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Uterine Focal Adhesions Are Retained at Implantation After Rat Ovarian Hyperstimulation

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Uterine Focal Adhesions Are Retained at Implantation After Rat Ovarian Hyperstimulation REPRODUCTIONRESEARCH PROOF ONLY Uterine focal adhesions are retained at implantation after rat ovarian hyperstimulation Laura A Lindsay, Samson N Dowland and Christopher R Murphy School of Medical Sciences (Anatomy and Histology), The University of Sydney, Sydney, New South Wales, Australia Correspondence should be addressed to L A Lindsay; Email: [email protected] Abstract Controlled ovarian hyperstimulation is an essential component of IVF techniques to ensure proliferation and development of multiple ovarian follicles, but the effects of these hormones on the endometrium are largely unknown. During normal pregnancy in rats, there are significant changes in the basal plasma membrane of uterine epithelial cells (UECs) at the time of receptivity, including loss of focal adhesions. This enables the UECs to be removed from the implantation chamber surrounding the blastocyst, thus allowing invasion into the underlying stroma. This study investigated the influence of ovarian hyperstimulation (OH) on the basal plasma membrane of UECs during early pregnancy in the rat. Immunofluorescence results demonstrate the presence of paxillin, talin, integrin β1 and phosphorylated FAK (Y397FAK) in the basal portion of UECs at the time of implantation in OH pregnancy. TEM analysis demonstrated a flattened basal lamina and the presence of focal adhesions on the basal surface at this time in OH pregnancy. Significantly low full-length paxillin, high paxillinδ and integrin β1 were seen at the time of implantation in OH compared with those in normal pregnancy. The increase in paxillin δ suggests that these cells are less mobile, whereas the increase in integrin β1 and Y397FAK suggests the retention of a stable FA complex. Taken together with the increase in morphological focal adhesions, this represents a cell type that is stable and less easily removed for blastocyst implantation. This may be one mechanism explaining lower implantation rates after fresh embryo transfers compared with frozen cycles. Reproduction (2016) 152 753–763 Introduction Focal adhesions (FAs) are key structural elements that play a role in cell-matrix adhesion. These specialised An essential part of the ability of uterine epithelial structures consist of several proteins that connect the cells (UECs) to become receptive to an implanting actin cytoskeleton to the extracellular matrix (ECM). blastocyst is the changes in all membrane domains of Intracellular proteins associated with FAs include the cell, including the basal plasma membrane (Shion & actopaxin, vinculin, paxillin and talin (Turner 2000) Murphy 1995). In species that have displacement and membrane-bound integrins, which span the plasma penetration such as rodents and humans (Schlafke & membrane and make contact with the ECM of the basal Enders 1975), the UECs must be removed for the lamina (Vuori 1998, Brown & Turner 2004) and all of implanting blastocyst to invade the underlying stroma. which have complex interactions (Zaidel-Bar et al. This removal was thought to be mediated via apoptotic 2007, Kuo et al. 2011). mechanisms (Parr et al. 1987); however, recent evidence Paxillin is a linker protein that connects to integrins suggests that luminal UECs surrounding the implanting via focal adhesion kinase (FAK) and actin stress fibres blastocyst undergo entosis (Li et al. 2015). Regardless via actopaxin and vinculin (Turner et al. 1990, Turner of the mode of removal of UECs, it is necessary for the 2000, Schaller 2001). There are four different isoforms of UECs to become less adherent to the underlying basal paxillin with paxillin α being the most widely expressed lamina, and then the UECs in the region immediately (Mazaki et al. 1997, Schaller 2001, Brown & Turner 2004). surrounding the implantation chamber are removed. Paxillin δ is an internal translation product of paxillin Previous studies in rat UECs during early pregnancy α that is found only in epithelial cells (Brown & Turner and in response to ovarian hormones showed that at the 2004, Tumbarello et al. 2005), and when overexpressed, time of implantation and in response to progesterone, leads to the inhibition of cell migration (Sorenson & the basal lamina thickens, becomes highly tortuous Sheibani 1999, Tumbarello et al. 2005). Talin is another and loses focal adhesions (Shion & Murphy 1995, intracellular protein associated with FAs and provides a Kaneko et al. 2008). direct link between the cytoplasmic domain of integrin © 2016 Society for Reproduction and Fertility DOI: 10.1530/REP-16-0331 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 10/04/2021 02:21:39AM via free access 10.1530/REP-16-0331 754 L A Lindsay and others and intracellular actin fibres (Nayal et al. 2004). Talin effect on the uterine lining, which impedes blastocyst is also involved in integrinPROOF activation (Tadokoro et al. implantation. ONLY This is confirmed in several studies 2003), which is essential in the establishment of FAs that show a significant decrease in implantation rate (Yamada & Geiger 1997, Critchley 2009). when embryos are transferred during the COH cycle, Integrins are large membrane-spanning proteins compared with those transferred in subsequent cycles composed of α and β subunits that mediate cell–cell and (Paulson et al. 1990, Check et al. 1994, 2010, Shapiro cell–extracellular matrix adhesion. In vitro studies have et al. 2011, 2014). However, the mechanisms underlying shown that integrin β1 and β3 colocalise and interact the effect of COH on uterine receptivity are currently with talin at FAs (Calderwood et al. 1999, Critchley 2000, unknown. Calderwood 2004). Previous studies in humans, rodents and other mammals have established that integrin αVβ3 is highly expressed at the time of implantation and is a well-known marker of uterine receptivity (Bowen et al. 1996, Fazleabas et al. 1997, Lessey 1997, Srinivasan et al. 2009). Integrin β1 has previously been localised to the basal plasma membrane of rat UECs on day 1 of pregnancy and in response to oestrogen stimulation (Kaneko et al. 2011). Focal adhesion kinase (FAK) is a cytosolic protein tyrosine kinase associated with the FA complex and is involved in many aspects of cell migration, invasion and signalling (Cary & Guan 1999, Hauck et al. 2002). Previous work in rat UECs during early pregnancy and in response to ovarian hormones has shown that FAK is found apically at the time of implantation and in response to progesterone. This apical localisation of FAK is suggested to be involved in integrin–integrin binding between UECs and blastocyst, which is an essential part of implantation (Kaneko et al. 2012). Human studies have also shown an increase in FAK staining within the luminal and glandular UECs in the mid-secretory phase of the menstrual cycle indicating a role in human uterine remodelling and implantation (Orazizadeh et al. 2009). For FAK to control intracellular signalling pathways, autophosphorylation must occur, and this can happen on a number of residues including tyrosine-397. This autophosphorylation then activates many of the kinase- dependant functions of FAK (Katz et al. 2003, Brami- Cherrier et al. 2014). Specifically, phosphorylation of FAK at Y397 (Y397FAK) leads to the activation of integrins in a variety of cell types in vitro and in vivo (Chan et al. 1994, Schaller et al. 1994, Eide et al. 1995, Schlaepfer et al. 1999). Although several studies have investigated FAK in the endometrium (Orazizadeh et al. 2009, Figure 1 Immunofluorescence micrographs and western blotting Kaneko et al. 2012), currently no studies have examined analysis of paxillin on days 1 and 3 of normal and OH pregnancy. the physiologically active form of FAK, Y397FAK, in Paxillin staining (green) is seen as a basal band below uterine epithelial cells (e) on day 1 of normal pregnancy (D1, arrowhead). the endometrium. However, in the myometrium, an Similar basal staining is also seen below UECs (e) on day 1 after OH increase in phosphorylation of FAK is associated with (OH1, arrowhead). Protein analysis from isolated UECs showed stretch-induced activation of human myometrial cells 68 kDa (full-length paxillin) and 46 kDa (paxillin δ) bands. When (Li et al. 2009). In human placental cytotrophoblast normalised to β-actin, there is no difference in either 68 kDa or cells, it was found that Y397FAK, rather than overall FAK 46 kDa isoforms of paxillin between normal and OH pregnancy on levels, is associated with invasion ability and that under day 1. On day 3 of normal (D3) and OH pregnancy (OH3), a small conditions of hypoxia in vitro and pre-eclampsia in vivo, amount of cytoplasmic paxillin staining is seen in UECs (e). Western blotting analysis demonstrates full-length paxillin (68 kDa) and a there was a reduction in Y397FAK (Ili et al. 2001). ć small amount of paxillin δ (46 kDa). However, there is no difference During IVF procedures, controlled ovarian in amount of either protein in isolated UECs of normal compared hyperstimulation (COH) is required to stimulate oocyte with OH pregnancy. Green staining, paxillin; blue, nuclei; scale production and maturation. COH has a detrimental bar = 20 μm. Reproduction (2016) 152 753–763 www.reproduction-online.org Downloaded from Bioscientifica.com at 10/04/2021 02:21:39AM via free access Uterine FAs in ovarian hyperstimulation 755 ion transport (Lindsay & Murphy 2014) and change in PROOFimportant ONLY uterine adhesion molecules (Sendag et al. 2010, Biyiksiz et al. 2011) are also observed in OH pregnancy compared with normal pregnancy. There are, however, no data investigating ultrastructural or molecular changes of the basal plasma membrane of UECS at the time of implantation after OH compared with normal pregnancy. Materials and methods Animals Adult female virgin Wistar rats were housed in a controlled environment with a 12-h light/darkness cycle and given food and water ad libitum. In one group of rats, vaginal smears were used to determine which animals were in pro-oestrus and these rats were mated overnight with a male of proven fertility.
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