Acta Histochemica 118 (2016) 137–143
Contents lists available at ScienceDirect
Acta Histochemica journal homepage: www.elsevier.de/acthis
The adherens junction is lost during normal pregnancy but not during ovarian hyperstimulated pregnancy Samson N. Dowland ∗ , Romanthi J. Madawala, Laura A. Lindsay, Christopher R. Murphy Cell & Reproductive Biology Laboratory, School of Medical Sciences (Discipline of Anatomy & Histology) and The Bosch Institute, The University of Sydney, Sydney, NSW 2006, Australia
a r t i c l e
i n f o
Article history: Received 3 December 2015 Received in revised form 15 December 2015 Accepted 15 December 2015 Keywords: Adherens junction Pregnancy Implantation Uterine receptivity Epithelium Ovarian hyperstimulation
a b s t r a c t 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. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction During invasive blastocyst implantation, which occurs in humans and rats, the trophoblast cells must breach the uterine epithelium and invade the underlying stroma to establish the placenta (Abrahamsohn and Zorn, 1993; Bazer et al., 2009; Lee and DeMayo, 2004). During the penetration stage of implantation, the trophoblast cells of the blastocyst extend processes between adjacent luminal uterine epithelial cells (UECs) (Schlafke and Enders, 1975; Tachi et al., 1970). These processes eventually lift the UECs from their basal lamina and they are internalised by the invading trophoblast (Finn and Lawn, 1968; Schlafke and Enders, 1975; Tachi et al., 1970). This allows the blastocyst to make direct contact with the newly exposed basal lamina, where it pauses until the decidual
∗ Corresponding author at: Cell & Reproductive Biology Laboratory, F13 Anderson Stuart Building, The University of Sydney, NSW 2006, Australia. E-mail address: [email protected] (S.N. Dowland). http://dx.doi.org/10.1016/j.acthis.2015.12.004 0065-1281/© 2015 Elsevier GmbH. All rights reserved.
cells breach through from the stromal side (Schlafke et al., 1985). Such disassociation and penetration of adjacent UECs represents a loss of the epithelial barrier function in this region (Kaneko et al., 2013; Murphy, 2000a). For this to occur, it is expected that the usual hallmarks of the barrier epithelium, in particular the lateral junctional complex, must be affected. To permit blastocyst implantation, the UECs must transform from a non-receptive to a receptive phenotype. This process is collectively termed “The Plasma Membrane Transformation” and is largely controlled by the maternal ovarian hormones oestrogen and progesterone (Murphy, 2004, 2000b, 1993). The Plasma Membrane Transformation includes changes to all three plasma membrane domains; apical, lateral and basal. In the apical domain, there is a loss of microvilli and the associated glycocalyx to permit the blastocyst to attach to the cell surface (Jones and Murphy, 1994; Png and Murphy, 1997). In the basal domain, there is an increase in basal plasma membrane tortuosity and a loss of morphological focal adhesions to “loosen” the adhesion of the UECs to the underlying extracellular matrix (Shion and Murphy, 1995). In the lateral
138
S.N. Dowland et al. / Acta Histochemica 118 (2016) 137–143
Fig. 1. Transmission electron microscopy of the lateral junctional complex of UECs during normal pregnancy. (a) On day 1 of normal pregnancy the adherens junction (AJ) is evident within the lateral junctional complex. The “fuzzy plaque” is visible on the intracellular surface of the junction and there is a uniform intercellular space of low electron-density. The adherens junction sits immediately below the tight junction (TJ), where the electron-dense plasma membranes are held close together, eliminating most of the intercellular space. (b) On day 6 of normal pregnancy the tight junction (TJ) becomes deepened as described previously. The adherens junction is no longer evident within the lateral junctional complex.
domain, there is an increase in the depth of the tight junction and a decrease in the number of desmosomes (Murphy et al., 1982; Preston et al., 2006). The increased depth of the tight junction is unexpected when considering that the blastocyst must penetrate between the UECs. However, the tight junction is primarily involved in controlling the paracellular permeability of epithelial cells and is thought to provide little mechanical adhesion (Alberts et al., 2008; Johnson, 2005; Lutz and Siahaan, 1997). The increased depth of the tight junction is required to strictly control the contents of the uterine
lumen during blastocyst implantation (Murphy, 2000a; Murphy et al., 1982). The decrease in the number of desmosomes (which are strong mechanical cell–cell contacts), represents a reduction in the lateral adhesiveness of the UECs that is suggested to allow the cells to disassociate and the trophoblast to penetrate between them (Preston et al., 2006; Schlafke and Enders, 1975; Tachi et al., 1970). However, the third component of the lateral junctional complex, the adherens junction, has been little investigated in rat UECs during early pregnancy. The adherens junction (zonula adherens) is the major lateral adhesive junction in epithelial cells and is primarily responsible for epithelial cell–cell adhesion (D’souza-Schorey, 2005; Niessen, 2007). The adherens junction forms a continuous band around the apical domain that links the actin cytoskeleton of adjacent cells, providing a strong structural attachment (Harris and Tepass, 2010; Vasioukhin and Fuchs, 2001). The lateral adhesion generated by the mechanical connection of the cells is essential for maintaining the integrity of the epithelial barrier (Meng and Takeichi, 2009; Niessen, 2007). Loss of the adherens junction represents a loss of cell–cell adhesion and acquisition of migratory potential (D’souzaSchorey, 2005). It is therefore suggested that the adherens junction would prevent the uterine epithelium from being breached by trophoblast cell processes; with the corollary that the adherens junction must be lost in order to permit the blastocyst to penetrate the uterine epithelium. The adherens junction is primarily composed of the single-pass transmembrane protein E-cadherin (Gumbiner, 2005; Hartsock and Nelson, 2008; Nagafuchi, 2001; Tsukita et al., 1992). The cadherin proteins are crucial for intercellular adhesion and cells lacking cadherin do not form strong cell–cell adhesions (Lutz and Siahaan, 1997). On the extracellular side, E-cadherin molecules within adherens junctions of adjacent cells are linked by calcium ions (Hartsock and Nelson, 2008; Tsukita et al., 1992). This calcium binding is essential for correct conformational organisation of the protein, which is crucial for E-cadherin (homotypic) adhesion (Baum and Georgiou, 2011; Hartsock and Nelson, 2008; Patel et al., 2006; Pokutta et al., 1994; Theard et al., 2008). As such, the adherens junction is calcium-dependent and removal of calcium causes disruption of the junction (Meng and Takeichi, 2009). Intracellularly, E-cadherin interacts with a range of proteins that mediate the functions of the adherens junction, including cell–cell adhesion, intracellular signalling, local control of the actin cytoskeleton and disassembly of the junction (Hartsock and Nelson, 2008). In particular, E-cadherin forms a complex with the cytoplasmic catenin proteins (␣,  and p120) on the intracellular face of the adherens junction that connects the junction to the actin cytoskeleton (Hartsock and Nelson, 2008; Rowlands et al., 2000; Yap et al., 1997). In the transmission electron microscope (TEM), the adherens junction is seen immediately below the tight junction on the lateral plasma membrane. In contrast to the tight junction, where adjacent plasma membranes are held close together (Farquhar and Palade, 1963; Niessen, 2007; Schneeberger and Lynch, 2004), in the adherens junction there is a uniform space between adjacent plasma membranes (Farquhar and Palade, 1963). This space is of low electron density and corresponds to the extracellular domains of E-cadherin molecules and the associated calcium ions (Farquhar and Palade, 1963; Miyaguchi, 2000; Niessen, 2007). On the intracellular surface, a “fuzzy plaque” characterises the adherens junction. This plaque is moderately electron dense and can be seen linking to the filaments of the actin cytoskeleton (Farquhar and Palade, 1963; Hirokawa and Heuser, 1981; Miyaguchi, 2000; Nagafuchi, 2001). This plaque corresponds to the E-cadherin-catenin complex that connects the adherens junction to the cytoskeleton (Miyaguchi, 2000; Nagafuchi, 2001).
S.N. Dowland et al. / Acta Histochemica 118 (2016) 137–143
While the adherens junction is yet to be studied ultrastructurally in rat UECs during early pregnancy, it has previously been observed in these cells. While studying the actin cytoskeleton, Luxford and Murphy (1992a,b) identified that the adherens junction is present in UECs at the time of fertilisation and in ovariectomised rats treated with oestrogen alone. Previous studies in mice (Potter et al., 1996) and rats (Li et al., 2002) have identified a reduction in Ecadherin in UECs at the time of receptivity. Further studies in humans have shown that while E-cadherin is present at distinct junctional regions during the non-receptive phase of the menstrual cycle, this protein spreads out into a diffuse lateral distribution specifically in the window of receptivity (Buck et al., 2012). These molecular biology studies indicate that the key adherens junction protein, E-cadherin, is likely removed from the adherens junction at the time of receptivity (Buck et al., 2012; Li et al., 2002; Potter et al., 1996). Therefore, it is hypothesised that the adherens junction is lost during early pregnancy, in preparation for blastocyst implantation. The actin cytoskeleton is an integral component of the adherens junction and abnormalities in this cytoskeleton prevent adherens junction assembly (Hong et al., 2010; Mège et al., 2006). Furthermore, cytoskeletal remodelling has been shown to cause a loss of adherens junctions (D’souza-Schorey, 2005). Therefore, as the terminal web (component of the actin cytoskeleton that anchors into the adherens junction) is disrupted at the time of receptivity in UECs (Luxford and Murphy, 1992b), it is expected that this causes the adherens junction to be lost at the time of receptivity. This study will investigate whether the loss of the terminal web affects the morphological adherens junction by artificially disrupting the actin cytoskeleton in UECs. As the actin cytoskeleton drives the formation of the adherens junction (Vasioukhin et al., 2000), it is hypothesised that artificial disruption of the actin cytoskeleton will cause a loss of the adherens junction. In assisted reproductive technologies, such as in vitro fertilisation (IVF), controlled ovarian hyperstimulation (COH) is used to increase oocyte yield (Arslan et al., 2005). However, COH has been found to cause a reduction in pregnancy and implantation rates, specifically due to its negative impact on the endometrium (Check et al., 1999; Paulson et al., 1990; Yeh et al., 2014). Recentlydeveloped rat ovarian hyperstimulation (OH) models emulate COH treatment used during IVF therapy, thereby enabling the experimental examination of the impact of such treatment on the endometrium (Jovanovic´ and Kramer, 2010; Kramer et al., 1990; Lindsay and Murphy, 2013). This study has utilised a rat OH model to investigate whether the OH procedure affects the adherens junction in UECs. Since COH induces a supraphysiological level of oestrogen that persists into the implantation window (Weinerman and Mainigi, 2014; Yeh et al., 2014), and the adherens junction has previously been observed in UECs under the influence of oestrogen (Luxford and Murphy, 1992a), it is predicted that the adherens junction will remain after OH.
2. Material and methods 2.1. Animals and mating Adult virgin female Wistar rats were used for this study and all procedures were approved by The University of Sydney Animal Ethics Committee. Rats were housed in plastic cages under a 12-h light–dark cycle with free access to food and water. Females were mated overnight with males of proven fertility and the presence of sperm in a vaginal smear the following morning indicated successful mating and was designated day 1 of pregnancy. Uterine horns were collected from 5 rats each of days 1 and 6 of pregnancy to study non-receptive and receptive conditions respectively.
139
Fig. 2. Transmission electron microscopy of the lateral junctional complex of UECs from day 1 pregnant rats treated with cytochalasin D, to artificially disrupt the actin cytoskeleton and vehicle controls. (a) Cytochalasin D treatment does not affect the lateral junctional complex of UECs, as the adherens junction (AJ) is still present immediately below the tight junction (TJ). Desmosomes (D) are also evident below the adherens junction, with electron-dense intracellular plaques connected to intermediate filaments. (b) In rats injected with saline alone, the lateral junctional complex is unaffected and appears similar to day 1 of normal pregnancy, with the adherens junction (AJ) evident below the tight junction (TJ) and desmosomes (D) are also present.
2.2. Ovarian hyperstimulation The ovarian hyperstimulation (OH) protocol developed by Lindsay and Murphy (2013) was used for this study. This involved daily vaginal smears to ensure rats were following a regular oestrous cycle. Once at least two continuous regular 4-day oestrous cycles were observed, 20IU of serum gonadotrophin (PMSG; Folligon, Intervet Australia, NSW, Australia) was administered via
140
S.N. Dowland et al. / Acta Histochemica 118 (2016) 137–143
intraperitoneal injection at noon mid-dioestrus. This was followed by 20IU of human chorionic gonadotrophin (hCG; Chorulon, Intervet Australia) 24 h later. These OH rats were then mated overnight with a male of proven fertility and the presence of sperm in a vaginal smear the following morning indicated successful mating and was designated day 1 of ovarian hyperstimulated pregnancy (OHD1). Uterine horns were collected from 5 rats each of OHD1 and OHD6 and tissue was processed as described below.
2.3. Cytochalasin D treatment Three day 1 pregnant rats were injected intraperitoneally with 30 g/kg of cytochalasin D (Sigma–Aldrich, MO, USA) in 0.2 ml sterile saline (Pfizer, NY, USA) as described previously (Luxford and Murphy, 1993). A further three day 1 pregnant rats were injected intraperitoneally with 0.2 ml sterile saline alone as a control. Tissue was collected 6 h later and processed for transmission electron microscopy. 2.4. Tissue collection Rats from normal pregnancy, OH pregnancy and after cytochalasin D treatment were injected with 20 mg/kg of sodium pentobarbitone (TROY Laboratories, NSW, Australia) and tissue was collected under deep anaesthesia, before euthanasia. Uterine horns were excised and processed for transmission electron microscopy (see below). 2.5. Transmission electron microscopy Uteri were excised, cut into 5 mm pieces and immediately immersed in Karnovsky’s fixative (2.5% glutaraldehyde (ProSciTech, Australia), 2% paraformaldehyde (ProSciTech, Australia) in 0.1 M Sorenson’s phosphate buffer (PB, pH 7.4)) for 45 mins at room temperature. Tissue was cut into 0.5–1 mm slices on dental wax under a droplet of fixative using a double-edged razor blade, and returned to fresh fixative for a further 45 mins. The tissue was washed in 0.1 M PB then postfixed for 1 hour in a solution of 1% osmium tetroxide (OsO4 ) in 0.1 M PB, containing 0.8% potassium ferricyanide to enhance the contrast of the plasma membrane and junctional complexes (Karnovsky, 1971). After a brief rinse in 0.1 M PB, the tissue was incubated in 2% OsO4 solution (in 0.1 M PB) for 10 min to remove any unreacted potassium ferricyanide (Hoshino et al., 1976). Tissue was washed extensively with MilliQ water before being dehydrated in a graded series of alcohols and infiltrated with Spurr’s resin (SPI supplies, Leicestershire, England, UK). Tissue slices were embedded in fresh Spurr’s resin in BEEM® capsules (ProSciTech, Australia) and polymerised at 60 ◦ C for 24 h. Two randomly-selected blocks per animal were cut using a Leica Ultracut T ultramicrotome (Leica, Heerbrugg, Switzerland) and 60–70 nm sections were mounted onto 400-mesh copper grids. Sections were post-stained with a saturated solution of uranyl acetate in 50% ethanol for 45 mins, followed by Reynold’s lead citrate for 10 mins. Sections were viewed with a Jeol 1011 transmission electron microscope (Jeol Ltd., Japan) at 80 kV and imaged with a Gatan SC200 Orius CCD Camera (Gatan Inc., USA). 3. Results 3.1. The adherens junction is lost during normal pregnancy The adherens junction was investigated throughout early pregnancy using TEM. On day 1 of pregnancy the adherens junction is present as part of the lateral junctional complex of UECs and immediately below the tight junction (Fig. 1a). On day 6 of pregnancy, the adherens junction is no longer evident between adjacent UECs (Fig. 1b).
Fig. 3. Transmission electron microscopy of the lateral junctional complex of UECs during OH pregnancy. (a) On day 1 of OH pregnancy the adherens junction appears similar to day 1 of normal pregnancy. The adherens junction (AJ) is present immediately below the tight junction (TJ) and desmosomes are evident (D). (b) In contrast to day 6 of normal pregnancy, on day 6 of OH pregnancy the adherens junction (AJ) remains. The lateral junctional complex is complete, with tight junctions (TJ), adherens junctions (AJ) and desmosomes (D) evident.
3.2. The adherens junction is unaffected by cytoskeletal disruption The effect of disrupting the actin cytoskeleton on the adherens junction was investigated by injecting the mycotoxin, cytochalasin D, which is known to disrupt the terminal web in UECs (Luxford and Murphy, 1993). Further rats were injected with saline alone
S.N. Dowland et al. / Acta Histochemica 118 (2016) 137–143
as a control. Day 1 pregnant rats injected with cytochalasin D retained morphological adherens junctions between adjacent UECs (Fig. 2a). Day 1 pregnant rats injected with saline alone also exhibited adherens junctions between UECs (Fig. 2b). 3.3. The adherens junction is not lost during OH pregnancy The effect of OH treatment on the adherens junction during early pregnancy was investigated using TEM. On day 1 of OH pregnancy, the adherens junction is visible in the lateral junctional complex between adjacent UECs (Fig. 3a). On day 6 of OH pregnancy, the adherens junction remains within the lateral junctional complex of UECs (Fig. 3b). 4. Discussion In preparation for blastocyst implantation, the lateral adhesiveness of UECs appears to be reduced, with a decrease in the number of desmosomes in the lateral plasma membrane (Preston et al., 2006). However, the other major component of lateral adhesiveness in epithelia, the adherens junction, has not yet been studied in UECs during early pregnancy. Previous molecular biology studies demonstrated a reduction in E-cadherin, a major component of the adherens junction, in UECs at the time of receptivity suggesting a change in the junctional composition at this time (Li et al., 2002; Potter et al., 1996). The present study aimed to examine whether the ultrastructural adherens junction is present during early pregnancy and whether it is affected by cytoskeletal disruption or OH treatment. This is the first study to demonstrate that the adherens junction is lost in rat UECs at the time of implantation during early pregnancy. The morphological adherens junction was found to be present at the time of fertilisation, which is consistent with previous observations (Luxford and Murphy, 1992b). However, at the time of receptivity, the adherens junction was no longer present in the lateral junctional complex. This confirms our hypothesis and is consistent with previous suggestions arising from molecular biology studies (Buck et al., 2012; Li et al., 2002; Potter et al., 1996). This loss of the adherens junction represents a decrease in cell–cell adhesion, which indicates that the integrity of the epithelial barrier is reduced (Meng and Takeichi, 2009; Niessen, 2007; Yap et al., 1997). This would promote dissociation of UECs and enable the processes extended by the blastocyst to penetrate between them (Nilsson, 1974; Schlafke and Enders, 1975; Schlafke et al., 1985; Tachi et al., 1970). These disassociated UECs would then be removed from the basal lamina and individual cells are internalised by the invading trophoblast (Finn and Lawn, 1968; Schlafke and Enders, 1975; Tachi et al., 1970). This study also demonstrates that while the actin cytoskeleton is required for adherens junction formation (Vasioukhin et al., 2000), disruption of the cytoskeleton does not necessarily result in the loss of this junction. Artificial disruption of the actin cytoskeleton with cytochalasin D did not result in a loss of the adherens junction and the junction was also unaffected in rats injected with saline alone. This indicates that the loss of the terminal web, which occurs during early pregnancy (Luxford and Murphy, 1992b), likely does not directly cause the loss of the adherens junction. We therefore suggest that more specific mechanisms are involved in removing the adherens junction components. One such possibility is that the loss of the adherens junction is driven by the deepening of the tight junction, which is known to occur at the time of implantation during normal pregnancy (Murphy et al., 1982). A recent study has demonstrated a reduction in E-cadherin mRNA expression in rat UECs at the time of implantation (Li et al., 2002). This coincided with a burst of calcitonin (a hormone involved
141
in calcium homeostasis) expression in rat UECs (Ding et al., 1994; Zhu et al., 1998b), which has also been found to increase sharply in the window of receptivity in humans (Kumar et al., 1998). It has also been shown that calcitonin treatment of Ishikawa cells (a human endometrial epithelial cell line) causes an increase in intracellular calcium and a loss of E-cadherin (Li et al., 2002). It is therefore suggested that the burst of calcitonin seen at the time of receptivity in rats (Li et al., 2002) causes a reduction in E-cadherin and a loss of the morphological adherens junction between UECs. Furthermore, as suppression of this calcitonin burst led to a decrease in implantation rate in rats (Zhu et al., 1998a), it is suggested that this loss of the adherens junction is important for blastocyst implantation. In contrast to our findings during normal pregnancy, this study demonstrated that during OH pregnancy, the adherens junction is not lost at the time of implantation. Previous studies have shown that oestrogen induces E-cadherin expression in UECs (Blaschuk et al., 1995), so the retention of the adherens junction during OH pregnancy may be due to the supraphysiological level of oestrogen that persists (Weinerman and Mainigi, 2014; Yeh et al., 2014). This indicates that at the time of implantation in OH pregnancy, adjacent UECs remain firmly attached to each other, thereby maintaining the epithelial barrier. This is likely to impede penetration of trophoblast processes between the UECs (Schlafke and Enders, 1975; Tachi et al., 1970). This then prevents such processes from extending laterally between the UECs and the underlying basal lamina, which is the cause of them being lifted off during normal pregnancy (Schlafke and Enders, 1975). Therefore the maintenance of the adherens junction at the time of implantation in OH pregnancy is suggested to impede blastocyst implantation. This may therefore be partially responsible for the decreased implantation and pregnancy rates that are seen after COH in IVF therapy (Check et al., 1999; Paulson et al., 1990; Yeh et al., 2014). Since high doses of oestrogen antagonise the production of calcitonin (Ding et al., 1994), this may also contribute to the retention of the adherens junction in OH pregnancy. This study has demonstrated that the adherens junction is lost in UECs at the time of implantation in normal pregnancy. This describes another component of The Plasma Membrane Transformation, which is required for UECs to become receptive to the implanting blastocyst. The study also provides further evidence to support the suggestion that alterations in the adherens junction are essential for blastocyst implantation. Author contributions S.N. Dowland and R.J. Madawala collected tissue. S.N. Dowland performed experiments and prepared the manuscript. S.N. Dowland, R.J. Madawala, L.A. Lindsay & C.R. Murphy designed the study and assisted with manuscript preparation. Acknowledgements The authors acknowledge the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility at the Australian Centre for Microscopy & Microanalysis, The University of Sydney. Financial support was provided by the Australian Research Council, The Ann Macintosh Foundation of the Discipline of Anatomy & Histology and the Murphy Laboratory. References Abrahamsohn PA, Zorn TM. Implantation and decidualization in rodents. J. Exp. Zool 1993;266:603–28, http://dx.doi.org/10.1002/jez.1402660610. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular Biology of the Cell. 5th ed. New York: Garland Science; 2008.
142
S.N. Dowland et al. / Acta Histochemica 118 (2016) 137–143
Arslan M, Bocca S, Mirkin S, Barroso G, Stadtmauer L, Oehninger S. Controlled ovarian hyperstimulation protocols for in vitro fertilization: two decades of experience after the birth of Elizabeth Carr. Fertil. Steril 2005;84:555––569, http://dx.doi.org/10.1016/j.fertnstert.2005.02.053. Baum B, Georgiou M. Dynamics of adherens junctions in epithelial establishment, maintenance, and remodeling. J. Cell Biol 2011;192:907–17, http://dx.doi.org/10.1083/jcb.201009141. Bazer FW, Spencer TE, Johnson GA, Burghardt RC, Wu G. Comparative aspects of implantation. Reproduction 2009;138:195–209, http://dx.doi.org/10.1530/REP-09-0158. Blaschuk OW, Munro SB, Farookhi R. Cadherins, steroids and cancer. Endocrine 1995;3:83–9, http://dx.doi.org/10.1007/BF02990057. Buck VU, Windoffer R, Leube RE, Classen-Linke I. Redistribution of adhering junctions in human endometrial epithelial cells during the implantation window of the menstrual cycle. Histochem. Cell Biol 2012;137:777–90, http://dx.doi.org/10.1007/s00418-012-0929-0. Check JH, Choe JK, Katsoff D, Summers-Chase D, Wilson C. Controlled ovarian hyperstimulation adversely affects implantation following in vitro fertilization-embryo transfer. J. Assist. Reprod. Genet 1999;16:416–20. D’souza-Schorey C. Disassembling adherens junctions: breaking up is hard to do. Trends Cell Biol 2005;15:19–26, http://dx.doi.org/10.1016/j.tcb.2004.11.002. Ding YQ, Zhu LJ, Bagchi MK, Bagchi IC. Progesterone stimulates calcitonin gene expression in the uterus during implantation. Endocrinology 1994;135:2265–74, http://dx.doi.org/10.1210/endo.135.5.7956949. Farquhar M, Palade G. Junctional complexes in various epithelia. J. Cell Biol 1963;17:375–412, http://dx.doi.org/10.1083/jcb.17.2.375. Finn CA, Lawn AM. Transfer of cellular material between the uterine epithelium and trophoblast during the early stages of implantation. Reproduction 1968;15:333–6, http://dx.doi.org/10.1530/jrf.0.0150333. Gumbiner BM. Regulation of cadherin-mediated adhesion in morphogenesis. Nat. Rev. Mol. Cell Biol 2005;6:622–34, http://dx.doi.org/10.1038/nrm1699. Harris TJC, Tepass U. Adherens junctions: from molecules to morphogenesis. Nat. Rev Mol. Cell Biol 2010;11:502–14, http://dx.doi.org/10.1038/nrm2927. Hartsock A, Nelson WJ. Adherens and tight junctions: structure, function and connections to the actin cytoskeleton. Biochim. Biophys. Acta 2008;1778:660–9, http://dx.doi.org/10.1016/j.bbamem.2007.07.012. Hirokawa N, Heuser JE. Quick-freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells. J. Cell Biol 1981;91:399–409. Hong S, Troyanovsky RB, Troyanovsky SM. Spontaneous assembly and active disassembly balance adherens junction homeostasis. Proc. Natl. Acad. Sci. U. S. A 2010;107:3528–33, http://dx.doi.org/10.1073/pnas.0911027107. Hoshino Y, Shannon WA, Seligman AM. Study of ferrocyanide-reduced osmium tetroxide as a stain and cytochemical agent. Acta Histochem. Cytochem 1976;9:125–36. Johnson L. Applications of imaging techniques to studies of epithelial tight junctions. Adv. Drug Deliv. Rev 2005;57:111–21, http://dx.doi.org/10.1016/j.addr.2004.08.004. Jones BJ, Murphy CR. A high resolution study of the glycocalyx of rat uterine epithelial cells during early pregnancy with the field emission gun scanning electron microscope. J. Anat 1994;185:443–6. Jovanovic´ A, Kramer B. The effect of hyperstimulation on transforming growth factor beta(1) and beta(2) in the rat uterus: possible consequences for embryo implantation. Fertil. Steril 2010;93:1509––1517, http://dx.doi.org/10.1016/j.fertnstert.2008.12.092. Kaneko Y, Day ML, Murphy CR. Uterine epithelial cells: serving two masters. Int. J. Biochem. Cell Biol 2013;45:359–63, http://dx.doi.org/10.1016/j.biocel.2012.10.012. Karnovsky MJ. Use of Ferrocyanide-Reduced Osmium Tetroxide in Electron Microscopy. Abstr. Am. Soc. Cell Biol. Elev. Annu. Meet. New Orleans 1971:146. Kramer B, Stein BA, Van der Walt LA. Exogenous gonadotropins–serum oestrogen and progesterone and the effect on endometrial morphology in the rat. J. Anat 1990;173:177–86. Kumar S, Zhu LJ, Polihronis M, Cameron ST, Baird DT, Schatz F, Dua A, Ying YK, Bagchi MK, Bagchi IC. Progesterone induces calcitonin gene expression in human endometrium within the putative window of implantation. J. Clin. Endocrinol. Metab 1998;83:4443–50, http://dx.doi.org/10.1210/jcem.83.12.5328. Lee KY, DeMayo FJ. Animal models of implantation. Reproduction 2004;128:679–95, http://dx.doi.org/10.1530/rep.1.00340. Li Q, Wang J, Armant DR, Bagchi MK, Bagchi IC. Calcitonin down-regulates E-cadherin expression in rodent uterine epithelium during implantation. J. Biol. Chem 2002;277:46447–55, http://dx.doi.org/10.1074/jbc.M203555200. Lindsay LA, Murphy CR. Ovarian hyperstimulation affects fluid transporters in the uterus: a potential mechanism in uterine receptivity. Reprod. Fertil. Dev 2013., http://dx.doi.org/10.1071/RD12396. Lutz KL, Siahaan TJ. Molecular structure of the apical junction complex and its contribution to the paracellular barrier. J. Pharm. Sci 1997;86:977–84, http://dx.doi.org/10.1021/js970134j. Luxford KA, Murphy CR. Cytoskeletal control of the apical surface transformation of rat uterine epithelium. Biol. Cell 1993;79:111–6.
Luxford KA, Murphy CR. Changes in the apical microfilaments of rat uterine epithelial cells in response to estradiol and progesterone. Anat. Rec 1992a;233:521–6, http://dx.doi.org/10.1002/ar.1092330405. Luxford KA, Murphy CR. Reorganization of the apical cytoskeleton of uterine epithelial-cells during early-pregnancy in the rat—a study with myosin subfragment-1. Biol. Cell 1992b;74:195–202. Mège R-M, Gavard J, Lambert M. Regulation of cell–cell junctions by the cytoskeleton. Curr. Opin. Cell Biol 2006;18:541–8, http://dx.doi.org/10.1016/j.ceb.2006.08.004. Meng W, Takeichi M. Adherens junction: molecular architecture and regulation. Cold Spring Harb. Perspect. Biol 2009;1., http://dx.doi.org/10.1101/cshperspect.a002899, a002899-a002899. Miyaguchi K. Ultrastructure of the zonula adherens revealed by rapid-freeze deep-etching. J. Struct. Biol 2000;132:169–78, http://dx.doi.org/10.1006/jsbi.2000.4244. Murphy CR. Uterine receptivity and the plasma membrane transformation. Cell Res 2004;14:259–67. Murphy CR. Junctional barrier complexes undergo major alterations during the plasma membrane transformation of uterine epithelial cells. Hum. Reprod 2000a;15(Suppl. 3):182–8. Murphy CR. The plasma membrane transformation of uterine epithelial cells during pregnancy. J. Reprod. Fertil. Suppl 2000b;55:23–8. Murphy CR. The plasma membrane of uterine epithelial cells: structure and histochemistry. Prog. Histochem. Cytochem 1993;27:1–66. Murphy CR, Swift JG, Mukherjee TM, Rogers AW. The structure of tight junctions between uterine luminal epithelial cells at different stages of pregnancy in the rat. Cell Tissue Res 1982;223:281–6, http://dx.doi.org/10.1007/bf01258489. Nagafuchi A. Molecular architecture of adherens junctions. Curr. Opin. Cell Biol 2001;13:600–3, http://dx.doi.org/10.1016/S0955-0674(00)00257-X. Niessen CM. Tight junctions/adherens junctions: basic structure and function. J. Invest. Dermatol 2007;127:2525–32, http://dx.doi.org/10.1038/sj.jid.5700865. Nilsson O. The morphology of blastocyst implantation. Reproduction 1974;39:187–94, http://dx.doi.org/10.1530/jrf.0.0390187. Patel SD, Ciatto C, Chen CP, Bahna F, Rajebhosale M, Arkus N, Schieren I, Jessell TM, Honig B, Price SR, Shapiro L. Type II cadherin ectodomain structures: implications for classical cadherin specificity. Cell 2006;124:1255–68, http://dx.doi.org/10.1016/j.cell.2005.12.046. Paulson RJ, Sauer MV, Lobo RA. Embryo implantation after human in vitro fertilization: importance of endometrial receptivity. Fertil. Steril 1990;53:870–4. Png FY, Murphy CR. The plasma membrane transformation does not last: microvilli return to the apical plasma membrane of uterine epithelial cells after the period of uterine receptivity. Eur. J. Morphol 1997;35:19–24, http://dx.doi.org/10.1076/ejom.35.1.19.13061. Pokutta S, Herrenknecht K, Kemler R, Engel J. Conformational changes of the recombinant extracellular domain of E-cadherin upon calcium binding. Eur. J Biochem 1994;223:1019–26, http://dx.doi.org/10.1111/j.1432-1033.1994.tb19080.x. Potter SW, Gaza G, Morris JE. Estradiol induces E-cadherin degradation in mouse uterine epithelium during the estrous cycle and early pregnancy. J. Cell Physiol 1996;169:1–14, 10.1002/(SICI)1097-4652(199610)169:13.0.CO;2-S. Preston AM, Lindsay LA, Murphy CR. Desmosomes in uterine epithelial cells decrease at the time of implantation: an ultrastructural and morphometric study. J. Morphol 2006;267:103–8, http://dx.doi.org/10.1002/jmor.10390. Rowlands T, Symonds JM, Farookhi R, Blaschuk OW. Cadherins: crucial regulators of structure and function in reproductive tissues. Rev. Reprod 2000;5:53–61, http://dx.doi.org/10.1530/ror.0.0050053. Schlafke S, Enders AC. Cellular basis of interaction between trophoblast and uterus at implantation. Biol. Reprod 1975;12:41–65, http://dx.doi.org/10.1095/biolreprod12.1.41. Schlafke S, Welsh AO, Enders AC. Penetration of the basal lamina of the uterine luminal epithelium during implantation in the rat. Anat. Rec 1985;212:47–56, http://dx.doi.org/10.1002/ar.1092120107. Schneeberger EE, Lynch RD. The tight junction: a multifunctional complex. Am. J. Physiol. Cell Physiol 2004;286:C1213–8, http://dx.doi.org/10.1152/ajpcell.00558.2003. Shion YL, Murphy CR. The basal plasma membrane and lamina densa of uterine epithelial cells are both altered during early pregnancy and by ovarian hormones in the rat. Eur. J. Morphol 1995;33:257–64. Tachi S, Tachi C, Lindner HR. Ultrastructural features of blastocyst attachment and trophoblastic invasion in the rat. Reproduction 1970;21:37–56, http://dx.doi.org/10.1530/jrf.0.0210037. Theard D, Raspe MA, Kalicharan D, Hoekstra D, van IJzendoorn SCD. Formation of E-cadherin/-catenin-based adherens junctions in hepatocytes requires serine-10 in p27(Kip1). Mol. Biol. Cell 2008;9:1605–13, http://dx.doi.org/10.1091/mbc.E07-07-0661. Tsukita S, Tsukita S, Nagafuchi A, Yonemura S. Molecular linkage between cadherins and actin filaments in cell–cell adherens junctions. Curr. Opin. Cell Biol 1992;4:834–9, http://dx.doi.org/10.1016/0955-0674(92) 90108-O. Vasioukhin V, Bauer C, Yin M, Fuchs E. Directed actin polymerization is the driving force for epithelial cell–cell adhesion. Cell 2000;100:209–19, http://dx.doi.org/10.1016/S0092-8674(00) 81559-7.
S.N. Dowland et al. / Acta Histochemica 118 (2016) 137–143 Vasioukhin V, Fuchs E. Actin dynamics and cell–cell adhesion in epithelia. Curr. Opin. Cell Biol 2001;13:76–84, http://dx.doi.org/10.1016/S0955-0674(00) 00177-0. Weinerman R, Mainigi M. Why we should transfer frozen instead of fresh embryos: the translational rationale. Fertil. Steril 2014;102:10–8, http://dx.doi.org/10.1016/j.fertnstert.2014.05.019. Yap AS, Brieher WM, Gumbiner BM. Molecular and functional analysis of cadherin-based adherens junctions. Annu. Rev. Cell Dev. Biol 1997;13:119–46, http://dx.doi.org/10.1146/annurev.cellbio.13.1.119. Yeh JS, Steward RG, Dude AM, Shah AA, Goldfarb JM, Muasher SJ. Pregnancy rates in donor oocyte cycles compared to similar autologous in vitro fertilization
143
cycles: an analysis of 26,457 fresh cycles from the Society for Assisted Reproductive Technology. Fertil. Steril 2014;102:399–404, http://dx.doi.org/10.1016/j.fertnstert.2014.04.027. Zhu L-J, Bagchi MK, Bagchi IC. Attenuation of calcitonin gene expression in pregnant rat uterus leads to a block in embryonic implantation 1. Endocrinology 1998a;139:330–9, http://dx.doi.org/10.1210/endo.139.1.5707. Zhu L-J, Cullinan-Bove K, Polihronis M, Bagchi MK, Bagchi IC. Calcitonin is a progesterone-regulated marker that forecasts the receptive state of endometrium during implantation 1. Endocrinology 1998b;139:3923–34, http://dx.doi.org/10.1210/endo.139.9.6178.
Contents lists available at ScienceDirect
Acta Histochemica journal homepage: www.elsevier.de/acthis
The adherens junction is lost during normal pregnancy but not during ovarian hyperstimulated pregnancy Samson N. Dowland ∗ , Romanthi J. Madawala, Laura A. Lindsay, Christopher R. Murphy Cell & Reproductive Biology Laboratory, School of Medical Sciences (Discipline of Anatomy & Histology) and The Bosch Institute, The University of Sydney, Sydney, NSW 2006, Australia
a r t i c l e
i n f o
Article history: Received 3 December 2015 Received in revised form 15 December 2015 Accepted 15 December 2015 Keywords: Adherens junction Pregnancy Implantation Uterine receptivity Epithelium Ovarian hyperstimulation
a b s t r a c t 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. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction During invasive blastocyst implantation, which occurs in humans and rats, the trophoblast cells must breach the uterine epithelium and invade the underlying stroma to establish the placenta (Abrahamsohn and Zorn, 1993; Bazer et al., 2009; Lee and DeMayo, 2004). During the penetration stage of implantation, the trophoblast cells of the blastocyst extend processes between adjacent luminal uterine epithelial cells (UECs) (Schlafke and Enders, 1975; Tachi et al., 1970). These processes eventually lift the UECs from their basal lamina and they are internalised by the invading trophoblast (Finn and Lawn, 1968; Schlafke and Enders, 1975; Tachi et al., 1970). This allows the blastocyst to make direct contact with the newly exposed basal lamina, where it pauses until the decidual
∗ Corresponding author at: Cell & Reproductive Biology Laboratory, F13 Anderson Stuart Building, The University of Sydney, NSW 2006, Australia. E-mail address: [email protected] (S.N. Dowland). http://dx.doi.org/10.1016/j.acthis.2015.12.004 0065-1281/© 2015 Elsevier GmbH. All rights reserved.
cells breach through from the stromal side (Schlafke et al., 1985). Such disassociation and penetration of adjacent UECs represents a loss of the epithelial barrier function in this region (Kaneko et al., 2013; Murphy, 2000a). For this to occur, it is expected that the usual hallmarks of the barrier epithelium, in particular the lateral junctional complex, must be affected. To permit blastocyst implantation, the UECs must transform from a non-receptive to a receptive phenotype. This process is collectively termed “The Plasma Membrane Transformation” and is largely controlled by the maternal ovarian hormones oestrogen and progesterone (Murphy, 2004, 2000b, 1993). The Plasma Membrane Transformation includes changes to all three plasma membrane domains; apical, lateral and basal. In the apical domain, there is a loss of microvilli and the associated glycocalyx to permit the blastocyst to attach to the cell surface (Jones and Murphy, 1994; Png and Murphy, 1997). In the basal domain, there is an increase in basal plasma membrane tortuosity and a loss of morphological focal adhesions to “loosen” the adhesion of the UECs to the underlying extracellular matrix (Shion and Murphy, 1995). In the lateral
138
S.N. Dowland et al. / Acta Histochemica 118 (2016) 137–143
Fig. 1. Transmission electron microscopy of the lateral junctional complex of UECs during normal pregnancy. (a) On day 1 of normal pregnancy the adherens junction (AJ) is evident within the lateral junctional complex. The “fuzzy plaque” is visible on the intracellular surface of the junction and there is a uniform intercellular space of low electron-density. The adherens junction sits immediately below the tight junction (TJ), where the electron-dense plasma membranes are held close together, eliminating most of the intercellular space. (b) On day 6 of normal pregnancy the tight junction (TJ) becomes deepened as described previously. The adherens junction is no longer evident within the lateral junctional complex.
domain, there is an increase in the depth of the tight junction and a decrease in the number of desmosomes (Murphy et al., 1982; Preston et al., 2006). The increased depth of the tight junction is unexpected when considering that the blastocyst must penetrate between the UECs. However, the tight junction is primarily involved in controlling the paracellular permeability of epithelial cells and is thought to provide little mechanical adhesion (Alberts et al., 2008; Johnson, 2005; Lutz and Siahaan, 1997). The increased depth of the tight junction is required to strictly control the contents of the uterine
lumen during blastocyst implantation (Murphy, 2000a; Murphy et al., 1982). The decrease in the number of desmosomes (which are strong mechanical cell–cell contacts), represents a reduction in the lateral adhesiveness of the UECs that is suggested to allow the cells to disassociate and the trophoblast to penetrate between them (Preston et al., 2006; Schlafke and Enders, 1975; Tachi et al., 1970). However, the third component of the lateral junctional complex, the adherens junction, has been little investigated in rat UECs during early pregnancy. The adherens junction (zonula adherens) is the major lateral adhesive junction in epithelial cells and is primarily responsible for epithelial cell–cell adhesion (D’souza-Schorey, 2005; Niessen, 2007). The adherens junction forms a continuous band around the apical domain that links the actin cytoskeleton of adjacent cells, providing a strong structural attachment (Harris and Tepass, 2010; Vasioukhin and Fuchs, 2001). The lateral adhesion generated by the mechanical connection of the cells is essential for maintaining the integrity of the epithelial barrier (Meng and Takeichi, 2009; Niessen, 2007). Loss of the adherens junction represents a loss of cell–cell adhesion and acquisition of migratory potential (D’souzaSchorey, 2005). It is therefore suggested that the adherens junction would prevent the uterine epithelium from being breached by trophoblast cell processes; with the corollary that the adherens junction must be lost in order to permit the blastocyst to penetrate the uterine epithelium. The adherens junction is primarily composed of the single-pass transmembrane protein E-cadherin (Gumbiner, 2005; Hartsock and Nelson, 2008; Nagafuchi, 2001; Tsukita et al., 1992). The cadherin proteins are crucial for intercellular adhesion and cells lacking cadherin do not form strong cell–cell adhesions (Lutz and Siahaan, 1997). On the extracellular side, E-cadherin molecules within adherens junctions of adjacent cells are linked by calcium ions (Hartsock and Nelson, 2008; Tsukita et al., 1992). This calcium binding is essential for correct conformational organisation of the protein, which is crucial for E-cadherin (homotypic) adhesion (Baum and Georgiou, 2011; Hartsock and Nelson, 2008; Patel et al., 2006; Pokutta et al., 1994; Theard et al., 2008). As such, the adherens junction is calcium-dependent and removal of calcium causes disruption of the junction (Meng and Takeichi, 2009). Intracellularly, E-cadherin interacts with a range of proteins that mediate the functions of the adherens junction, including cell–cell adhesion, intracellular signalling, local control of the actin cytoskeleton and disassembly of the junction (Hartsock and Nelson, 2008). In particular, E-cadherin forms a complex with the cytoplasmic catenin proteins (␣,  and p120) on the intracellular face of the adherens junction that connects the junction to the actin cytoskeleton (Hartsock and Nelson, 2008; Rowlands et al., 2000; Yap et al., 1997). In the transmission electron microscope (TEM), the adherens junction is seen immediately below the tight junction on the lateral plasma membrane. In contrast to the tight junction, where adjacent plasma membranes are held close together (Farquhar and Palade, 1963; Niessen, 2007; Schneeberger and Lynch, 2004), in the adherens junction there is a uniform space between adjacent plasma membranes (Farquhar and Palade, 1963). This space is of low electron density and corresponds to the extracellular domains of E-cadherin molecules and the associated calcium ions (Farquhar and Palade, 1963; Miyaguchi, 2000; Niessen, 2007). On the intracellular surface, a “fuzzy plaque” characterises the adherens junction. This plaque is moderately electron dense and can be seen linking to the filaments of the actin cytoskeleton (Farquhar and Palade, 1963; Hirokawa and Heuser, 1981; Miyaguchi, 2000; Nagafuchi, 2001). This plaque corresponds to the E-cadherin-catenin complex that connects the adherens junction to the cytoskeleton (Miyaguchi, 2000; Nagafuchi, 2001).
S.N. Dowland et al. / Acta Histochemica 118 (2016) 137–143
While the adherens junction is yet to be studied ultrastructurally in rat UECs during early pregnancy, it has previously been observed in these cells. While studying the actin cytoskeleton, Luxford and Murphy (1992a,b) identified that the adherens junction is present in UECs at the time of fertilisation and in ovariectomised rats treated with oestrogen alone. Previous studies in mice (Potter et al., 1996) and rats (Li et al., 2002) have identified a reduction in Ecadherin in UECs at the time of receptivity. Further studies in humans have shown that while E-cadherin is present at distinct junctional regions during the non-receptive phase of the menstrual cycle, this protein spreads out into a diffuse lateral distribution specifically in the window of receptivity (Buck et al., 2012). These molecular biology studies indicate that the key adherens junction protein, E-cadherin, is likely removed from the adherens junction at the time of receptivity (Buck et al., 2012; Li et al., 2002; Potter et al., 1996). Therefore, it is hypothesised that the adherens junction is lost during early pregnancy, in preparation for blastocyst implantation. The actin cytoskeleton is an integral component of the adherens junction and abnormalities in this cytoskeleton prevent adherens junction assembly (Hong et al., 2010; Mège et al., 2006). Furthermore, cytoskeletal remodelling has been shown to cause a loss of adherens junctions (D’souza-Schorey, 2005). Therefore, as the terminal web (component of the actin cytoskeleton that anchors into the adherens junction) is disrupted at the time of receptivity in UECs (Luxford and Murphy, 1992b), it is expected that this causes the adherens junction to be lost at the time of receptivity. This study will investigate whether the loss of the terminal web affects the morphological adherens junction by artificially disrupting the actin cytoskeleton in UECs. As the actin cytoskeleton drives the formation of the adherens junction (Vasioukhin et al., 2000), it is hypothesised that artificial disruption of the actin cytoskeleton will cause a loss of the adherens junction. In assisted reproductive technologies, such as in vitro fertilisation (IVF), controlled ovarian hyperstimulation (COH) is used to increase oocyte yield (Arslan et al., 2005). However, COH has been found to cause a reduction in pregnancy and implantation rates, specifically due to its negative impact on the endometrium (Check et al., 1999; Paulson et al., 1990; Yeh et al., 2014). Recentlydeveloped rat ovarian hyperstimulation (OH) models emulate COH treatment used during IVF therapy, thereby enabling the experimental examination of the impact of such treatment on the endometrium (Jovanovic´ and Kramer, 2010; Kramer et al., 1990; Lindsay and Murphy, 2013). This study has utilised a rat OH model to investigate whether the OH procedure affects the adherens junction in UECs. Since COH induces a supraphysiological level of oestrogen that persists into the implantation window (Weinerman and Mainigi, 2014; Yeh et al., 2014), and the adherens junction has previously been observed in UECs under the influence of oestrogen (Luxford and Murphy, 1992a), it is predicted that the adherens junction will remain after OH.
2. Material and methods 2.1. Animals and mating Adult virgin female Wistar rats were used for this study and all procedures were approved by The University of Sydney Animal Ethics Committee. Rats were housed in plastic cages under a 12-h light–dark cycle with free access to food and water. Females were mated overnight with males of proven fertility and the presence of sperm in a vaginal smear the following morning indicated successful mating and was designated day 1 of pregnancy. Uterine horns were collected from 5 rats each of days 1 and 6 of pregnancy to study non-receptive and receptive conditions respectively.
139
Fig. 2. Transmission electron microscopy of the lateral junctional complex of UECs from day 1 pregnant rats treated with cytochalasin D, to artificially disrupt the actin cytoskeleton and vehicle controls. (a) Cytochalasin D treatment does not affect the lateral junctional complex of UECs, as the adherens junction (AJ) is still present immediately below the tight junction (TJ). Desmosomes (D) are also evident below the adherens junction, with electron-dense intracellular plaques connected to intermediate filaments. (b) In rats injected with saline alone, the lateral junctional complex is unaffected and appears similar to day 1 of normal pregnancy, with the adherens junction (AJ) evident below the tight junction (TJ) and desmosomes (D) are also present.
2.2. Ovarian hyperstimulation The ovarian hyperstimulation (OH) protocol developed by Lindsay and Murphy (2013) was used for this study. This involved daily vaginal smears to ensure rats were following a regular oestrous cycle. Once at least two continuous regular 4-day oestrous cycles were observed, 20IU of serum gonadotrophin (PMSG; Folligon, Intervet Australia, NSW, Australia) was administered via
140
S.N. Dowland et al. / Acta Histochemica 118 (2016) 137–143
intraperitoneal injection at noon mid-dioestrus. This was followed by 20IU of human chorionic gonadotrophin (hCG; Chorulon, Intervet Australia) 24 h later. These OH rats were then mated overnight with a male of proven fertility and the presence of sperm in a vaginal smear the following morning indicated successful mating and was designated day 1 of ovarian hyperstimulated pregnancy (OHD1). Uterine horns were collected from 5 rats each of OHD1 and OHD6 and tissue was processed as described below.
2.3. Cytochalasin D treatment Three day 1 pregnant rats were injected intraperitoneally with 30 g/kg of cytochalasin D (Sigma–Aldrich, MO, USA) in 0.2 ml sterile saline (Pfizer, NY, USA) as described previously (Luxford and Murphy, 1993). A further three day 1 pregnant rats were injected intraperitoneally with 0.2 ml sterile saline alone as a control. Tissue was collected 6 h later and processed for transmission electron microscopy. 2.4. Tissue collection Rats from normal pregnancy, OH pregnancy and after cytochalasin D treatment were injected with 20 mg/kg of sodium pentobarbitone (TROY Laboratories, NSW, Australia) and tissue was collected under deep anaesthesia, before euthanasia. Uterine horns were excised and processed for transmission electron microscopy (see below). 2.5. Transmission electron microscopy Uteri were excised, cut into 5 mm pieces and immediately immersed in Karnovsky’s fixative (2.5% glutaraldehyde (ProSciTech, Australia), 2% paraformaldehyde (ProSciTech, Australia) in 0.1 M Sorenson’s phosphate buffer (PB, pH 7.4)) for 45 mins at room temperature. Tissue was cut into 0.5–1 mm slices on dental wax under a droplet of fixative using a double-edged razor blade, and returned to fresh fixative for a further 45 mins. The tissue was washed in 0.1 M PB then postfixed for 1 hour in a solution of 1% osmium tetroxide (OsO4 ) in 0.1 M PB, containing 0.8% potassium ferricyanide to enhance the contrast of the plasma membrane and junctional complexes (Karnovsky, 1971). After a brief rinse in 0.1 M PB, the tissue was incubated in 2% OsO4 solution (in 0.1 M PB) for 10 min to remove any unreacted potassium ferricyanide (Hoshino et al., 1976). Tissue was washed extensively with MilliQ water before being dehydrated in a graded series of alcohols and infiltrated with Spurr’s resin (SPI supplies, Leicestershire, England, UK). Tissue slices were embedded in fresh Spurr’s resin in BEEM® capsules (ProSciTech, Australia) and polymerised at 60 ◦ C for 24 h. Two randomly-selected blocks per animal were cut using a Leica Ultracut T ultramicrotome (Leica, Heerbrugg, Switzerland) and 60–70 nm sections were mounted onto 400-mesh copper grids. Sections were post-stained with a saturated solution of uranyl acetate in 50% ethanol for 45 mins, followed by Reynold’s lead citrate for 10 mins. Sections were viewed with a Jeol 1011 transmission electron microscope (Jeol Ltd., Japan) at 80 kV and imaged with a Gatan SC200 Orius CCD Camera (Gatan Inc., USA). 3. Results 3.1. The adherens junction is lost during normal pregnancy The adherens junction was investigated throughout early pregnancy using TEM. On day 1 of pregnancy the adherens junction is present as part of the lateral junctional complex of UECs and immediately below the tight junction (Fig. 1a). On day 6 of pregnancy, the adherens junction is no longer evident between adjacent UECs (Fig. 1b).
Fig. 3. Transmission electron microscopy of the lateral junctional complex of UECs during OH pregnancy. (a) On day 1 of OH pregnancy the adherens junction appears similar to day 1 of normal pregnancy. The adherens junction (AJ) is present immediately below the tight junction (TJ) and desmosomes are evident (D). (b) In contrast to day 6 of normal pregnancy, on day 6 of OH pregnancy the adherens junction (AJ) remains. The lateral junctional complex is complete, with tight junctions (TJ), adherens junctions (AJ) and desmosomes (D) evident.
3.2. The adherens junction is unaffected by cytoskeletal disruption The effect of disrupting the actin cytoskeleton on the adherens junction was investigated by injecting the mycotoxin, cytochalasin D, which is known to disrupt the terminal web in UECs (Luxford and Murphy, 1993). Further rats were injected with saline alone
S.N. Dowland et al. / Acta Histochemica 118 (2016) 137–143
as a control. Day 1 pregnant rats injected with cytochalasin D retained morphological adherens junctions between adjacent UECs (Fig. 2a). Day 1 pregnant rats injected with saline alone also exhibited adherens junctions between UECs (Fig. 2b). 3.3. The adherens junction is not lost during OH pregnancy The effect of OH treatment on the adherens junction during early pregnancy was investigated using TEM. On day 1 of OH pregnancy, the adherens junction is visible in the lateral junctional complex between adjacent UECs (Fig. 3a). On day 6 of OH pregnancy, the adherens junction remains within the lateral junctional complex of UECs (Fig. 3b). 4. Discussion In preparation for blastocyst implantation, the lateral adhesiveness of UECs appears to be reduced, with a decrease in the number of desmosomes in the lateral plasma membrane (Preston et al., 2006). However, the other major component of lateral adhesiveness in epithelia, the adherens junction, has not yet been studied in UECs during early pregnancy. Previous molecular biology studies demonstrated a reduction in E-cadherin, a major component of the adherens junction, in UECs at the time of receptivity suggesting a change in the junctional composition at this time (Li et al., 2002; Potter et al., 1996). The present study aimed to examine whether the ultrastructural adherens junction is present during early pregnancy and whether it is affected by cytoskeletal disruption or OH treatment. This is the first study to demonstrate that the adherens junction is lost in rat UECs at the time of implantation during early pregnancy. The morphological adherens junction was found to be present at the time of fertilisation, which is consistent with previous observations (Luxford and Murphy, 1992b). However, at the time of receptivity, the adherens junction was no longer present in the lateral junctional complex. This confirms our hypothesis and is consistent with previous suggestions arising from molecular biology studies (Buck et al., 2012; Li et al., 2002; Potter et al., 1996). This loss of the adherens junction represents a decrease in cell–cell adhesion, which indicates that the integrity of the epithelial barrier is reduced (Meng and Takeichi, 2009; Niessen, 2007; Yap et al., 1997). This would promote dissociation of UECs and enable the processes extended by the blastocyst to penetrate between them (Nilsson, 1974; Schlafke and Enders, 1975; Schlafke et al., 1985; Tachi et al., 1970). These disassociated UECs would then be removed from the basal lamina and individual cells are internalised by the invading trophoblast (Finn and Lawn, 1968; Schlafke and Enders, 1975; Tachi et al., 1970). This study also demonstrates that while the actin cytoskeleton is required for adherens junction formation (Vasioukhin et al., 2000), disruption of the cytoskeleton does not necessarily result in the loss of this junction. Artificial disruption of the actin cytoskeleton with cytochalasin D did not result in a loss of the adherens junction and the junction was also unaffected in rats injected with saline alone. This indicates that the loss of the terminal web, which occurs during early pregnancy (Luxford and Murphy, 1992b), likely does not directly cause the loss of the adherens junction. We therefore suggest that more specific mechanisms are involved in removing the adherens junction components. One such possibility is that the loss of the adherens junction is driven by the deepening of the tight junction, which is known to occur at the time of implantation during normal pregnancy (Murphy et al., 1982). A recent study has demonstrated a reduction in E-cadherin mRNA expression in rat UECs at the time of implantation (Li et al., 2002). This coincided with a burst of calcitonin (a hormone involved
141
in calcium homeostasis) expression in rat UECs (Ding et al., 1994; Zhu et al., 1998b), which has also been found to increase sharply in the window of receptivity in humans (Kumar et al., 1998). It has also been shown that calcitonin treatment of Ishikawa cells (a human endometrial epithelial cell line) causes an increase in intracellular calcium and a loss of E-cadherin (Li et al., 2002). It is therefore suggested that the burst of calcitonin seen at the time of receptivity in rats (Li et al., 2002) causes a reduction in E-cadherin and a loss of the morphological adherens junction between UECs. Furthermore, as suppression of this calcitonin burst led to a decrease in implantation rate in rats (Zhu et al., 1998a), it is suggested that this loss of the adherens junction is important for blastocyst implantation. In contrast to our findings during normal pregnancy, this study demonstrated that during OH pregnancy, the adherens junction is not lost at the time of implantation. Previous studies have shown that oestrogen induces E-cadherin expression in UECs (Blaschuk et al., 1995), so the retention of the adherens junction during OH pregnancy may be due to the supraphysiological level of oestrogen that persists (Weinerman and Mainigi, 2014; Yeh et al., 2014). This indicates that at the time of implantation in OH pregnancy, adjacent UECs remain firmly attached to each other, thereby maintaining the epithelial barrier. This is likely to impede penetration of trophoblast processes between the UECs (Schlafke and Enders, 1975; Tachi et al., 1970). This then prevents such processes from extending laterally between the UECs and the underlying basal lamina, which is the cause of them being lifted off during normal pregnancy (Schlafke and Enders, 1975). Therefore the maintenance of the adherens junction at the time of implantation in OH pregnancy is suggested to impede blastocyst implantation. This may therefore be partially responsible for the decreased implantation and pregnancy rates that are seen after COH in IVF therapy (Check et al., 1999; Paulson et al., 1990; Yeh et al., 2014). Since high doses of oestrogen antagonise the production of calcitonin (Ding et al., 1994), this may also contribute to the retention of the adherens junction in OH pregnancy. This study has demonstrated that the adherens junction is lost in UECs at the time of implantation in normal pregnancy. This describes another component of The Plasma Membrane Transformation, which is required for UECs to become receptive to the implanting blastocyst. The study also provides further evidence to support the suggestion that alterations in the adherens junction are essential for blastocyst implantation. Author contributions S.N. Dowland and R.J. Madawala collected tissue. S.N. Dowland performed experiments and prepared the manuscript. S.N. Dowland, R.J. Madawala, L.A. Lindsay & C.R. Murphy designed the study and assisted with manuscript preparation. Acknowledgements The authors acknowledge the facilities, and the scientific and technical assistance, of the Australian Microscopy & Microanalysis Research Facility at the Australian Centre for Microscopy & Microanalysis, The University of Sydney. Financial support was provided by the Australian Research Council, The Ann Macintosh Foundation of the Discipline of Anatomy & Histology and the Murphy Laboratory. References Abrahamsohn PA, Zorn TM. Implantation and decidualization in rodents. J. Exp. Zool 1993;266:603–28, http://dx.doi.org/10.1002/jez.1402660610. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular Biology of the Cell. 5th ed. New York: Garland Science; 2008.
142
S.N. Dowland et al. / Acta Histochemica 118 (2016) 137–143
Arslan M, Bocca S, Mirkin S, Barroso G, Stadtmauer L, Oehninger S. Controlled ovarian hyperstimulation protocols for in vitro fertilization: two decades of experience after the birth of Elizabeth Carr. Fertil. Steril 2005;84:555––569, http://dx.doi.org/10.1016/j.fertnstert.2005.02.053. Baum B, Georgiou M. Dynamics of adherens junctions in epithelial establishment, maintenance, and remodeling. J. Cell Biol 2011;192:907–17, http://dx.doi.org/10.1083/jcb.201009141. Bazer FW, Spencer TE, Johnson GA, Burghardt RC, Wu G. Comparative aspects of implantation. Reproduction 2009;138:195–209, http://dx.doi.org/10.1530/REP-09-0158. Blaschuk OW, Munro SB, Farookhi R. Cadherins, steroids and cancer. Endocrine 1995;3:83–9, http://dx.doi.org/10.1007/BF02990057. Buck VU, Windoffer R, Leube RE, Classen-Linke I. Redistribution of adhering junctions in human endometrial epithelial cells during the implantation window of the menstrual cycle. Histochem. Cell Biol 2012;137:777–90, http://dx.doi.org/10.1007/s00418-012-0929-0. Check JH, Choe JK, Katsoff D, Summers-Chase D, Wilson C. Controlled ovarian hyperstimulation adversely affects implantation following in vitro fertilization-embryo transfer. J. Assist. Reprod. Genet 1999;16:416–20. D’souza-Schorey C. Disassembling adherens junctions: breaking up is hard to do. Trends Cell Biol 2005;15:19–26, http://dx.doi.org/10.1016/j.tcb.2004.11.002. Ding YQ, Zhu LJ, Bagchi MK, Bagchi IC. Progesterone stimulates calcitonin gene expression in the uterus during implantation. Endocrinology 1994;135:2265–74, http://dx.doi.org/10.1210/endo.135.5.7956949. Farquhar M, Palade G. Junctional complexes in various epithelia. J. Cell Biol 1963;17:375–412, http://dx.doi.org/10.1083/jcb.17.2.375. Finn CA, Lawn AM. Transfer of cellular material between the uterine epithelium and trophoblast during the early stages of implantation. Reproduction 1968;15:333–6, http://dx.doi.org/10.1530/jrf.0.0150333. Gumbiner BM. Regulation of cadherin-mediated adhesion in morphogenesis. Nat. Rev. Mol. Cell Biol 2005;6:622–34, http://dx.doi.org/10.1038/nrm1699. Harris TJC, Tepass U. Adherens junctions: from molecules to morphogenesis. Nat. Rev Mol. Cell Biol 2010;11:502–14, http://dx.doi.org/10.1038/nrm2927. Hartsock A, Nelson WJ. Adherens and tight junctions: structure, function and connections to the actin cytoskeleton. Biochim. Biophys. Acta 2008;1778:660–9, http://dx.doi.org/10.1016/j.bbamem.2007.07.012. Hirokawa N, Heuser JE. Quick-freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells. J. Cell Biol 1981;91:399–409. Hong S, Troyanovsky RB, Troyanovsky SM. Spontaneous assembly and active disassembly balance adherens junction homeostasis. Proc. Natl. Acad. Sci. U. S. A 2010;107:3528–33, http://dx.doi.org/10.1073/pnas.0911027107. Hoshino Y, Shannon WA, Seligman AM. Study of ferrocyanide-reduced osmium tetroxide as a stain and cytochemical agent. Acta Histochem. Cytochem 1976;9:125–36. Johnson L. Applications of imaging techniques to studies of epithelial tight junctions. Adv. Drug Deliv. Rev 2005;57:111–21, http://dx.doi.org/10.1016/j.addr.2004.08.004. Jones BJ, Murphy CR. A high resolution study of the glycocalyx of rat uterine epithelial cells during early pregnancy with the field emission gun scanning electron microscope. J. Anat 1994;185:443–6. Jovanovic´ A, Kramer B. The effect of hyperstimulation on transforming growth factor beta(1) and beta(2) in the rat uterus: possible consequences for embryo implantation. Fertil. Steril 2010;93:1509––1517, http://dx.doi.org/10.1016/j.fertnstert.2008.12.092. Kaneko Y, Day ML, Murphy CR. Uterine epithelial cells: serving two masters. Int. J. Biochem. Cell Biol 2013;45:359–63, http://dx.doi.org/10.1016/j.biocel.2012.10.012. Karnovsky MJ. Use of Ferrocyanide-Reduced Osmium Tetroxide in Electron Microscopy. Abstr. Am. Soc. Cell Biol. Elev. Annu. Meet. New Orleans 1971:146. Kramer B, Stein BA, Van der Walt LA. Exogenous gonadotropins–serum oestrogen and progesterone and the effect on endometrial morphology in the rat. J. Anat 1990;173:177–86. Kumar S, Zhu LJ, Polihronis M, Cameron ST, Baird DT, Schatz F, Dua A, Ying YK, Bagchi MK, Bagchi IC. Progesterone induces calcitonin gene expression in human endometrium within the putative window of implantation. J. Clin. Endocrinol. Metab 1998;83:4443–50, http://dx.doi.org/10.1210/jcem.83.12.5328. Lee KY, DeMayo FJ. Animal models of implantation. Reproduction 2004;128:679–95, http://dx.doi.org/10.1530/rep.1.00340. Li Q, Wang J, Armant DR, Bagchi MK, Bagchi IC. Calcitonin down-regulates E-cadherin expression in rodent uterine epithelium during implantation. J. Biol. Chem 2002;277:46447–55, http://dx.doi.org/10.1074/jbc.M203555200. Lindsay LA, Murphy CR. Ovarian hyperstimulation affects fluid transporters in the uterus: a potential mechanism in uterine receptivity. Reprod. Fertil. Dev 2013., http://dx.doi.org/10.1071/RD12396. Lutz KL, Siahaan TJ. Molecular structure of the apical junction complex and its contribution to the paracellular barrier. J. Pharm. Sci 1997;86:977–84, http://dx.doi.org/10.1021/js970134j. Luxford KA, Murphy CR. Cytoskeletal control of the apical surface transformation of rat uterine epithelium. Biol. Cell 1993;79:111–6.
Luxford KA, Murphy CR. Changes in the apical microfilaments of rat uterine epithelial cells in response to estradiol and progesterone. Anat. Rec 1992a;233:521–6, http://dx.doi.org/10.1002/ar.1092330405. Luxford KA, Murphy CR. Reorganization of the apical cytoskeleton of uterine epithelial-cells during early-pregnancy in the rat—a study with myosin subfragment-1. Biol. Cell 1992b;74:195–202. Mège R-M, Gavard J, Lambert M. Regulation of cell–cell junctions by the cytoskeleton. Curr. Opin. Cell Biol 2006;18:541–8, http://dx.doi.org/10.1016/j.ceb.2006.08.004. Meng W, Takeichi M. Adherens junction: molecular architecture and regulation. Cold Spring Harb. Perspect. Biol 2009;1., http://dx.doi.org/10.1101/cshperspect.a002899, a002899-a002899. Miyaguchi K. Ultrastructure of the zonula adherens revealed by rapid-freeze deep-etching. J. Struct. Biol 2000;132:169–78, http://dx.doi.org/10.1006/jsbi.2000.4244. Murphy CR. Uterine receptivity and the plasma membrane transformation. Cell Res 2004;14:259–67. Murphy CR. Junctional barrier complexes undergo major alterations during the plasma membrane transformation of uterine epithelial cells. Hum. Reprod 2000a;15(Suppl. 3):182–8. Murphy CR. The plasma membrane transformation of uterine epithelial cells during pregnancy. J. Reprod. Fertil. Suppl 2000b;55:23–8. Murphy CR. The plasma membrane of uterine epithelial cells: structure and histochemistry. Prog. Histochem. Cytochem 1993;27:1–66. Murphy CR, Swift JG, Mukherjee TM, Rogers AW. The structure of tight junctions between uterine luminal epithelial cells at different stages of pregnancy in the rat. Cell Tissue Res 1982;223:281–6, http://dx.doi.org/10.1007/bf01258489. Nagafuchi A. Molecular architecture of adherens junctions. Curr. Opin. Cell Biol 2001;13:600–3, http://dx.doi.org/10.1016/S0955-0674(00)00257-X. Niessen CM. Tight junctions/adherens junctions: basic structure and function. J. Invest. Dermatol 2007;127:2525–32, http://dx.doi.org/10.1038/sj.jid.5700865. Nilsson O. The morphology of blastocyst implantation. Reproduction 1974;39:187–94, http://dx.doi.org/10.1530/jrf.0.0390187. Patel SD, Ciatto C, Chen CP, Bahna F, Rajebhosale M, Arkus N, Schieren I, Jessell TM, Honig B, Price SR, Shapiro L. Type II cadherin ectodomain structures: implications for classical cadherin specificity. Cell 2006;124:1255–68, http://dx.doi.org/10.1016/j.cell.2005.12.046. Paulson RJ, Sauer MV, Lobo RA. Embryo implantation after human in vitro fertilization: importance of endometrial receptivity. Fertil. Steril 1990;53:870–4. Png FY, Murphy CR. The plasma membrane transformation does not last: microvilli return to the apical plasma membrane of uterine epithelial cells after the period of uterine receptivity. Eur. J. Morphol 1997;35:19–24, http://dx.doi.org/10.1076/ejom.35.1.19.13061. Pokutta S, Herrenknecht K, Kemler R, Engel J. Conformational changes of the recombinant extracellular domain of E-cadherin upon calcium binding. Eur. J Biochem 1994;223:1019–26, http://dx.doi.org/10.1111/j.1432-1033.1994.tb19080.x. Potter SW, Gaza G, Morris JE. Estradiol induces E-cadherin degradation in mouse uterine epithelium during the estrous cycle and early pregnancy. J. Cell Physiol 1996;169:1–14, 10.1002/(SICI)1097-4652(199610)169:13.0.CO;2-S. Preston AM, Lindsay LA, Murphy CR. Desmosomes in uterine epithelial cells decrease at the time of implantation: an ultrastructural and morphometric study. J. Morphol 2006;267:103–8, http://dx.doi.org/10.1002/jmor.10390. Rowlands T, Symonds JM, Farookhi R, Blaschuk OW. Cadherins: crucial regulators of structure and function in reproductive tissues. Rev. Reprod 2000;5:53–61, http://dx.doi.org/10.1530/ror.0.0050053. Schlafke S, Enders AC. Cellular basis of interaction between trophoblast and uterus at implantation. Biol. Reprod 1975;12:41–65, http://dx.doi.org/10.1095/biolreprod12.1.41. Schlafke S, Welsh AO, Enders AC. Penetration of the basal lamina of the uterine luminal epithelium during implantation in the rat. Anat. Rec 1985;212:47–56, http://dx.doi.org/10.1002/ar.1092120107. Schneeberger EE, Lynch RD. The tight junction: a multifunctional complex. Am. J. Physiol. Cell Physiol 2004;286:C1213–8, http://dx.doi.org/10.1152/ajpcell.00558.2003. Shion YL, Murphy CR. The basal plasma membrane and lamina densa of uterine epithelial cells are both altered during early pregnancy and by ovarian hormones in the rat. Eur. J. Morphol 1995;33:257–64. Tachi S, Tachi C, Lindner HR. Ultrastructural features of blastocyst attachment and trophoblastic invasion in the rat. Reproduction 1970;21:37–56, http://dx.doi.org/10.1530/jrf.0.0210037. Theard D, Raspe MA, Kalicharan D, Hoekstra D, van IJzendoorn SCD. Formation of E-cadherin/-catenin-based adherens junctions in hepatocytes requires serine-10 in p27(Kip1). Mol. Biol. Cell 2008;9:1605–13, http://dx.doi.org/10.1091/mbc.E07-07-0661. Tsukita S, Tsukita S, Nagafuchi A, Yonemura S. Molecular linkage between cadherins and actin filaments in cell–cell adherens junctions. Curr. Opin. Cell Biol 1992;4:834–9, http://dx.doi.org/10.1016/0955-0674(92) 90108-O. Vasioukhin V, Bauer C, Yin M, Fuchs E. Directed actin polymerization is the driving force for epithelial cell–cell adhesion. Cell 2000;100:209–19, http://dx.doi.org/10.1016/S0092-8674(00) 81559-7.
S.N. Dowland et al. / Acta Histochemica 118 (2016) 137–143 Vasioukhin V, Fuchs E. Actin dynamics and cell–cell adhesion in epithelia. Curr. Opin. Cell Biol 2001;13:76–84, http://dx.doi.org/10.1016/S0955-0674(00) 00177-0. Weinerman R, Mainigi M. Why we should transfer frozen instead of fresh embryos: the translational rationale. Fertil. Steril 2014;102:10–8, http://dx.doi.org/10.1016/j.fertnstert.2014.05.019. Yap AS, Brieher WM, Gumbiner BM. Molecular and functional analysis of cadherin-based adherens junctions. Annu. Rev. Cell Dev. Biol 1997;13:119–46, http://dx.doi.org/10.1146/annurev.cellbio.13.1.119. Yeh JS, Steward RG, Dude AM, Shah AA, Goldfarb JM, Muasher SJ. Pregnancy rates in donor oocyte cycles compared to similar autologous in vitro fertilization
143
cycles: an analysis of 26,457 fresh cycles from the Society for Assisted Reproductive Technology. Fertil. Steril 2014;102:399–404, http://dx.doi.org/10.1016/j.fertnstert.2014.04.027. Zhu L-J, Bagchi MK, Bagchi IC. Attenuation of calcitonin gene expression in pregnant rat uterus leads to a block in embryonic implantation 1. Endocrinology 1998a;139:330–9, http://dx.doi.org/10.1210/endo.139.1.5707. Zhu L-J, Cullinan-Bove K, Polihronis M, Bagchi MK, Bagchi IC. Calcitonin is a progesterone-regulated marker that forecasts the receptive state of endometrium during implantation 1. Endocrinology 1998b;139:3923–34, http://dx.doi.org/10.1210/endo.139.9.6178.
