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Focal Adhesion Proteins, Vinculin and Integrin [beta]5, During Early Pregnancy in Rat Uterine Epithelial Cells: Anastrozole Favors their Normal Distribution/Proteinas de Adhesion Focales, Vinculina e Integrina [beta]5, Durante el Embarazo Temprano en Celulas Epiteliales Uterinas de Rata: Anastrozol Favorece Su Distribucion Normal.

INTRODUCTION

Cytoskeletal reorganization is an ongoing process when cells adhere, move or invade extracellular substrates. In order for cells to adhere, they employ focal adhesions (Fabry et al., 2011). Focal adhesion (FA) is a specialized structure formed where bundles of actin filaments are anchored to transmembrane receptors of the integrin family through a complex of adaptor and signaling proteins enabling cells to adhere, spread and migrate (Gilmore & Burridge, 1996). The binding of ligands to integrins on the extracellular side promotes recruitment of various intracellular proteins to the cytoplasmic tails of integrins that mechanically link them to the actin cytoskeleton (Hirata et al., 2014). These signaling events culminate in reorganization of the actin cytoskeleton; a prerequisite for changes in cell shape and motility, gene expression and pregnancy (Lele et al., 2008; Fabry et al.; Kaneko et al, 2011). Moreover, the implantation of blastocysts into the endometrial stroma cells is regulated by the Rho GTPases RhoA, Rac 1 and Cdc42 (Grewal et al., 2010).

Other studies have provided evidence to suggest that the binding of vinculin to actin and talin can be regulated by phosphatidyl-inositol-4-5-bisphosphate (Gilmore & Burridge) and inhibited by acidic phospholipids. Clearly, the structural links between actin filaments and integrins are regulated in at least some cell types, and it would not be unreasonable to hypothesize that agents known to alter cell behavior may do so by affecting the expression or function of actin binding proteins such as vinculin, talin, paxillin and integrins that are recruited to focal adhesions (Mwakikunga et al., 2011). Focal adhesions are dynamically assembled and disassembled by cells, including uterine luminal epithelial cells (Hosie et al., 2008; Kaneko et al., 2008, 2011), and these mechanisms are likely governed by the modulation of cross-talk between their constituent proteins, which influences their gene expression and morphology.

Gonadotropin injections are medically dangerous and invasive, and their compliance is not easy. They are also associated with multiple pregnancies (Bruna-Catalan et al., 2011). Similarly, polycystic ovarian syndrome patients are resistant to chlomiphine citrate (Casper & Mitwally, 2011). Aromatase inhibitors (Als) such as letrozole and anastrozole, which are used to treat breast cancer, have been suggested as alternatives to gonadotropins and chlomiphine citrate due to their easy compliance and oral administration (Lee & Ledger, 2011). The use of letrozole as a first line therapy for women with anovulation is not fully conclusive and still remains deba (Kamath & George, 2011). Anastrozole is clinically effective in ovulation induction, but it has not been well researched (Tredway & Schertz, 2011). Investigating and understanding the effects of anastrozole on the endometrium at the time of implantation in vivo will enable manipulation of uterine receptivity to control fertility and to improve the outcome of assisted reproductive procedures (Paria et al., 2001; Hosie et al., 2003; Karaer et al, 2005).

Since blastocyst adhesion onto the endometrium is at the center of the implantation process (Enders et al., 1986; Murphy, 2004), this means that focal adhesion proteins are employed. The expression and distribution of the focal adhesion proteins in the rat uterine epithelial cells are associated with uterine receptivity (Kaneko et al., 2008). In other words, the dynamics of focal adhesions are crucial at the time of implantation. Disassembly of the basal focal adhesions along the uterine epithelial cells makes cells less adherent to the underlying basement membrane, and this facilitates their removal, enabling embryonic trophoblast cells to invade the endometrial decidual cells beneath. Since previous studies demonstrated that focal adhesion proteins are more likely regulated in uterine epithelia to change cell behavior (Kaneko et al., 2009), and that super-ovulatory drugs can alter expression of key molecules in the uterine epithelial cells during implantation (Hosie et al., 2003), anastrozole may act on focal adhesion proteins during implantation. To date, anastrozole regulation of focal adhesion proteins in the uterine epithelial cells during implantation has not been established.

The aim of this study was to determine the gene and protein expression levels and localization of the focal adhesion proteins vinculin and integrin [beta]5 in the rat uterine epithelial cells during implantation in vivo.

MATERIAL AND METHOD

Animals. The study was approved by the University of the Witwatersrand Animal Ethics Committee with a clearance certificate number: 2012/11/03.

Thirty five female virgin inbred Wistar rats were used in the preliminary dose response study (Mwakikunga & Hosie, 2016) to determine the optimal dose of anastrozole required to superovulate rats in the main study. On day 6 pregnant rats were used in the preliminary study (Mwakikunga & Hosie). A chlomiphene citrate (CC) treatment group was also used as a control to compare with the anastrozole treatment groups. Rats were housed in plastic cages at 21[degrees]C under a controlled 12 hour light-dark cycle. They were provided with water and food ad libitum (Dukes et al., 1996). Vaginal smears were obtained from all female rats to confirm that they had regular cycles for two cycles before drug administration (Singletary et al., 2005; Jaramillo et al., 2012). Thirty five mature female rats, 12 to 14 weeks old and weighing 200-250 g, were randomly divided into 7 groups of 5 rats each. Vaginal smear was done in the late afternoon (Kaneko et al., 2008) and rats in pro-oestrus were treated with the drugs or placebo and then caged overnight with males of proven fertility. The morning of finding the vaginal plug or presence of spermatozoa in the smear was designated as day 1 of pregnancy (Kaneko et al., 2008).

Rationale for the doses. A daily dose of 1mg/kg anastrozole (Sigma-Aldrich Co., St. Louis, MO, USA) for 5 days is the standard recommended dose to achieve ovulation in humans (Franik et al., 2014). A 12-14 week mature female Wistar rat weighs between 200-250 g and has a 4-day estrous cycle, so a single dose of anastrozole administered at pro-estrus was deemed appropriate. Kilic-Okman et al. (2003) employed a similar regime using letrozole. Earlier studies have suggested that the standard 1 mg/kg anastrozole of body weight dose is too low for optimal follicle recruitment and ovulation, so higher doses are recommended (Al-Omari et al., 2004; Casper & Mitwally, 2012). A single 25 mg/kg anastrozole dose has been successful in inducing ovulation in mice and is associated with favorable embryo development (Karaer et al.). However, in our preliminary work, a drug concentration study with anastrozole was carried out to determine the optimal dose to be used to superovulate and achieve pregnancy in the Wistar rats (Mwakikunga & Hosie). This was found to be dose-specific, and the 15 mg/kg anastrozole dose superovulated and achieved pregnancy the most; therefore, this dose was used in the subsequent experiments in this study. Dose rates in the preliminary work were started at 1 mg/kg. The chlomiphene citrate dose of 1.25 mg/kg (Sigma-Aldrich Co., St. Louis, MO, USA) was adopted from earlier studies (Hosie et al., 2003) and used as a comparison.

Drug preparation and administration

Treatment regimes for the dose response study. Anastrozole has moderate aqueous solubility and previous studies used normal saline as a vehicle for anastrozole (Karaer et al.; Fatum et al., 2006). In this study, the vehicle for all of the drugs was normal saline and each injection consisted of 0.2 ml normal saline (Fatum et al.) including the drug per injection per rat. All injections were intraperitoneal. The four anastrozole dose regimes for the preliminary dose response study were: 25 mg, 15 mg, 10 mg, and 1 mg/kg body weight.

Treatment regimes: Group 1 was left untreated (negative control). Group 2 (carrier control group) received the vehicle sterile saline. Groups 3, 4, 5 and 6 received a single dose of 1 mg/kg, 10 mg/kg, 15 mg/kg and 25 mg/kg of body weight anastrozole respectively in the pro-estrous phase intraperitoneally. Group 7 received 1.25 mg/kg of body weight CC; n=5 in each group, all 0.2 ml intraperitoneal injections (I).

Tissue preparation. Pregnant rats were sacrificed on day 6 at the time of implantation using a lethal dose (0.35 ml) of Euthanase, intraperitoneally (1 ml/kg is recommended; Kyron Labs, South Africa). Once deeply unconscious, the abdominal cavity was opened and the rats to be sacrificed on day 6 of pregnancy were injected intravenously using the inferior vena cava with 0.1 ml of 1 % high molecular weight vital dye Pontamine sky blue (Sigma, St. Louis, MO, USA) in normal saline (0.9 % NaCl) (Pakrasi & Tiwari, 2007), in order to distinguish implantation sites from non-implantation sites. This dye quickly localizes in the implantation sites due to increased vascularization and vascular permeability during early pregnancy (Psychoyos, 1986). After removal of the uterine horns, implantation sites were counted and recorded. The uterus was cut into 1cm pieces separating implantation and non-implantation sites and prepared for scanning electron microscopy (SEM), confocal microscopy and qPCR.

Animals for the main study (confocal microscopy and qPCR). A further twenty similar female rats to be sacrificed on day 1 of pregnancy were used in themain study. Five rats were treated with 15 mg/kg anastrozole (as this was found to be the optimal dose) (Mwakikunga & Hosie), five were treated with 1.25 mg/kg CC, five were treated with saline alone, and five were untreated. These matched the groups in the earlier study where the animals were sacrificed on day 6 of pregnancy.

Immunofluorescence and confocal microscopy

Tissue processing and sectioning. All histological glass slides that were used in this study were gelatin-coated (Rajamohamedsait & Sigurdsson, 2012) in order to stick the tissue sections to the slides during the staining and washing process. Using standard procedures, samples for immunofluorescence and confocal microscopy were fixed in 10 % buffered formalin for 48 hours (Karaer et al.), then placed in an automatic tissue processor (Shandon Citadel 1000, Labotek, South Africa) in which samples were incubated in a series of 70 %, 95 %, 95 %, 95 %, 100 %, 100 %, 100 % ethanol, then in chloroform and finally in paraffin wax. Samples were then embedded in paraffin wax. Histological sections (5 [micro]m) were cut using a Leica 2035 Biocut microtome (Leica, Nussloch, Germany) fitted with a disposable blade and then dewaxed overnight by placing them in an oven at 60[degrees]C, then immersed in histoclear for 5 minutes and then repeated in fresh histoclear for 5 minutes, rehydrated through a graded series of ethanol (100 %, 100 %, 95 %, 80 % 70 % and 60 %) for 30 seconds each, then washed in running water for 5 minutes (Mohan et al, 2008; Rajamohamedsait & Sigurdsson, 2012).

Immunofluorescence. The immunolocalization and double labeling protocol was adopted and modified from previous studies (Mohan et al.; Kaneko et al., 2011; Mwakikunga, et al.). De-waxed sections of uterus from each group were washed 3 times, 2 min each with PBS. This was followed by a 10 min incubation in 30 % [H.sub.2][O.sub.2] (Sigma-Aldrich Co., St. Louis, MO, USA) in methanol to block endogenous peroxidase activity (Mwakikunga et al.), and then washed 3 times, 2 min each with PBS. Sections were then permeabilized for 30 minutes in 0.1 % Triton-X 100 (Sigma-Aldrich Co., St. Louis, MO, USA) in PBS, and incubated for 30 minutes in blocking solution 5 % (v/v) normal goat serum (Sigma-Aldrich Co., St. Louis, MO, USA) in PBS. All primary and secondary antibodies were diluted in blocking solution (1:100). Sections were incubated with mouse monoclonal anti-Vinculin, rabbit polyclonal anti-Integrin (Abcam, Cambridge, MA, USA) for 24 hours at 4[degrees]C (double staining: vinculin with integrin [beta]5) (Mohan et al.; Kaneko et al., 2011). After washing with PBS 3 times 5 min each, sections were incubated with fluorescein isothiocyanate (FITC) conjugated to AffiniPure Goat anti-mouse IgG secondary antibody (Abcam) and rodamine conjugated Goat anti-rabbit IgG secondary (Abcam) at a dilution of 1:100 [10] for 30 min in the dark followed by 3 PBS washes of 5 min each. Sections were also counterstained with DAPI to label nuclei. Expression and localization of vinculin and integrin [beta]5 were examined under the Zeiss LSM 780 confocal microscope (Carl Zeiss, Jena, Germany) and images were acquired using the Zeiss LSM software (Carl Zeiss, Jena, Germany). The focal adhesion protein expression levels and localization in the confocal images were scored using a scoring method modified from previous studies (Englund et al., 2001) as shown in Table II. JMP10 software (SAS Institute, Cary, NC, USA) was used for the statistical comparisons of means of focal adhesion protein expression of vinculin and integrin [beta]5 among treatment groups using a one way ANOVA followed by a Tukey-Kramer post hoc analysis. Differences were considered statistically significant when p [less than or equal to] 0.05.

Real time quantitative polymerase chain reaction (qPCR). In this study, the qPCR was performed for the quantitation of gene expression of the focal adhesion proteins vinculin and integrin P5 in the uterine epithelial cells from day 1 and day 6 pregnant rats of all treatment regimes. The house keeping genes b-actin, 18Sr RNA and Lactate dehydrogenase A (Ldha) were used as reference genes (Al-Bader & Al-Sarraf, 2005; Hong et al., 2006; Li et al., 2014).

The RNA from the uterine epithelial cells was prepared with the GeneJET RNA purification kit (Thermo Scientific Inc., 2011), and genomic DNA was removed according to the manufacturer's instructions (Thermo Scientific Inc., 2011). For qPCR, amplification was performed in a 7500 real-time PCR cycler (Applied Biosystems, Foster City, CA) and revealed with a QuantiFast SYBR Green PCR kit (Thermo Fisher Scientific Inc., 2011). The amplification cycling conditions were as follows: 95[degrees]C for 10min, (95[degrees]C for 15 s, 60[degrees]C for 1min in 40 cycles), 95[degrees]C for 15 s, 60[degrees]C for 1min, 95[degrees]C for 30 s, 60[degrees]C for 15 s. Table III shows the primer sequences for the real-time qPCR.

SYBR green qPCR data analysis. The [2.sup.-[DELTA][DELTA]CT] method was used to determine the gene expression fold change relative to the control (calibrator sample) following drug treatment regime (Schmittgen & Livak, 2008). The data (threshold cycle or ct values) were first normalized using the three reference genes ([beta]-actin, 18S rRNA and lactate dehydrogenase A); then the fold change was calculated (Schmittgen & Livak, 2008). JMP10 software was used to conduct statistical analyses. A one way ANOVA, followed by a Tukey-Kramer post hoc analysis, was performed to compare the means of normalized relative quantities (NRQ) between treatment groups. Differences were considered statistically significant when p [less than or equal to] 0.05.

RESULTS

Vinculin and integrin [beta]5 localization. Vinculin and integrin [beta]5 are co-localized at the base of the uterine epithelium at day 1 of pregnancy whereas at day 6, they disassemble from the basal focal adhesions and co-localize and significantly increase their expression apically (p [less than or equal to] 0.0001) as noted in Figs. 1, 2, 3, 4 and Table IV. Additionally, vinculin is expressed in the perinuclear region at day 1 and relocates apically at day 6 of pregnancy as seen in Figs. 1, 2, 3, 4. Moreover, there is a significant difference (p [less than or equal to] 0.0001) in the expression levels of both vinculin and integrin b5 between untreated (control) and chlomiphene citrate (CC) treated rats, anastrozole and CC treated rats at both day 1 and day 6 as seen in Figs. 1, 2, 3, 4. Although there is no significant difference in the expression levels between untreated and anastrozole treated rats at day 1 and day 6 (p [less than or equal to] 0.05), the means for vinculin and integrin [beta]5 expression are higher in the anastrozole treated groups. The non-immune controls (not shown) were used to validate and determine non-specific binding of the secondary antibody on other targets within the epithelium; there is no non-specific staining.

Relative quantification (RQ) analysis of vinculin and integrin [beta]5 gene expression in the uterine epithelial cells from day 1 and day 6 pregnant rats. At day 1 of pregnancy, vinculin gene expression in uterine luminal epithelial cells is fairly similar (1.2-fold increase) in the anastrozole treated rats relative to the calibrator sample (day 1 untreated rats). This is also true for integrin [beta]5 gene expression (1.01-fold increase) (p < 0.05). Vinculin and integrin [beta]5 gene expression in uterine luminal epithelial cells, however, is down-regulated (0.77-fold decrease) and (0.79-fold decrease) respectively with chlomiphene citrate (CC) treatment at day 1 of pregnancy relative to the calibrator sample as shown in Tables V and VI and Figure 5. Interestingly, like the immunofluorescence experiments, at day 6 of pregnancy vinculin and integrin P5 gene expression are significantly up-regulated (more than 1.5-fold increase) (p [less than or equal to] 0.0001) in uterine luminal epithelial cells in the implantation and non-implantation sites in the anastrozole treated group relative to the calibrator sample (Tables V and VI and Fig. 5). A significant increase in the integrin [beta]5 gene expression is also noted in uterine luminal epithelial cells of day 6 implantation sites in the untreated group (more than 1.5-fold increase) (p [less than or equal to] 0.0001) while it has remained similar (1.04-fold increase) (p [less than or equal to] 0.05) in the non-implantation sites relative to the calibrator sample (Tables V and VI and Fig. 5). However, vinculin gene expression in uterine luminal epithelial cells is down-regulated with CC treatment at day 6 in implantation sites (0.96-fold decrease) and non-implantation sites (0.93-fold decrease) relative to the calibrator sample. This is also true for integrin b5 gene expression (0.86-fold decrease and 0.42-fold decrease) respectively. In general, there is an increase in vinculin and integrin b5 gene expression at day 6 of pregnancy in uterine luminal epithelial cells in untreated and anastrozole treated groups, as reflected in the immunofluorescence experiments.

DISCUSSION

In this study, the focal adhesion proteins vinculin and integrin [beta]5 are co-localized at the base of the uterine epithelium at day 1 of pregnancy whereas at day 6, they disassemble from the basal focal adhesions and co-localize and significantly increase their expression apically (p [less than or equal to] 0.0001). Additionally, vinculin is also expressed in the perinuclear region at day 1 and relocates apically at day 6 of pregnancy. Moreover, there is a significant difference in the protein expression levels of both vinculin and integrin b5 between untreated (control) and chlomiphene citrate (CC) treated rats (p [less than or equal to] 0.0001), anastrozole and chlomiphene citrate (CC) treated rats at day 6 (p [less than or equal to] 0.0001) suggesting the interpretation that CC seems to decrease their expression. Although there is no significant difference in the expression levels between untreated and anastrozole treated rats at day 1 and day 6 (p [less than or equal to] 0.05), the means for vinculin and integrin P5 protein expression are higher in the anastrozole treated groups suggesting perhaps, that anastrozole seems to enhance their expression in order to assist in the implantation process of the embryo.

Moreover, vinculin and integrin [beta]5 gene expression in uterine luminal epithelial cells at day 1 of pregnancy agrees with the protein expression. Vinculin gene expression is fairly similar (1.2-fold increase) (p [less than or equal to] 0.05) in the anastrozole treated rats relative to the calibrator sample (day 1 untreated rats). This is also true for integrin [beta]5 gene expression (1.01-fold increase) (p [less than or equal to] 0.05) suggesting the interpretation that anastrozole seems not to retard vinculin and integrin [beta]5 gene expression in the process of endometrial readiness for implantation. Vinculin and integrin b5 gene expression in uterine luminal epithelial cells, however, is down-regulated (0.77-fold decrease) and (0.79-fold decrease) respectively with chlomiphene citrate (CC) treatment at day 1 of pregnancy relative to the calibrator sample, which may suggest that CC perhaps decreases their expression. Interestingly, like the immunofluorescence experiments, at day 6 of pregnancy, vinculin and integrin P5 gene expression are significantly up-regulated (more than 1.5-fold increase) (p [less than or equal to] 0.0001) in uterine luminal epithelial cells in the implantation and non-implantation sites in the anastrozole treated group relative to the calibrator sample. A significant increase in the integrin [beta]5 gene expression is also noted in uterine luminal epithelial cells of day 6 implantation sites in the untreated group (more than 1.5-fold increase) (p [less than or equal to] 0.0001) while it has remained similar (1.04-fold increase) (p [less than or equal to] 0.05) in the non- implantation sites relative to the calibrator sample suggesting the interpretation that anastrozole appears to enhance their expression to probably assist in blastocyst implantation. However, vinculin gene expression in uterine luminal epithelial cells is down-regulated with chlomiphene citrate (CC) treatment at day 6 in implantation sites (0.96-fold decrease) relative to the calibrator sample. This is also true for integrin [beta]5 gene expression (0.86-fold decrease), which may suggest that chlomiphene citrate (CC) may lag endometrial receptivity. In general, there is an increase in vinculin and integrin [beta]5 gene expression at day 6 of pregnancy in uterine luminal epithelial cells in untreated and anastrozole treated groups, as reflected in the immunofluorescence experiments, in order to perhaps assist in implantation.

Vinculin is one of the core and best characterized focal adhesion (FA) proteins (Humphries et al., 2007; Carisey et al, 2013). Tyrosine phosphorylation of vinculin modifies focal adhesion dynamics and cell tractions (Kupper et al., 2010). Vinculin regulates the recruitment and release of other core focal adhesion proteins in a force dependent manner and interacts with the talin-integrin complex and thus drives the recruitment and release of core FA components (Humphries et al.; Carisey et al.). Earlier studies demonstrated that lack of cytoskeletal connecting proteins like vinculin, talin and focal adhesion kinase (FAK) reduces the overall cellular binding strength (Carisey et al.). As mentioned earlier, focal adhesions disassemble from the base of 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 during early pregnancy in the rat (Kaneko et al., 2009). Kaneko et al. (2009) showed a major core FA protein talin distributional change between different hormone regimes. Talin is highly concentrated along the basal cell surface of uterine luminal epithelial cells in response to estrogen treatment which is observed at day 1 of pregnancy. However, this prominent staining of talin is absent in response to progesterone alone or progesterone in combination with estrogen, which is also observed at the time of implantation (Kaneko et al., 2009). This supports one of the important findings of the present study as noted in the immunofluorescence experiments in which FA proteins vinculin and integrin b5 disassemble from the base of the uterine epithelium at day 1 of pregnancy and co-localize and increase their expression apically at day 6 of pregnancy to facilitate the implantation process.

Of particular significance, the disassembly of vinculin and integrin [beta]5 from the basal focal adhesions and their apical expression increase at day 6 of pregnancy in the anastrozole treated rats means that anastrozole may favor implantation. Moreover, integrin aVb3 is required for endometrial receptivity (Zhang et al., 2011). Other studies demonstrated that human preimplantation embryos constitutively express integrin b3 and integrin [beta]5 subunit proteins (Bloor et al., 2002). Put together, this means that integrins play a role in the implantation process and could be regulated by agents that are known to alter cell behavior such as anastrozole and chlomiphene citrate (CC).

On the other hand, the decrease in the apical vinculin and integrin 05 expression with chlomiphene citrate (CC) treatment at day 6 in the current study may suggest the interpretation that CC might negatively affect the implantation process in this regard. This agrees with earlier studies that also observed a decrease in uterine integrin 03 expression in chlomiphene citrate (CC) treated patients during the window of implantation, suggesting that chlomiphene citrate (CC) might affect the expression of uterine receptivity markers (Palomino et al, 2005). On the contrary, other studies noted that chlomiphene citrate (CC) does not affect the secretion of integrin [alpha]3, integrin aV and integrin b1 during the implantation window in patients with unexplained infertility (Lacin et al., 2001). All in all, chlomiphene citrate (CC) appears to negatively affect the implantation process by down-regulating the expression of key implantation markers such as vinculin and integrin [beta]5 and thus being associated with low pregnancy rates.

Additionally, other studies done on the effects of letrozole and chlomiphene citrate (CC) on the expression of integrin [[alpha].sub.V] [[beta].sub.3] during implantation in rats demonstrated that the expression of integrin [[alpha].sub.V] [[beta].sub.3] in the CC group is significantly lower than in the letrozole and saline groups (Bao et al., 2009) suggesting that CC suppresses uterine receptivity more than letrozole (Bao et al.). Similar observations are made by previous studies in which the expression of the molecular markers of endometrial receptivity, integrin [[alpha].sub.V] [[beta].sub.3] and glycodelin, are decreased in endometrial biopsy specimens from women with PCOS when ovulation is induced with clomiphene citrate (Gonzalez et al., 2001; Jakubowicz et al., 2001). Interestingly, these observations agree with the results of the current study in which integrin [beta]5 expression in the uterine luminal epithelial cells of CC treated rats is significantly lower than anastrozole and untreated groups during implantation (p [less than or equal to] 0.0001). However, in the present study, integrin [beta]5 disassembles from the basal focal adhesions and later get localized at the apical plasma membrane of the rat uterine luminal epithelial cells at the time of implantation as also noted by (Kaneko et al., 2011) in their studies with integrin [beta]3. Moreover, integrin [beta]5, [beta]3 and aV are apically distributed in uterine epithelial cells during implantation (Aplin & Kimber, 2004) suggesting their crucial role in the implantation process. Similarly, Creus et al. (2003) have shown that the human endometrial maturity is characterized by integrin aVb3 expression and pinopod formation. Therefore, this clearly shows that integrins (including integrins [beta]5) are pivotal with regard to blastocyst-uterus interaction during early pregnancy. Viewed in its totality, anastrozole seems to enhance the focal adhesion protein vinculin and integrin [beta]5 expression at day 6 suggesting the interpretation that anastrozole is implantation friendly as also evidenced by its high implantation rates (Mwakikunga & Hosie).

CONCLUSIONS

The immunofluorescence and gene expression results for the uterine receptivity markers FA proteins vinculin and integrins [beta]5 appear to demonstrate that anastrozole is implantation friendly. It is also important to note that anastrozole is a competitive inhibitor of aromatase in the conversion of androgens to estrogens ([E.sub.2]); therefore, the 15 mg/kg anastrozole dose, which is effective in causing ovulation, may allow just enough of the intermediates and [E.sub.2] to be present with regard to the hormone profiles for ovulation and successful implantation of the embryo. Essentially, it may appear that there is blocking of E2 and increasing of androgens in anastrozole treated rats compared to controls. Progesterone (P) is probably also high as [E.sub.2] is low. In chlomiphene citrate (CC) treated rats, estrogen receptors might be blocked, which increases [E.sub.2]. The effect of anastrozole in the level of [E.sub.2] and P may change the E2/P ratio at day 6 of pregnancy, suggesting its effect on endometrial maturation and therefore improving receptivity. If this is true, the advantage of anastrozole therapy over chlomiphene citrate (CC) may be the lower [E.sub.2]/P ratio and increased levels of FSH and LH. Therefore, measuring parameters such as the intermediates, estrogens and androgens in future studies may also be of use in determining hormonal profiles for implantation. Anastrozole may be a suitable replacement or alternative drug used for hyperovulation or just to promote fertility in patients, particularly those suffering from polycystic ovary syndrome and those that are unresponsive to chlomiphene citrate (CC).

ACKNOWLEDGMENTS

We thank the staff of the Central Animal Unit and the School of Anatomical Sciences of the University of the Witwatersrand, South Africa and the staff and management of Newcastle University, NuMed Malaysia Campus for their support.

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Corresponding author:

Gbenga A Adefolaju

Department of Pre-clinical Sciences

School of Health Care Sciences

University of Limpopo

Private Bag x1106

Sovenga 0727

SOUTH AFRICA

E-mail: gbenga.adefolaju@ul.ac.za

anton.ul2016@gmail.com

Received: 08-02-2017

Accepted: 03-05-2017

Anthony Mwakikunga (1), Gbenga A. Adefolaju (2, 3), Lynne Schepartz (1) & Margot J. Hosie (1, 3)

(1) School of Anatomical Sciences, University of the Witwatersrand Medical School, 7, York road, Parktown 2193, Johannesburg, South Africa.

(2) Department of Pre-Clinical Sciences, Faculty of Health Sciences, University of Limpopo, Sovenga 0727, South Africa.

(3) Newcastle University Medicine Malaysia Campus, Nusajaya, Johor, Malaysia. Funding: This work was funded by grants from the National Research Foundation and Medical Research Council of South Africa, the government of Norway (NORAD project) and College of Medicine, University of Malawi, Blantyre, Malawi.

Caption: Fig. 1. Vinculin and intergrin [beta]5 expression in the surface luminal uterine epithelial cells of day 1 and day 6 pregnant rats. There is a significant difference in the expression of vinculin and integrin [beta]5 between anastrozole day 1 and day 6 (p [less than or equal to] 0.0001), untreated (control) day 1 and day 6 (p [less than or equal to] 0.0001), untreated day 6 and CC day 6 (p [less than or equal to] 0.0001). No significant difference in vinculin and integrin b5 expression is noted between chlomiphene citrate day 1 and chlomiphene citrate day 6 (p [less than or equal to] 0.05), untreated day 6 and anastrozole day 6 (p <0.05). Generally, chlomiphene citrate (CC) seems to decrease their expression while anastrozole seems to enhance their expression as pregnancy progresses. Note: numerical scale on the left represents the scoring system average scores as described previously in Table II.

Caption: Fig. 2. Vinculin and intergrin [beta]5 uterine epithelial localization. Vinculin and integrin [beta]5 are co-localized at the base of the uterine epithelium at day 1 of pregnancy whereas at day 6, they disassemble from the basal focal adhesions and co-localize and significantly increase their expression apically (p [less than or equal to] 0.0001) as also noted in Figs. 3 and 4. Note: vinculin is also expressed in the perinuclear region at day 1 and relocates apically at day 6 of pregnancy in the untreated and anastrozole treated rats as seen in Figs. 3 and 4. Note: numerical scale on the left represents the scoring system average scores as described previously in Table II (1, basal; 2, apical; 3, lateral; 4, basolateral; 5, basal and apical).

Caption: Fig. 3. Micrograph showing vinculin and integrin [beta]5 expression and co-localization in luminal uterine epithelial cells from day 1 of pregnancy in rats. (A), (B) and (C) show nuclei stained with DAPI (blue). (D) and (E) A distinct band of vinculin (green, FITC) expression at the base of the uterine epithelium (yellow arrow) is noted in both untreated and anastrozole treated rats. (F) The basal expression of vinculin in chlomiphene citrate (CC) treated rat is not as high as in (D) and (E). (G) and (H) A distinct band of integrin [beta]5 (red, rodamine) expression at the base of the uterine epithelium (yellow arrow) is noted in both untreated and anastrozole treated rats. (I) The basal expression of integrin [beta]5 in CC treated rat is not as high as in (G) and (H). (J), (K) and (L) Vinculin and integrin [beta]5 co-localize basally on day 1 of pregnancy. All images are representative of staining from the 5 rats in each of the treatment regimes.

Caption: Fig. 4. Micrograph showing vinculin and integrin [beta]5 expression and co-localization in luminal uterine epithelial cells from implantation sites of day 6 pregnant rats. (A), (B) and (C) show nuclei stained with DAPI (blue); orange arrow shows the embryo. (D) and (E) Vinculin (green, fluorescein isothiocyanate (FITC) disassemble from the base of the epithelium (yellow arrows) and become highly expressed apically (white arrows) in both untreated and anastrozole treated rats. (F) The apical expression of vinculin in chlomiphene citrate (CC) treated rat is down-regulated. (G) and (H) Integrin [beta]5 (red, rodamine) disassemble from the base of the epithelium (yellow arrows) and become highly expressed apically (white arrows) in both untreated and anastrozole treated rats. (I) The apical expression of integrin P5 in CC treated rat is down-regulated. (J), (K) and (L) Vinculin and integrin [beta]5 co-localize apically on day 6 of pregnancy. All images are representative of staining from the 5 rats in each of the treatment regimes.

Caption: Fig. 5. Graphical representation of vinculin and intergrin 05 gene expression in the surface luminal uterine epithelial cells of day 1 and day 6 pregnant rats. There is a significant difference in the gene expression of vinculin and integrin [beta]5 between anastrozole day 1 and day 6 (p [less than or equal to] 0.0001), untreated (control) day 1 and day 6 (p [less than or equal to] 0.0001), untreated day 6 and CC day 6 (p [less than or equal to] 0.0001). No significant difference is noted between CC day 1 and CC day 6 (p [less than or equal to] 0.05), untreated day 6 and anastrozole day 6 (p [less than or equal to] 0.05). Note: Generally, chlomiphene citrate (CC) seems to decrease their expression while anastrozole seems to enhance their expression as pregnancy progresses.'
Table I: Anastrozole and CC dose regimes made up to 0.2 ml normal
saline for the 7 groups of mature female Wistar rats undergoing
ovulation stimulation.

Groups   Treatments                         Number
                                            of rats

1        Untreated *                           5
2        Saline (vehicle) *                    5
3        1 mg/kg anastrozole                   5
4        10 mg/kg anastrozole                  5
5        15 mg/kg anastrozole *                5
6        25 mg/kg anastrozole                  5
7        1.25 mg/kg chlomiphene citrate *      5

Table II: Description Scoring system
protein expression and localization in the confocal images among
treatment groups adopted from previous studies (Englund et al., 2001;
Mwakikunga et al., 2011). Epithelial immunohistochemical
characteristics

Protein expression    Whether the epithelium is positively stained or
                      not:

                      (0, negative staining, intensity absent)

                      (1, positive staining and 1 to 10 % positively
                      stained epithelium, low intensity)

                      (2, positive staining and 11 to 50 % positively
                      stained epithelium, medium intensity)

                      (3, positive staining and 51 to 100 % positively
                      stained epithelium, high intensity)Protein
                      localization in the epithelium)

Main epithelial       (1, basal; 2, apical; 3,lateral; 4, basolateral;
domain protein        5, basal and apical)
localization

Additional protein    (1, cytoplasmic; 2, perinuclear; 3, evenly
localization          distributed in epithelial cell; 4, stroma).

Table III: Description of the real tim.
characteristics for the genes of interest and reference genes used in
the study.

Gene       Gene      RefSeq GenBank   5'-3' primer sequence
symbol     name       Accessi on
                         Number

Vcl      Vinculin    NM 001107248.1   F: 5'-GTT CCC GGT TTT CTG TTG
                                      CC -3' (20 bases)

                                      R: 5'- GCC CTC GTG CAT AAT CA
                                      -3' (20 bases)

Itgb5    Integrin,    NM 147139.2     F: 5'- GTG CGA CAG CTT TTC
                                      CTG TG -3' (20 bases)

          beta 5                      R: 5'- AAT GTA ACC GAC GTG
                                      GCA CT -3' (20bases)

Actb      Actin,      NM 031144.3     F: 5'--GCA GGA GTA CGA TGA
                                      GTC CG -3' (20 bases)

           beta                       R: 5'--ACG CAG CTC AGT AAC
                                      AGT CC -3' (20 bases)

Ldha      Lactate     NM 017025.1     F: 5'--CCG TTA CCT GAT GGG
                                      AGA

         dehydro-                     AA -3' (20 bases)
          genase
             A                        R: 5'--ACG TTC ACA CCA CTC
                                      CAC AC -3' (20 bases)

18s      18Sr RNA       X01117.1      F: 5'--GTT GGT TTT CGG AAC
                                      TGA GGC -3' (21 bases)

                                      R: 5'--GTC GGC ATC GTT TAT
                                      GGT CG -3' (20 bases)

Gene       Gene         Position of         GC       Amplicon
symbol     name      primer on template   content   length (bp)

Vcl      Vinculin          40-59            55%         139

                          178-159           55%

Itgb5    Integrin,       1694-1713          55%         97

          beta 5         1790-1771          50%

Actb      Actin,         1155-1174          60%         74

           beta          1228-1209          55%

Ldha      Lactate         613-632           50%         108

         dehydro-
          genase
             A            720-701           55%

18s      18Sr RNA         895-915          52.4%        204

                         1098-1079          55%

Table IV: A summary of vinculin and integrin P5 expression and
localization.

                         Vinculin               Integrin p 5

Day 1      Untreated     Medium intensity       Medium intensity
                         expression in focal    expression in focal
                         adhesions at the       adhesions at the
                         base of t he           base of the
                         epithelium with some   epithelium
                         sparse perinuclear
                         region expression

           Anastrozole   Medium to high         Medium to high
                         intensity expression   intensity
                         in the basal regio n   expression in the
                         of the epithelium      basal region of the
                         with some sparse       epithelium
                         perinuclear region
                         expression

           CC            Medium intensity       Medium intensity
                         expression in focal    expression in focal
                         adhesions at the       adhesions at the
                         base of the            base of the
                         epithelium             epithelium

Day 6 IP   Untreated     High                   High intensity
                         intensity apical       apical cytoplasmic
                         cytoplasmic            expression
                         expression

           Anastrozole   High intensity         High intensity
                         apical cytoplasmic     apical cytoplasmic
                         expression             expression

           CC            Low intensity apical   Low intensity
                                                apical cytoplasmic
                                                expression

Day 6 NP   Untreated     cytoplasmic            High intensity
                         expression High        apical cytoplasmic
                         intensity apical       expression
                         cytoplasmic
                         expression

           Anastrozole   High intensity         High intensity
                         apical cytoplasmic     apical cytoplasmic
                         expression             expression

           CC            Low intensity apical   Low intensity
                         cytoplasmic            apical cytoplasmic
                         expression             expression

Table V: Relative quantification (RQ) analysis of vinculin gene
expression in the luminal surface uterine epithelial cells of day 1
and day 6 of pregnant rats. A fold change of 1.5 or more represents a
significant difference in gene expression (p < 0.05).

Sample       Vin Ave Ct           * RFG Ave Ct

** 1U    24.40 [+ or -] 0.01   22.81 [+ or -] 0.03
1A       24.14 [+ or -] 0.06   22.75 [+ or -] 0.04
1CC      24.81 [+ or -] 0.13   22.82 [+ or -] 0.06
6U IP    23.96 [+ or -] 0.03   22.59 [+ or -] 0.09
6U NP    23.91 [+ or -] 0.07   22.37 [+ or -] 0.04
6A IP    23.17 [+ or -] 0.03   22.19 [+ or -] 0.02
6A NP    24.19 [+ or -] 0.07   23.17 [+ or -] 0.03
6CC IP   25.18 [+ or -] 0.28   23.52 [+ or -] 0.05
6CC NP   25.26 [+ or -] 0.11   23.56 [+ or -] 0.05

Sample    [[DELTA]C.sub.t]    [[DELTA][DELTA]C.sub.t]

** 1U    1.59 [+ or -] 0.03       0 [+ or -] 0.03
1A       1.39 [+ or -] 0.07     -0.2 [+ or -] 0.07
1CC      1.99 [+ or -] 0.14      0.4 [+ or -] 0.14
6U IP    1.37 [+ or -] 0.09     -0.22 [+ or -] 0.09
6U NP    1.54 [+ or -] 0.08     -0.05 [+ or -] 0.08
6A IP    0.98 [+ or -] 0.04     -0.61 [+ or -] 0.04
6A NP    1.02 [+ or -] 0.08     -0.57 [+ or -] 0.08
6CC IP   1.66 [+ or -] 0.08     0.07 [+ or -] 0.08
6CC NP   1.7 [+ or -] 0.12      0.11 [+ or -] 0.12

Sample   [Log.sub.10] ([2.sup.-   Fold change [2.sup.-
           [DELTA][DELTA]CT])     [DELTA][DELTA]CT]

** 1U              0              1 (0.97 to 1.02)
1A                0.08            1.2 (1.09 to 1.21)
1CC              -0.11            0.77 (0.69 to 0.84)
6U IP             0.08            1.2 (1.09 to 1.24)
6U NP             0.02            1.04 (0.98 to 1.09)
6A IP             0.20            1.6 (1.5 to 1.6)
6A NP             0.18            1.5 (1.4 to 1.5)
6CC IP           -0.02            0.96 (0.90 to 1.01)
6CC NP           -0.03            0.93 (0.85 to 1.01)
CT-threshold cycle, SEM-standard error of the mean, *-endogenous
control, ** -calibrator sample. [DELTA]CT = CT (Target)-CT (Endogenous
control). [DELTA][DELTA]CT = [DELTA]CT (Target-[DELTA]CT (Calibrator).

Table VI: RQ analysis of integrin 05 gene expression in the luminal
surface uterine epithelial cells of day 1 and day 6 of pregnant rats.
A fold change of 1.5 or more represents a significant difference in
gene expression (p < 0.05).

Sample   Int 5 Ave [C.sub.T]   * RFG Ave [C.sub.T]

** 1U    27.8 [+ or -] 0.02    22.81 [+ or -] 0.03
1A       27.64 [+ or -] 0.07   22.75 [+ or -] 0.04
1CC      27.39 [+ or -] 0.05   22.05 [+ or -] 0.06
6U IP    26.88 [+ or -] 0.06   22.59 [+ or -] 0.09
6U NP    26.96 [+ or -] 0.02   22.37 [+ or -] 0.04
6A IP    26.30 [+ or -] 0.06   22.19 [+ or -] 0.02
6A NP    27.41 [+ or -] 0.02   23.17 [+ or -] 0.03
6CC IP   28.74 [+ or -] 0.08   23.52 [+ or -] 0.05
6CC NP   29.83 [+ or -] 0.01   23.56 [+ or -] 0.05

Sample    [[DELTA]C.sub.T]    [[DELTA][DELTA]C.sub.T]

** 1U    4.99 [+ or -] 0.04       0 [+ or -] 0.04
1A       4.89 [+ or -] 0.08     -0.1 [+ or -] 0.08
1CC      5.34 [+ or -] 0.08     0.35 [+ or -] 0.08
6U IP    4.29 [+ or -] 0.11     -0.7 [+ or -] 0.11
6U NP    4.59 [+ or -] 0.04     -0.4 [+ or -] 0.04
6A IP    4.11 [+ or -] 0.06     -0.88 [+ or -] 0.06
6A NP    4.24 [+ or -] 0.04     -0.75 [+ or -] 0.04
6CC IP   5.22 [+ or -] 0.09     0.23 [+ or -] 0.09
6CC NP   6.27 [+ or -] 0.05     1.28 [+ or -] 0.05

Sample       [Log.sub.10](2-            Fold change 2-
         [[DELTA][DELTA]C.sub.T])   [[DELTA][DELTA]C.sub.T]

** 1U               0                  1 (0.97 to 1.03)
1A                 0.03               1.07 (1.01 to 1.13)
1CC               -0.10               0.79 (0.74 to 0.83)
6U IP              0.21               1.63 (1.51 to 1.75)
6U NP              0.12               1.32 (1.29 to 1.35)
6A IP              0.27               1.85 (1.77 to 1.92)
6A NP              0.23               1.68 (1.64 to 1.73)
6CC IP            -0.07               0.86 (0.8 to 0.91)
6CC NP            -0.38               0.42 (0.4 to 0.43)

CT-threshold cycle, SEM--standard error of the mean, *-endogenous
control, ** -calibrator sample.[DELTA]CT = CT (Target) -CT (Endogenous
control). [DELTA][DELTA]CT = [DELTA]CT (Target-[DELTA]CT (Calibrator).
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Author:Mwakikunga, Anthony; Adefolaju, Gbenga A.; Schepartz, Lynne; Hosie, Margot J.
Publication:International Journal of Morphology
Date:Mar 1, 2018
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