly reduced the number of implantation sites, implying that calpain 2 plays an important role in implantation. The present study suggests a role for calpain 2 in the disassembly of focal adhesions, which has been previously shown to play a key role in uterine receptivity for implantation.     

Focal adhesion kinase localizes to sites of cell‐to‐cell contact in vivo and increases apically in rat uterine luminal epithelium and the blastocyst at the time of implantation

Focal adhesions play an important role in promoting embryo invasion; in particular, focal adhesions disassemble at the time of implantation in the rat, facilitating the detachment of the uterine luminal epithelium to allow the embryo to invade the endometrium. This study investigated focal adhesion protein, focal adhesion kinase (FAK) in the rat uterine luminal, and glandular epithelial cells to understand the dynamics of focal adhesions during early pregnancy. FAK undergoes extensive distributional change during early pregnancy, and surprisingly, FAK was not localized at the site of focal adhesions, instead being localized to the site of cell‐to‐cell contact and colocalizing with ZO‐1 on day 1 of pregnancy. At the time of implantation, FAK increases in the apical region of the uterine luminal epithelial cells which was regulated by progesterone. Using an in vitro co‐culture model of rat blastocysts attached to Ishikawa cells, FAK was present apically both in the rat blastocyst and the Ishikawa cells, suggesting a role in attachment andin mediating signal transduction between these two genetically different cell types. J. Morphol., 2012. © 2012 Wiley Periodicals, Inc.     

Production of an endothelial cell migratory signal in rat endometrium during early pregnancy

Rat endometrial explants were cultured in a three-dimensional collagen/endothelial cell matrix to measure angiogenic activity, as represented by migration of vascular endothelial cells towards the explants. Minimal endothelial cell migratory activity was observed with endometrial explants taken during the four-day oestrous cycle and days 3 and 4 of pregnancy. In contrast, a surge of endothelial cell migration occurred in response to endometrial explants taken from day-5-pregnant rats. Activity was found in explants taken approximately 5 h prior to implantation, but returned to minimal levels by day 6 of pregnancy. Endothelial cell migration remained minimal in response to both implantation and intersite tissue explants taken from days 6 and 7 of pregnancy. Endometrium from ovariectomised rats produced no endothelial cell migratory activity as measured by this technique. However, near preimplantation levels of endothelial cell migratory activity could be induced in ovariectomised rat endometrium by administering progesterone for 72 hours. Oestrogen given in conjunction with progesterone had no additional effect. These results demonstrate the presence of an endometrial signal that controls endothelial cell migration, and demonstrate this activity can be induced by progesterone without the addition of oestrogen.     

Regulation of gene expression and cellular localization of prostaglandin synthase by oestrogen and progesterone in the ovine uterus

Expression of the gene for prostaglandin synthase (PGS) was examined in whole endometrial tissue derived from ewes during the oestrous cycle (Days 4-14), on Day 15 of pregnancy and following ovariectomy and treatment with ovarian steroid hormones. Whilst no significant differences were seen in PGS mRNA concentrations analysed by Northern blot analysis in endometrial tissue during the oestrous cycle or in early pregnancy, treatment of ovariectomized (OVX) ewes with oestradiol-17 beta markedly reduced endometrial PGS mRNA concentration. There was no difference in PGS mRNA concentration in ewes treated with progesterone, either alone or in conjunction with oestrogen, from that in OVX controls. In contrast, differences in immunolocalization of PGS observed in uterine tissue from OVX-steroid-treated ewes were much more marked and reflected similar changes seen previously in the immunocytochemical distribution of endometrial PGS during the oestrous cycle. In OVX ewes and those treated with oestrogen, immunocytochemical staining for PGS was seen in stromal cells, but little immunoreactive PGS was located in the endometrial epithelial cells. However, in ewes treated with progesterone alone or with oestrogen plus progesterone, PGS was found in luminal and glandular epithelial cells and in stromal cells. Intensity of immunostaining for PGS in endothelial cells and myometrium did not differ between the treatments. Thus, whilst oestrogen lowers PGS mRNA in the endometrium, presumably in stroma, it may also increase the stability of the enzyme itself in the stromal cells. Although oestradiol-17 beta has no effect on PGS in endometrial epithelium, progesterone stimulates the production of PGS in endometrial epithelial cells without altering the overall abundance of PGS mRNA in the endometrium as a whole. Conceptus-induced changes in PGF-2 alpha release by ovine endometrium would not appear to be mediated via effects on PGS gene expression or protein synthesis.     

Epithelial cell function during blastocyst implantation

In response to the ovarian secretion of progesterone and estrogen during early pregnancy, the mammalian uterus develops the capacity to perform complex cellular activities which occur before and after blastocyst implantation. Luminal epithelial cells participate in regulation of the metabolism of the blastocyst through the control of its humoral environment, provide an appropriate matrix for changes to occur at the interface between trophoblast and epithelium, and appear to transmit information from the blastocyst to the underlying stroma to initiate decidualization. With the completion of these functions during implantation in rodents, the epithelial cells self-destruct and are removed by phagocytic activity of the trophoblast. Control of both the endocytotic and secretory activity of luminal epithelial cells and their eventual self-destruction would require regulation of the Golgi-endoplasmic reticulum-lysosomes system within these cells. Progesterone secretion during early pseudo-pregnancy increases levels of cathepsin D, a lysosomal proteinase, in luminal epithelial cells by increasing the rate of enzyme synthesis. Progesterone pretreatment of ovariectomized rats followed by estradiol treatment results in the development of uterine sensitivity to deciduogenic stimuli. The number of proteins which are synthesized by luminal epithelial cells in response to estradiol to achieve this sensitivity has been determined. Epithelial cytosol proteins from rats treated with medroxyprogesterone acetate (3.5 mg sc) or medroxyprogesterone acetate plus estradiol (200 ng sc) were separated by two dimensional polyacrylamide gel electrophoresis. The synthesis of two proteins increased after 8 h of estradiol treatment and the synthesis of another three was increased by 12 h. The increased synthesis of these proteins could be related to changes in the capacity of the luminal epithelial cell for prostaglandin synthesis. The epithelial capacity for prostaglandin synthesis increases during pseudopregnancy to maximum levels at the time of maximum sensitivity to deciduogenic stimuli. Epithelial prostaglandin synthetic capacity may also depend upon the accumulation of prostaglandin precursors within these cells. Estradiol treatment of medroxyprogesterone acetate pretreated ovariectomized rats increased the arachidonic acid content and composition of epithelial phosphatidyl choline but the increases were not statistically significant. These changes in protein and lipid synthesis controlled by progesterone and estrogen would appear to contribute to the cellular activities of the luminal epithelium during early pregnancy.     

Hormonal control of enzyme activity during the plasma membrane transformation of uterine epithelial cells

Ultrastructural and light microscopic catalytic histochemical methods were used to study the distribution and changes in distribution of four phosphatase enzymes; alkaline phosphatase, 5'-nucleotidase, thiamine pyrophosphatase and adenosine triphosphatase in uterine epithelial cells in response to the ovarian hormones, oestrogen, progesterone or a combination of both used in different regimes on ovariectomised rats. Reaction product for all four enzymes was clearly localised in the epithelial cells, especially with oestrogen priming. However, the four enzymes showed markedly different patterns of organisation of reaction product in response to other hormonal treatments.Our findings clearly show that the expression of these enzymes is under ovarian hormonal control. However, while all of the enzymes are upregulated by oestrogen, the response to progesterone is variable, which can upregulate or downregulate different enzymes. The findings are particularly obvious at the electron microscopic level on the apical plasma membrane of the uterine epithelial cells, which was the main focus of our study.     

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

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.     

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.     

Occlusion and reformation of the rat uterine lumen during pregnancy

Implantation sites were obtained from rats at various stages of pregnancy and were studied by light microscopy and scanning electron microscopy. Early in pregnancy the uterine luminal epithelium and the decidual cells in the implantation site formed an implantation chamber containing the conceptus. The epithelial cells lining the chamber and the mouth of the chamber degenerated, and the uterine lumen that was mesometrial to the conceptus was obliterated such that the uterine lumen became discontinuous, and the luminal epithelia of intersite areas were isolated. As the conceptus continued to grow, the decidua-conceptus unit bulged into the intersite areas and was partially covered by an epithelium that eventually became discontinuous and degenerated. Once this had occurred, the luminal epithelium of the intersite areas reestablished contact antimesometrial to the decidua-conceptus unit, and the uterine lumen was again continuous. However, the epithelium lining the lumen was not complete in the mesometrial region because of the vascular connections between the uterine stroma and the placenta. Factors influencing the restructuring of the uterine luminal epithelium were discussed.     

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.     

GRP78 expression and regulation in the mouse uterus during embryo implantation

The aim of this study was to investigate the spatiotemporal expression and regulation of GRP78 in the mouse uterus during the peri-implantation period. The GRP78 protein was mainly detected in the luminal and glandular epithelia on days 1–4 of pregnancy. On day 5 of pregnancy, the GRP78 protein was more highly observed around the implanted embryo at the implantation site. There was no detectable GRP78 protein signal on day 5 of pseudopregnancy. GRP78 mRNA and protein levels gradually increased on days 6–8 of pregnancy, and the expression pattern was also expanded, coinciding with the development of decidua. Similarly, GRP78 expression was also strongly expressed in decidualised cells following artificial decidualisation. Compared with the results obtained with the delayed uterus, a high level of GRP78 expression was detected in the implantation-activated uterus. In the uteri of ovariectomised mice, GRP78 expression increased and reached its highest level after injection of oestrogen, and progesterone seemed to have an antagonistic effect on oestrogen up-regulation of GRP78 expression. Our data indicate that GRP78 might play an important role during the process of mouse embryo implantation, and GRP78 expression was mainly regulated by active blastocysts and maternal oestrogen.     

Arachidonic acid in uterine phospholipid during early pregnancy and following hormone treatment

Evidence that prostaglandins are involved in intercellular communication during blastocyst implantation suggested that development and loss of uterine sensitivity to deciduogenic stimuli during early pregnancy might depend upon changes in uterine capacity to mobilize arachidonic acid from phospholipid. We measured levels of arachidonic acid in lipid fractions on Day 6 of pregnancy in uterine segments containing implantation sites, in uterine segments between implantation sites, and in luminal epithelial cells after a deciduogenic stimulus. Arachidonic acid in uterine phospholipid was depleted at implantation sites. With an intrauterine deciduogenic stimulus of hormonally primed ovariectomized rat uteri, the arachidonic acid content of the luminal epithelium decreased. When the fatty acid composition of the luminal epithelium was examined during pseudopregnancy and after progestin-estrogen treatment, however, no changes in arachidonic acid composition and content were observed. These data suggest that during blastocyst implantation, luminal epithelial cells at implantation sites mobilize arachidonic acid from phospholipid for prostaglandin synthesis, but that uterine sensitivity and the capacity to synthesize prostaglandins in response to the blastocyst does not depend upon changes in arachidonic acid levels in uterine phospholipid.     

沙眼衣原体感染对大鼠着床期间子宫内膜上皮及其β-连接素表达的影响

目的研究沙眼衣原体(Chlamydiatrachomatis,Ct)感染后大鼠着床期间子宫内膜形态学改变及β-连接素(β-catenin,β-cat)的变化。方法大鼠雌雄合笼建立早孕模型,取正常妊娠和Ct感染后妊娠大鼠子宫,采用扫描电镜(SEM)观察子宫内膜超微结构改变及免疫组化检测β-cat的表达。结果SEM显示对照组子宫内膜腔上皮细胞排列紧密,细胞间隙较窄;实验组腔上皮细胞排列疏松,间隙变宽;免疫组化定性结果示β-cat妊娠4-6d主要定位于内膜上皮细胞膜,妊娠7d在蜕膜细胞膜;半定量结果示在感染组与正常组妊娠第5d其表达量达到高峰,但感染组弱于正常组,且两者之间有显著性差异(P<0·05)。结论Ct感染后可能通过影响子宫内膜黏附分子的表达,破坏着床的微环境;同时影响上皮细胞之间的连接,从而干扰早期妊娠。     

Actin binding protein expression is altered in uterine luminal epithelium by clomiphene citrate, a synthetic estrogen receptor modulator

Clomiphene citrate (CC), a synthetic oestrogen, is often prescribed as a superovulator in treating infertility. Although CC works efficiently, pregnancy rates following CC treatment are approximately 10 times lower than "natural" rates. This study investigates how a dose of 1.25 mg CC given to ovariectomized rats before the implantation priming hormones (a single dose of progesterone for 3 days and a dose of estradiol-17beta on d3, P-P-PE), alters the expression and distribution of alpha-actinin, gelsolin and vinculin. Actin binding proteins show a specific distribution within the uterine epithelium during implantation, linking the actin cytoskeleton to integrin expression on the uterine surface and in this way aiding "adhesiveness" for blastocyst apposition to the uterine epithelium. In this study, immunocytochemistry on frozen uterine sections using mouse monoclonal antibodies against alpha-actinin, gelsolin and vinculin and peroxidase-conjugated secondary antibodies, show that CC, administered before the P-P-PE regimen, down-regulates the expression of vinculin, does not alter the expression of gelsolin and up-regulates alpha-actinin on the uterine apical surface, when compared to P-P-PE treated animals. All three proteins are down-regulated on the apical surface of the luminal epithelium and glands in all groups when compared to pregnant controls. Vinculin was only localized in the basolateral compartment of the uterine epithelial cells in the CC treated groups. By down-regulating these proteins on the uterine surface and up-regulating vinculin on the basolateral membrane of the epithelium, CC may impede adhesion and invasion of blastocysts at implantation. These results may aid the exogenous manipulation of uterine tissue to control fertility and improve assisted reproductive out-comes.     

Hormonal control of pinocytosis in the uterine epithelium of the rat.

Ovariectomized rats were treated with oestradiol-17 beta and/or progesterone to mimic the hormonal parameters inducing uterine sensitivity for implantation. The degree of pinocytosis of trypan blue and ferritin in the endometrial cells was examined. Significant epithelial pinocytosis of trypan blue occurred after a 3-day treatment of progesterone, and uptake was independently increased by priming with oestrogen and by oestradiol given on the 3rd day of progesterone treatment. Progesterone treatment caused uptake of ferritin by the epithelial cells; in control animals epithelial and stromal cells were involved. Oestrogen priming enhanced ferritin absorption, while 'nidatory' oestrogen had no effect. Oestradiol given alone completely blocked pinocytosis of both intraluminally injected substances.     

Expression of carbohydrate antigens in the goat uterus during early pregnancy and on steroid-treated polarized uterine epithelial cells in vitro

Our objectives were to determine whether specific fucosylated carbohydrate antigens, associated with uterine receptivity in rodents, are expressed in pregnant caprine uterine tissues and polarized uterine luminal epithelial (ULE) cells in culture. Immunofluorescence microscopy on frozen endometrium revealed that expression of the H-type 1 antigen, confined to epithelial cells, was regulated during early pregnancy. Staining was high on Day 5 and low on Days 11 and 13. Strong, uniform apical staining was characteristic of ULE cells between Days 15 and 19 but declined markedly by Day 25. Immunofluorescence analysis of the apical surface of polarized ULE cells cultured in steroid-free medium revealed weak and diffuse staining for the H-type 1 antigen, while progesterone (P(4)) treatment resulted in the formation of aggregates of punctate staining along the apical surface. Domain-specific biotinylation of polarized ULE cells, coupled with streptavidin precipitation and Western blotting, revealed that six apical surface proteins (31, 33, 42, 55, 60, and 70 kDa) carry the H-type 1 antigen. Therefore, H-type 1 antigen expression is up-regulated in vivo during the periimplantation period, stimulated by P(4) on polarized ULE cells in culture, and may be a useful marker for uterine receptivity in this species.     

Effects of leukaemia inhibitory factor on endometrial receptivity and its hormonal regulation in rabbits

The effects of hormones on production of leukaemia inhibitory factor (LIF) and the uterine receptivity in rabbits were studied. In ovariectomised rabbits, LIF protein was not detected in control but upregulated by progesterone alone. Oestrogen had a slightly negative effect when the rabbits were treated with both oestrogen and progesterone. Mifepristone (Mi) inhibited the progesterone-stimulated production of LIF in rabbit uterus. The transfer of embryos to LIF-treated recipients significantly increased pregnancy rate (70%) and implantation rate (27%) as compared with control (pregnancy rate=40% and implantation rate=17%). The transfer of embryos to LIF and mifepristone-treated recipients significantly decreased pregnancy rate (30%) and implantation rate (9%). The results indicated that LIF protein had a beneficial effect on uterine receptivity and mifepristone prevented this effect.