158 1

REPRODUCTIONRESEARCH

Microtubule depolymerization attenuates WNT4/CaMKIIα signaling in mouse uterus and leads to implantation failure

Vinay Shukla1,2, Jyoti Bala Kaushal1,2, Rohit Kumar1, Pooja Popli1, Promod Kumar Agnihotri3, Kalyan Mitra2,4 and Anila Dwivedi1,2 1Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow, India, 2Academy of Scientific and Innovative Research (AcSIR), CSIR-CDRI Campus, Lucknow, India, 3Division of Toxicology & Experimental Medicine, CSIR-Central Drug Research Institute, Lucknow, India and 4Electron Microscopy Unit, SAIF, CSIR-Central Drug Research Institute, Lucknow, India Correspondence should be addressed to A Dwivedi; Email: [email protected]

Abstract

Microtubule (MT) dynamics plays a crucial role in fertilization and early embryonic development; however its involvement in uterus during embryo implantation remains unclear. Herein, we report the effect of microtubule depolymerization during embryo implantation in BALB/c mice. Intrauterine treatment with depolymerizing agent nocodazole at pre-implantation phase (D4, 07:00 h) in mice resulted into mitigation in receptivity markers viz. LIF, HoxA10, -β3, IHH, WNT4 and led to pregnancy failure. MT depolymerization in endometrial epithelial cells (EECs) also inhibited the blastocyst attachment and the adhesion. The decreased expression of MT polymerization-related proteins TPPP and α/β-tubulin in luminal and glandular epithelial cells along with the alteration in morphology of pinopodes in the luminal epithelium was observed in nocodazole receiving uteri. Nocodazole treatment also led to increased intracellular Ca+2 levels in EECs, which indicated that altered Ca+2 homeostasis might be responsible for implantation failure. Microtubule depolymerization inhibited WNT4 and Fz-2 interaction, thereby suppressing the downstream WNT4/CaMKIIα signaling cascades calmodulin and calcineurin which led to attenuation of NF-κB transcriptional promoter activity in EECs. MT depolymerization or CaMKIIα knockdown inhibited the transcription factor NFAT and NF-κB expression along with reduced secretion of prostaglandins PGE2 and PGF2α in mouse EECs. Overall, MT depolymerization impaired the WNT4/CaMKIIα signaling and suppressed the secretion of PGE2 and PGF2α in EECs which may be responsible for implantation failure in mice. (2019) 158 47–59

Introduction secretion of (Sawyer et al. 1979). Although MT dynamics plays a crucial role in early embryonic For successful pregnancy, a blastocyst competent development and fertilization (Wu et al. 1996, Yan for implantation needs to be synchronized with the et al. 2006, Watanabe et al. 2016), its involvement in proliferation and differentiation of specific uterine cell embryo implantation is not completely understood. types under the influence of steroids mainly Earlier studies show that uterine tubulin levels rise and progesterone (Wang & Dey 2006, Bazer et al. 2009, rapidly in the endometrium and myometrium during Huang et al. 2017, Kaczyński et al. 2018). Impaired pre-implantation period in rabbits (Fujimoto & Saldana embryo implantation and/or decidual aberrations are 1976). In mice, the MT-associated protein HURP plays thought to be responsible for infertility and recurrent a crucial role in embryo implantation (Tsai et al. 2008). pregnancy loss (Cha et al. 2012). Although several Other MT-regulator protein, stathmin, has been reported attempts have been made to explain the molecular in rat during embryo implantation (Tamura et al. 2003) aspects of embryo implantation failure in past decade, and also in human endometrium during pre-receptive the underlying mechanisms still remain unclear. (LH + 2) and receptive (LH + 7) phases (Domínguez et al. The ability of the cytoskeleton to deform and reform is 2009) as well as in uterine fluid at secretory phase of critical for at the time of embryo menstrual cycle (Bhutada et al. 2014). In our earlier invasion (Paule et al. 2010). Microtubules (MTs) are study, we have also demonstrated the expression of structural components and their dynamic instability tubulin polymerization promoting protein 3 (TPPP3) can lead to sub-cellular movement, mitotic block, cell during endometrial receptivity in human (Manohar et al. cycle arrest, protein trafficking, vesicle transport, axonal 2014a) and have recently demonstrated its functional extension and even cell death (Lopez & Valentine role in mice and hESCs (Shukla et al. 2018, 2019). 2015). MTs are also involved in the transport and

© 2019 Society for Reproduction and Fertility https://doi.org/10.1530/REP -18-0611 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via https://rep.bioscientifica.com Downloaded from Bioscientifica.com at 09/26/2021 07:57:48AM via free access

-18-0611 48 V Shukla and others

Though the literature indicates that MT polymerization Microtubule depolymerization in mouse uterus with the might be important for endometrial functions, its help of microtubule-depolymerizing agent, nocodazole significance particularly in embryo implantation is not We used microtubule-depolymerizing agent nocodazole to yet known. The current study was undertaken to explore evaluate the role of MT polymerization on early pregnancy the significance and functional role of MT polymerization events (Lagos-Cabré & Moreno 2008). The intrauterine during early implantation phase in mice. Nocodazole injection surgery was done on pre-implantation stage i.e. on disrupts MTs by binding to β-tubulin (Mitchison & Kirschner D4 (07:00 h) of pregnancy (Maurya et al. 2013). Nocodazole 1984) and has been used as a MT-depolymerizing agent (300 nM, 2 μL) or vehicle was injected into uterine horn in in several studies (Choi et al. 2011, Guo et al. 2012, mice. We first optimized the concentration of effective dose Isshiki et al. 2015, Signoretto et al. 2016). Therefore, to of nocodazole by assessing the effect of nocodazole at varying prove our hypothesis, the effect of MT-depolymerizing concentrations in mice. At 300 nM, nocodazole suppressed agent (nocodazole) was studied on morphological the embryo implantation by ~90%. Whereas at lower characteristics, receptivity markers and downstream concentrations, the suppressing effect on implantation was not signaling mechanisms regulating implantation and significant. Therefore, we selected the optimized effective dose pregnancy establishment in mouse uterus.

Materials and methods Reagents and chemicals To induce MT depolymerization, nocodazole (Calbiochem, Merck Millipore) was used. Lysis buffer, Bradford reagent, Collagenase, DNase, protease inhibitor cocktail (PIC), PIPES,

MgCl2, EGTA, PBS, cell culture media, FBS, osmium tetroxide, penicillin streptomycin antibiotics and Cy3 secondary antibodies were purchased from Sigma-Aldrich. Anti-fade reagent with DAPI from Life Technologies, Thermo Fisher Scientific, nylon cell strainer from BD Biosciences (NJ, USA) and fluorescein isothiocyanate (FITC) were procured from Santa Cruz Biotechnology. Immunoblot PVDF membrane was purchased from Merck Millipore. ECL reagent was purchased from GE Healthcare.

Antibodies

Antibodies for TPPP (sc-98687), α-tubulin (sc-8035), β-tubulin (sc-5274), WNT4 (sc-376279), LIF (sc-20087), IHH (sc-13088), Fz-2 (sc-68328), HoxA10 (sc-17159), Integrin-β3 (sc-52685), NF-κB p50 (sc-53744), Cytokeratin (sc-57004), Vimentin (sc- 32322), ERα (sc-543), PR (sc-538), JNK (sc-572) and GAPDH Figure 1 Investigation of TPPP expression during window of (sc-32233) were procured from Santa Cruz Biotechnology. implantation. (A) The expression analysis of TPPP was done in uterine Antibody for NF-κB p65 (#8242) was procured from Cell protein fraction through immunoblotting. Representative immunoblot Signaling Technology and STAT3 (610189) was procured from images showing the expression of TPPP on different days of BD Biosciences. pregnancy. GAPDH was used as a control to correct for loading (upper panel). Densitometric quantitation of protein expression levels is shown as fold changes (lower panel). Number of animals per Mouse implantation model group = 5. (B) The mRNA expression of Tppp genes in mouse early pregnancy was analyzed by real-time PCR. Number of animals per Adult female virgin BALB/c mice (3 months old, ~26 g) were group = 5. (C) Hormonal regulation of TPPP using delayed used in this study. All the animal protocols were approved implantation model. To maintain delayed implantation, by Institutional Animal Ethical Committee of CSIR-Central ovariectomized mouse was injected subcutaneously with P4 Drug Research Institute, Lucknow, India. Female mice were (1 mg/0.1 mL sesame oil/mouse) from D4 to D7. E2 (25 ng/0.1 mL of co-caged with fertile (2:1) and were checked next morning sesame oil/mouse) was given to P4-primed mouse to terminate delayed implantation on D8. TPPP protein expression in vehicle, for copulation plug. The day on which copulatory plug was delayed (P4) and activated uterus (E2 + P4) was analyzed by observed, was designated as D1 of pregnancy. Uterine tissues immunoblotting (left panel). Number of animals per group = 5. were collected from these animals during different days of Densitometric quantitation of protein expression levels is shown the pre-implantation period. The excised uterine horn (D5, as fold changes (right panel). Three replicates (individual animal 08:00 h) was flushed gently through the oviductal end with as a replicate) were used in each group. P values: aP < 0.001, 1 mL sterile PBS to obtain embryo (Shukla et al. 2018). bP < 0.01, cP < 0.05 and dP > 0.05 vs. D1/vehicle-treated-group.

Reproduction (2019) 158 47–59 https://rep.bioscientifica.com

Downloaded from Bioscientifica.com at 09/26/2021 07:57:48AM via free access MT depolymerization inhibits implantation 49 of nocodazole of 300 nM for our study. The implantation sites the remaining tissues were incubated in 3 mL of DMEM/F-12 (IS) in the uterus were visualized on D8 (10:00 h) (Fig. 2A). containing 0.15 mg/mL collagenase I (Sigma-Aldrich) at 37°C for 30 min. The digested uteri were shaken and filtered through a 70 m wire gauze filter and collected cells having more Primary culture of mouse EECs μ than 70 μm cell size. The purity of EECs was confirmed with For mouse EECs isolation, uterine horns were cut longitudinally anti-cytokeratin (epithelial cell marker) and anti-vimentin and washed with DMEM/F-12 and digested with 1% (w/v) (stromal cell marker), respectively. Immunofluorescence trypsin (Sigma-Aldrich) and 4 mg/mL dispase (Sigma-Aldrich) experiment showed that the EECs were positive for cytokeratin in DMEM/F-12 for 1 h at 4°C followed by 1 h at 25°C and and negative for vimentin. The isolated EECs were cultured at 10 min at 37°C. After rinsing three times with DMEM/F-12, 37°C and 5% CO2. Prior to experiment, cells were cultured in phenol red-free MEM supplemented with 10% charcoal- stripped FBS (Shukla et al. 2018).

Co-culture of mouse blastocysts and primary mouse EECs Attachment experiment was performed by co-culture of mouse blastocysts and mouse EECs. The EECs were treated with nocodazole (300 nM, 10 μL) or the vehicle control in serum medium (containing 10% FBS) for 24 h prior to receiving blastocysts. The medium was changed after 24 h of nocodazole treatment. Blastocysts were isolated from seven mice in each experiment and were used randomly for co-culture with nocodazole or the vehicle control mouse EECs. Initially, co-cultures were incubated undisturbed and blastocyst attachment to mouse EECs was determined at 24 h and their position was examined under a microscope (Nikon Eclipse TE2000-S). Embryos that did not float away were considered to have attached. Upon moving the plate, unattached embryos floated or rolled over the epithelial surface. Attached embryos were then examined for tandem movement when the microscope stage was tapped. Co-culture experiments were performed in triplicate and data were pooled from the three separate experiments and counted as the proportion of blastocysts that had attached to the epithelial cells out of the total number of blastocysts added to the epithelial cells (Green et al. 2015, Shukla et al. 2018).

Delayed implantation model Delayed implantation was induced in pregnant mouse which were bilaterally ovariectomized on D3. To maintain delayed implantation, ovariectomized mouse was injected subcutaneously with progesterone (1 mg/0.1 mL sesame oil/ Figure 2 Microtubule depolymerization by nocodazole (300 nM) at mouse) from D4 to D7. Estradiol-17 (25 ng/0.1 mL of sesame D4 (07:00 h) reduced the implantation sites at D5 (10:00 h) of β oil/mouse) was administered to progesterone-primed mouse to pregnancy in mice. Representative image of uterus on D8 from the nocodazole-treated group. (A) The intrauterine injection surgery was terminate delayed implantation on D8. The delayed group and done on pre-implantation stage i.e. on D4 (07:00 h) of pregnancy and activation group was confirmed by flushing blastocysts from nocodazole (300 nM, 2 μL) or vehicle was injected into uterine horn. the uterine horns (Liang et al. 2014, Shukla et al. 2018). The IS in the uterus were visualized on D8 (10:00 h) (A). The arrow indicates blastocyst implantation. Number of animals per group = 5. (B) Number of implantation sites on D8 (10:00 h). (C) Nocodazole Immunoblot analysis treatment decreased the expression of α-tubulin, β-tubulin, TPPP at Tissue (in vivo experiments) or cells (in vitro experiments) peri-implantation stage i.e. D5 (08:00 h). (D) Effect of nocodazole in were lysed in lysis buffer (Sigma-Aldrich). Briefly, uterine the expression of ERα, PR, JNK and STAT3 on D5 (08:00 h) (left panel). GAPDH was used as a control to correct for loading (left tissue was homogenized in ice-cold RIPA lysis buffer (150 mM panel). The each experiment was performed three times with three NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% tissue samples. Densitometric quantitation of protein expression SDS, 50 Mm Tris, pH 8.0) (Sigma-Aldrich). The homogenate levels is shown as fold changes (right panel). P values: aP < 0.001, was supplemented with a protease inhibitor cocktail (PIC) bP < 0.01, cP < 0.05 and dP > 0.05 vs vehicle group. (AEBSF 2 mM, Aprotinin 0.3 μM, Bestatin 116 μM, E-64 14 μM, https://rep.bioscientifica.com Reproduction (2019) 158 47–59

Downloaded from Bioscientifica.com at 09/26/2021 07:57:48AM via free access 50 V Shukla and others

Leupeptin 1 μM and EDTA 1 mM) (Sigma-Aldrich) for 4 h at 4°C Gapdh. Comparative cycle threshold (2−ΔΔCt) method was used and then kept overnight at −20°C. The protein was obtained for relative quantification. The PCR system was programmed as supernatant by centrifuging at 12000 g for 50 min at 4°C. according to the manufacturer's instructions. All measurements Protein estimation was done by Bradford reagent (Sigma- were performed in triplicate (Shukla et al. 2015). Aldrich). Equal amounts of protein (20 µg) were separated by SDS-PAGE and transferred to Immunoblot PVDF membrane. The membrane was blocked for 2 h in 5% skimmed milk Hematoxylin and eosin staining and dissolved in TBST (Tris-buffered saline, 0.1% Tween 20, 7.5 immunofluorescence imaging by confocal microscopy pH) and incubated with primary antibody overnight at 4°C. Formalin-fixed tissues of nocodazole- or vehicle-treated The membranes were incubated with secondary antibody mouse uterus on D5 (IS) were sectioned. Tissues were for 1 h. Antibody binding was detected by using enhanced dehydrated and thereafter embedded in paraffin wax and chemiluminescence detection system (BIO-RAD ChemiDoc stained with hematoxylin & eosin (H&E) and examined under XRS+). After developing, the membrane was stripped and light microscope. Images were captured with NIS-Elements F re-probed with GAPDH antibodies. Densitometry of band 3.0 camera (Nikon). density was performed by using Quantity One Software In immunofluorescence experiment, formalin-fixed tissues (v.4.5.1). The density of a given band was measured as the were dehydrated and thereafter embedded in paraffin wax. total volume for each group and normalized to GAPDH as an Paraffin sections of 5 μm were cut from each experimental internal control (Manohar et al. 2014b, Kaushal et al. 2018). group. Mouse uterine tissue sections were then fixed in methanol and acetone in 1:1 ratio at 4°C for 2 h and Transmission electron microscopy (TEM) permeabilized with 0.1% Triton X-100 at 25°C for 10 min and then mixed with microtubule stabilizing buffer (100 mM For TEM, samples were washed with phosphate buffer, and PIPES, 1 mM MgCl2, 5 mM EGTA, pH 6.8). Tissues sections then cut into small pieces (1 mm3) followed by fixation in 4% were washed with PBS and blocked with 1% BSA in distilled paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium water and incubated with and TPPP-α/β tubulin antibody phosphate buffer (pH 7.4) for 4 h at room temperature (24°C). for overnight followed by 1 h incubation with fluorescence- The tissues were then post fixed in 2% osmium tetroxide in tagged secondary anti-rabbit/mouse antibody, then 0.1 M phosphate buffer (pH 7.4) for 2 h at room temperature counterstained with DAPI for 5 min. Images were captured and dehydrated in an ascending grade of ethanol followed by at 40× with the using Carl Zeiss LSM 510 META microscope embedding in Epon 812 and polymerized at 60°C for 24 h. and analyzed using LSM Image- Examiner Software to detect Ultra-thin sections (50–70 nm) were obtained using an ultracut fluorescence and DAPI emissions Kaushal( et al. 2017). In Ultra-microtome (Leica Microsystems GmbH) and picked up negative control, sections/cells were incubated with IgG in onto 200 mesh copper grids. The sections were double-stained place of primary antibody. with uranyl acetate and lead citrate and analyzed under a Jeol JEM-1400 electron microscope (Jeol, Japan) fitted with a GatanOrius SC200 CCD camera. Images were acquired using Co-immunoprecepitation Gatan Digital Micrograph software at 80 kV (Kathuria et al. Interaction between WNT4 and Fz-2 proteins was studied 2014). Imaging was performed at the Electron Microscopy by co-immunoprecipitation of the complex followed by Unit, CSIR-Central Drug Research Institute, Lucknow. immunoblotting. Briefly, 2 μg anti-Fz-2 antibody were added to 300 μg of cell lysate and samples were incubated for Real-time polymerase chain reaction overnight at 4°C. In negative control, cell lysate was incubated with IgG instead of anti-Fz-2. Next, 100 μL of protein Total RNA from tissue was extracted using TRIzol reagent A-sepharose beads (Sigma-Aldrich) suspension were added (Invitrogen, Thermo Fisher Scientific) by following the and samples were incubated for 1 h at 4°C with constant manufacturer's instructions. The concentration of RNA was rocking. Immunoprecipitated complexes were collected by measured using Nanodrop (Thermo Fisher Scientific). The centrifugation at 3000 g for 2 min at 4°C and then washed three isolated RNA was treated with RNase-free DNase to remove times with RIPA buffer (Sigma-Aldrich), then resuspended in any residual genomic DNA. First-strand of DNA (cDNA) Laemmli sample buffer to a final concentration and heated was prepared from total RNA (1 μg) at each group using for 5 min at 95°C. The supernatants were collected by high-capacity cDNA reverse transcription kit, according to centrifugation at 12000 g for 30 s at room temperature. Equal the manufacturer’s protocol (Thermo Fisher Scientific). The amounts of immunoprecipitated proteins were separated quantification of the genes by Real-TimePCR was performed by 12% SDS-PAGE and transferred on PVDF membrane with a Light Cycler (Roche Life Science). Quantitative PCR (Millipore). The proteins were probed with anti-WNT4 and analyses were performed using appropriate primers (Mus anti-Fz-2, followed by the related secondary peroxidase- musculus) (Tppp Forward CACACAGTGGCCTCAGGATA, conjugated antibody. Antibody binding was detected by Reverse: AAAATGTCCCACCCTCAACA; Gapdh Forward using enhanced chemiluminescence detection system (GE AGCTTGTCATCAACGGGAAG, Reverse TTTGATGTTAGTGG Healthcare). Bands were detected by Gel Doc imaging system GGTCTCG). Expression of the investigated gene was (Bio-Rad) and analyzed by densitometry using Quantity One normalized to the steady expression of a housekeeping gene Software (v. 4.5.1) (Shukla et al. 2018).

Reproduction (2019) 158 47–59 https://rep.bioscientifica.com

Downloaded from Bioscientifica.com at 09/26/2021 07:57:48AM via free access MT depolymerization inhibits implantation 51

Flow cytometric analysis for cytosolic free Statistical analysis Ca+2 measurement Statistical analysis was performed using GraphPad Prism Primary mouse EECs were seeded (2 × 105 cells/well) into v6.0, all statistical tests are described in their respective 6-well plate and maintained overnight in phenol red-free figure legends. Briefly, data are presented as mean ± s.e.m. for MEM containing 10% stripped FBS. Next day, EECs were at least three separate determinations for each experiment. treated/transfected with nocodazole (300 nM) or cyclosporine One-way ANOVA in combination with Tukey test was A (0.5 μM) or CaMKIIα siRNA (30 nM) in serum-free MEM done to compare the multiple group’s comparison (three according to manufacturer’s protocol. Cells were washed with to four groups) and the Student’s ‘t’ test was performed PBS and incubated in serum-free MEM medium for 24 h. After for comparing the two groups. P value less than 0.05 was 24 h, cells were collected by trypsinization and resuspended in considered as significant. PBS. Fluo-3AM dye (2 μM) was added in each EECs groups for 30 min at 37°C in the dark with continuous shaking. Cytosolic Results free Ca+2 measurement was detected using a FACScan flow cytometer (BD Biosciences) with excitation and emission TPPP is highly expressed during peri-implantation settings at 506 nm and 526 nm, respectively (Zhang et al. phase and ovarian hormones modestly influence 2004, Gupta et al. 2018). TPPP expression Immunoblotting analysis was performed to analyze the expression of TPPP in all the stages of window/period Annexin-V/propidium iodide labeling and flow of implantation (Fig. 1A). We observed approximately cytometry assay for fourfold (P < 0.001) increase in expression of TPPP at IS Primary mouse EECs (2 × 105 cells per well) were cultured in on peri-implantation (D5, 08:00 h) as compared to pre- six-well plates and were treated with nocodazole (300 nM) implantation (D1), which indicates its contribution in for 48 h. After trypsinization, cells were probed with FITC- the embryo implantation. TPPP protein expression was conjugated Annexin-V and PI for 10 min. The fluorescence increased significantly in uterus on D4 compared to D1 staining profiles were determined through FACScan and Cell- of pre-implantation period and this high expression level Quest software. Staurosporin (1 μM) was used as a positive was maintained until receptivity/D5 (08:00 h) (Fig. 1A). control. The experiments were performed three times. On D5 (08:00 h), the expression of TPPP was prominent at the IS than that at respective non-implantation sites Transactivation assay (non-IS) (Fig. 1A). Apart from this, our study on the uterine transcript also revealed a low level of Tppp Mouse EECs were seeded and allowed to attain confluency of transcript at D1, which was significantly elevated on D5 70–80%. In transactivation assay, all plasmids were prepared (P < 0.001) (Fig. 1B). using QIAGEN plasmid DNA preparation kits. EECs were To investigate whether the process of uterine MT then transfected with 400 ng of pNF-kB-luc (Stratagene) using polymerization is a subject to ovarian steroid hormones Lipofectamine RNAiMAX transfection reagent (Invitrogen) regulation, we analyzed the hormonal regulation of TPPP as per manufacturer’s protocol. To normalize for transfection protein expression after subcutaneous administration of efficiencies, 200 ng of pRL-SV40-luc (Promega) was and progesterone (E2 + P4; active group)- and co-transfected. After 24 h of nocodazole (300 nM) treatment progesterone (P4; delayed group)-treated groups in or cyclosporine A (0.5 M) or transfection of CaMKII siRNA μ α bilaterally ovariectomized mouse in delayed embryo (30 nM), medium was changed and EECs were treated with cyclosporine A (calcineurin inhibitor). Next, EECs were lysed implantation model (Fig. 1C). It was found that treatment with lysis buffer and luciferase activity was measured using of E2 along with P4 caused an increase in TPPP protein Dual Luciferase Assay System (Promega) according to the expression (by ~1.8-fold) in the uterus. On the contrary, manufacturer’s protocol to detect the transcriptional activity of the administration of P4 resulted into decreased the transfected promoter. The firefly luciferase activity for each TPPP protein expression in the uterus (P < 0.001) as group was normalized with transfection efficiency determined compared to E2 + P4 active group (Fig. 1C). Notably, this by Renilla luciferase activity (Popli et al. 2015). information indicated that P4 could negatively affect the regulation of TPPP (Fig. 1C). These results showed that TPPP is upregulated by E2 in the uterus during the Measurement of PGE2 and PGF2α levels embryo implantation process. EESc were seeded in six-well plates and grown to confluence. EESc were washed with PBS and incubated in serum-free Uterine microtubule depolymerization impaired DMEM medium for 24 h, and then treated/transfected with embryo implantation and inhibited expression of nocodazole (300 nM) or cyclosporine A (0.5 μM) or CaMKIIα associated proteins siRNA (30 nM). The culture supernatant was collected to measure PGE2 and PGF2α concentration using a monoclonal To analyze the specific role of MT polymerization antibody in an ELISA kit as specified by the manufacturer in uterus, we used nocodazole, a microtubule (Abcam, Enzo Lifesciences). de-polymerizing agent (Lagos-Cabré & Moreno 2008). https://rep.bioscientifica.com Reproduction (2019) 158 47–59

Downloaded from Bioscientifica.com at 09/26/2021 07:57:48AM via free access 52 V Shukla and others

Accordingly, intrauterine injection of the optimized Microtubule depolymerization inhibits ERα and PR concentration of nocodazole (300 nM, 2 μL) in right expression during peri-implantation period uterine horn and vehicle in the left uterine horn was The receptors for estrogen (ER) and progesterone (PR) given on D4 (07:00 h). The significantly reduced are important for successful blastocyst implantation number of embryos (~90%) (P < 0.001) was observed (Bulun 2017). Hence, we determined the levels of on D8 in nocodazole-treated uterine horn compared ER and PR protein expression in nocodazole-treated to that of vehicle-treated horn (Fig. 2A and B). MT uteri at D5. The immunoblot analysis showed that remains stiff and with help of GTP, -tubulin and α nocodazole treatment caused reduction in ERα by ~0.3- -tubulin modestly regulate MT polymerization β fold (P < 0.001) PR-A by ~0.65-fold (P < 0.001) and PR-B (Mitchison & Kirschner 1984, Paule et al. 2010). In by ~0.6-fold (P < 0.01) (Fig. 2D). These results showed immunoblotting experiment, we found a decrease in that MT depolymerization inhibits ERα and PR protein the expression of α-tubulin by ~0.5-fold, β-tubulin expression in mice uteri. by ~0.55-fold and TPPP by ~0.5-fold in the uteri of Apart from microtubule-associated proteins, we nocodazole-treated group as compared to vehicle- checked the effect of nocodazole on some other proteins treated control group (P < 0.001) (Fig. 2C). Results viz. JNK and STAT3 and our results showed that MT revealed that MT depolymerization suppressed the depolymerization did not alter the expression of these expression of -tubulin, -tubulin and TPPP in peri- α β proteins (P > 0.05) (Fig. 2D). These results indicated implantation phase (D5) of pregnancy. that microtubule depolymerization affects specifically

Figure 3 The effect of nocodazole on the mouse co-culture (EECs and blastocyst) and spatiotemporal expression of TPPP, α-tubulin and β-tubulin in the mouse uterus during peri-implantation (D5, 08:00 h). (A) Nocodazole (300 nM) treatment in primary mouse endometrial epithelial cells mitigates mouse blastocyst attachment. Representative images were taken after 24 h of co-culture. Each experiment was performed three times with three different samples. Data are presented as mean ± s.e.m. P values: P < 0.001 vs vehicle control. (B) H&E staining was performed in the uteri of D5 (IS) from the vehicle- or nocodazole-treated horns (magnification× 10). Asterisk indicates an embryo. Each experiment was performed three times with three different samples. (C and D) Tissue sections were incubated with and TPPP-α/β tubulin antibody for overnight followed by 1-h incubation with fluorescence-tagged secondary anti-rabbit/mouse antibody, and then counterstained with DAPI for 5 min. The expression of α-tubulin and TPPP β-tubulin and TPPP protein was analyzed in the LE, GE and stromal cells (Str) of the endometrium by confocal microscope at ×40. Three replicates (individual animal as a replicate) were used in each group.

Reproduction (2019) 158 47–59 https://rep.bioscientifica.com

Downloaded from Bioscientifica.com at 09/26/2021 07:57:48AM via free access MT depolymerization inhibits implantation 53 the microtubule polymerization-associated proteins Pinopodes as an indicator of endometrial receptivity (Fig. 2C) and receptivity markers (Fig. 4A) in the uterus. arise from the apical surface of the uterine LE during the window of receptivity both in rodents and humans (Murphy 2004, Montazeri et al. 2015). Hence, we Microtubule depolymerization suppresses attachment evaluated the pinopodes structure in mice uteri after of mouse blastocyst to primary EECs the vehicle or nocodazole treatment on D5. The TEM For in vitro implantation experiment, we analyzed results showed that the pinopodes were present on peri- the effect of nocodazole on primary mouse EECs and implantation phase (D5) in vehicle-treated group which mouse blastocysts co-culture attachment reaction. were deformed in nocodazole-treated uterine horn due Mouse blastocysts were transferred on EECs treated to MT depolymerization (Fig. 4B). with nocodazole or vehicle control and the blastocysts attachment were assessed. The proportion of blastocysts Microtubule depolymerization induced mild apoptosis attached to nocodazole-treated mouse EECs was found in primary mouse EECs to be significantly reduced (7 attached out of 50) as compared to that of vehicle-treated cells (38 attached To determine whether nocodazole causes apoptosis in out of 50) (P < 0.001) (Fig. 3A). These results showed primary mouse EECs, we did flow cytometric analysis that MT depolymerization in EECs inhibits in vitro embryo attachment.

Microtubule depolymerization caused morphological defects in luminal epithelium in mouse uterus and impaired uterine receptivity Prior to implantation, luminal epithelium (LE) undergoes the ovarian steroid hormones-induced structural and functional changes that make it competent for embryo invasion and attachment (Hantak et al. 2014). Hence, to evaluate the changes in LE, we performed the H&E staining in nocodozole- or vehicle- treated horn at D5 (IS). The luminal epithelial cell closure was not observed in MT de-polymerized (nocodazole-treated) horn. Moreover, no embryo attached was seen in cross-section of nocodazole-treated horn, whereas vehicle-treated horn showed normally attached embryo (Fig. 3B). Further, we also checked the spatiotemporal expression of microtubule polymerization-related proteins during peri-implantation phase (D5). The immunoflourecence imaging confirmed that the expression ofα -tubulin, β-tubulin and TPPP proteins were highly expressed in the luminal (LE) and glandular epithelium (GE) of vehicle- Figure 4 Uterine receptivity was distorted by intrauterine nocodazole treatment in mice. (A) Immunoblotting of receptivity markers (LIF, treated uterus, whereas at peri-implantation phase (D5), HoxA10, Integrin β-3, IHH and WNT4) at peri-implantation stage approximately 50% reduction in expression of TPPP and (D5, 08:00 h). GAPDH was used as a control to correct for loading α-/β-tubulin was observed in nocodazole-treated uterus (right panel). Densitometric quantitation of protein expression levels as compared to vehicle-treated uterus (Fig. 3C and D). is shown as fold changes (right panel). The results are presented as Further, we analyzed the expression of receptivity mean ± s.e.m. of three independent experiments. Three replicates markers in mouse uterus (D5). The protein expression (individual animal as a replicate) were used in each group. P values: a b c d of embryo implantation/receptivity markers (LIF, P < 0.001, P < 0.01, P < 0.05 and P > 0.05 vs vehicle control. (B) TEM of the luminal epithelium showing clear pinopodes (P) on D5 in HoxA10, Integrin β-3, IHH and WNT4) was found to the vehicle- and distorted in nocodazole-treated horn. Three be drastically decreased in uteri of nocodazole-treated replicates (individual animal as a replicate) were used in each group. group as compared to vehicle-treated group (Fig. 4A). (C) Flow cytometric analysis of apoptosis in vehicle- and nocodazole- The densitometric analysis showed that nocodazole treated cells stained with Annexin-V/PI(AV+/PI -intact cells; AV−/PI+ treatment caused reduction in LIF by ~0.3-fold -nonviable/necrotic cells; AV+/PI− and AV+/PI+ –apoptotic cells). Representative images of flow cytometry of treated cells are shown in (P < 0.001), in HoxA10 by ~0.4-fold (P < 0.001), in Integrin 3 by ~0.2-fold (P 0.001), in IHH and WNT4 the upper panel and the percentage of apoptosis with mean ± s.e.m. is β < shown in the lower panel. P values: aP < 0.001, bP < 0.01, cP < 0.05 by ~0.5-fold (P 0.01) (Fig. 4A). These results showed d < and P > 0.05 vs vehicle-treated group. Staurosporin (1 μM) was used that MT depolymerization inhibits uterine receptivity as a positive control. Three replicates (individual animal as a in mouse. replicate) were used in each group. https://rep.bioscientifica.com Reproduction (2019) 158 47–59

Downloaded from Bioscientifica.com at 09/26/2021 07:57:48AM via free access 54 V Shukla and others of Annexin-V/PI staining after 48 h of nocodazole treatment. Nocodazole significantly increased the number of apoptotic cells (by ~18%) in primary mouse EECs (P < 0.01) as compared to vehicle-treated cells (Fig. 4C), whereas 78% cells were found to be live. These results indicated that only a mild induction of apoptosis is caused by microtubule depolymerization in mouse EECs.

Microtubule depolymerization inhibits WNT4/CaMKIIα signaling during peri-implantation in mice WNT4 has been suggested to play a crucial role in the embryo implantation and is also known to non-canonically regulate calcium signaling, (Li et al. 2007, Angers & Moon 2009). In our previous experiment on uterine receptivity, nocodazole caused suppression in WNT4 protein expression (Fig. 4A). Thus, co-immunoprecipitation studies were performed to analyze the effect of nocodazole on WNT4 and Fz-2 interaction. Results indicated that nocodazole significantly decreased the interaction of WNT4/Fz-2 during peri-implantation (Fig. 5A). Further, we assessed the downstream cascades and found reduction in the expression of Fz-2 by ~0.55-fold (P < 0.01), DVL-1 by ~0.4-fold (P 0.001), PKC by ~0.5-fold (P 0.001), Figure 5 Microtubule depolymerization attenuates WNT4/CaMKIIα < α < signaling during peri-implantation, D5. (A) Interaction between calmodulin by ~0.4-fold, CaMKIIα and calcineurin by WNT4 ligand and Fz-2 was determined by co-immunoprecipitation. ~0.5-fold (P < 0.001), transcription factor NFAT by ~0.4- Tissue lysates were immunoprecipitated with anti-Fz-2 and fold (P < 0.001), NF-κB p50 by ~0.6-fold (P < 0.001) subsequently immunoblotted with anti-WNT4. NC is the negative and in NF-κB p65 by ~0.5-fold (P < 0.001) as observed control in which cell lysate was incubated with IgG instead of by densitometry analysis (Fig. 5B and C). Thus, our anti-Fz-2. Representative blots are shown in the left panel and results showed that MT depolymerization reduced densitometric quantitation of relative protein expression levels are a the WNT4-mediated CaMKII signaling during peri- shown as fold changes in the right panel. P values: P < 0.001, α bP 0.01, cP 0.05 and dP 0.05 vs vehicle. (B) Effect of nocodazole implantation phase. < < > the WNT4/CaMKIIα signaling and its downstream effectors calcineurin and NFAT during peri-implantation. Three replicates Microtubule depolymerization increased the (individual animal as a replicate) were used in each group. (C) MT +2 depolymerization suppressed NF-κB protein expression during intracellular Ca level in mouse EECs peri-implantation. Each experiment was performed three times with MT stability is related to calcium homeostasis (Ciani three tissue samples. GAPDH was used as a control to correct for et al. 2004, Salinas 2007), thus, our next aim was to loading. Representative blots are shown in the left panel and densitometric quantitation of protein expression levels are shown as assess the effect of microtubule depolymerization on fold changes in the right panel. P values: aP 0.001, bP 0.01, +2 < < intracellular Ca in EECs. The results of flow cytometric cP < 0.05 and dP > 0.05 vs control. Three replicates (individual animal analysis revealed that nocodazole treatment in EECs as a replicate) were used in each group. enhanced the influx of intracellular Ca+2 (~3.5-fold; P < 0.001) as compared to that in vehicle-treated transcription factor NFAT by ~0.4-fold (P < 0.001), EECs (Fig. 6A). NF-κB p50 by ~0.5-fold (P < 0.001) and NF-κB p65 by ~0.4-fold (P < 0.001) (Fig. 6B). The NF-κB -Luc reporter gene was significantly inhibited by nocodazole or Microtubule depolymerization suppressed CaMKII - α cyclosporine A treatment or CaMKII siRNA knockdown mediated NF- B and NFAT expression and inhibited α κ (Fig. 6C). Overall, these results consistently defined that PGE2/PGF2α release microtubule depolymerization inhibits WNT4/CaMKIIα Further, to assess whether calcineurin and downstream signaling and suppressed the transcription factor NFAT proteins NFAT and NF-κB are regulated via CaMKIIα, we and NF-κB via CaMKIIα during peri-implantation functionally blocked the CaMKIIα through siRNA and in mice. measured the expression of these downstream cascades. Previous reports confirmed that prostaglandins The immunoblotting analysis showed the reduction in regulate Ca+2 homeostasis (Harks et al. 2003, Sales & CaMKIIα and calcineurin by ~0.4-fold (P < 0.001), Jabbour 2003) and PGE2 and PGF2α have been reported

Reproduction (2019) 158 47–59 https://rep.bioscientifica.com

Downloaded from Bioscientifica.com at 09/26/2021 07:57:48AM via free access MT depolymerization inhibits implantation 55

Figure 6 Microtubule depolymerization attenuates WNT4/CaMKIIα singnaling with inhibition of NFAT and NF-κB in EECs. (A) Effect of microtubule depolymerization on cytosolic free Ca+2. Fluorescence was measured from EECs loaded with 2 μM Fluo-3-AM dye. Exposure of primary mouse EECs to 5 μM ionomycin induced a Ca+2 transient. Each experiment was performed three times with three different samples. (B) Nocodazole (300 nM)- or cyclosporine A (0.5 μM)- treatment or CaMKIIα (30 nM) siRNA in EECs suppressed CaMKIIα, calcineurin, NFAT and NF-κB expression during peri-implantation. Each experiment was performed three times with three different samples. GAPDH was used as a control to correct for loading. Representative blots are shown in the left panel and densitometric quantitation of protein expression levels are shown as fold changes in the right panel. (C) Transcriptional activation of the NF-κB promoter in primary mouse EECs transiently transfected with pNF-κB-luc reporter plasmids or incubated with nocodazole (300 nM) or cyclosporine A (calcineurin inhibitor; 0.5 μM) or transfected with CaMKIIα siRNA (30 nM). pRL-luc plasmid was used as internal control and fold change of normalized relative luciferase activity was determined. Each experiment was performed three times with three different samples. (D) Mouse EECs were treated with nocodazole (300 nM) or cyclosporine A (0.5 μM) or transfected with CaMKIIα siRNA (30 nM). Conditioned media were collected to measure PGE2 levels and PGF2α levels by ELISA. Three replicates (individual animal as a replicate) were used in each group. Data are presented as mean ± s.e.m. P values: aP < 0.001, bP < 0.01, cP < 0.05 and dP > 0.05 vs control group. https://rep.bioscientifica.com Reproduction (2019) 158 47–59

Downloaded from Bioscientifica.com at 09/26/2021 07:57:48AM via free access 56 V Shukla and others to play a significant role in embryo implantation et al. 2015). PR-A is the leading functional isoform in the (Matsumoto et al. 2001, Parent et al. 2003). Thus, we uterus (Large & DeMayo 2012). In our study, microtubule sought to examine whether MT depolymerization depolymerization suppressed the expression of ERα, PR-A impaired PGE2 and PGF2α biosynthesis via CaMKIIα in and PR-B indicates that microtubule depolymerization mouse EECs. For this, we determined PGE2 and PGF2α can affect the action of ovarian hormones which may levels by ELISA. A significant decrease in level of PGE2 be responsible for suppressed levels of TPPP. Previous and PGF2α was observed in all three conditions i. e. after reports indicate that nocodazole does not significantly nocodazole (P < 0.001) or cyclosporine A (P < 0.001) alter the expression of JNK and STAT3 in different types treatment or CaMKIIα siRNA knockdown (P < 0.01) of cells (Chen et al. 2002, Zhang et al. 2002, Shi et al. (Fig. 6D). 2006, Zou et al. 2008, Guo et al. 2012). Our data clearly indicated that microtubule depolymerization by nocodazole did not alter the expression of JNK and Discussion STAT3; however, it affected specifically the microtubule The physiological, cellular, micro-structural and polymerization-associated proteins and receptivity molecular mechanisms of uterine receptivity are markers in the uterus. Our results also indicated that the implicated in malfunction of embryo implantation in effect of nocodazole was primarily due to microtubule mammals. The ability of the cytoskeleton to deform depolymerization and not because of cell death. and reform is critical for cellular differentiation in the The successful implantation requires simultaneous uterine endometrium at the time of embryo invasion communication between LE and the synchronous (Paule et al. 2010). More importantly, understanding the development of the endometrial stroma in mouse mechanism(s) of microtubule dynamics is of particular (Wang et al. 2015, Lindsay et al. 2016). The LE plasma interest in the context of embryo implantation. Herein, membrane transformation and cytoskeletal changes are we explored the functional significance of microtubule essential for uterine receptivity in rodents and mammals polymerization in the embryo implantation using mouse (Murphy 2004, Montazeri et al. 2015). Previous report as an experimental model. on comprehensive analysis suggested that spindle- Our study revealed that microtubule polymerization microtubules-related proteins CDC2, KIF11, PRC1 has functional significance in regulating crucial cellular and KIF4A are important for receptivity in human functions of the endometrium. TPPP was found to be endometrium (Diaz-Gimeno et al. 2011). The failure expressed throughout the window of implantation of embryo attachment in nocodazole-treated mouse and was highly detected on D5 of pregnancy. During uterus was due to altered cellular structure and failed menstrual cycle, the differentiation and growth of the remodeling of the uterine LE cells into a state that is not endometrium is controlled by E2 and P4 (Wang & Dey receptive for embryo implantation. The epithelial plasma 2006). The uterine response to E2 is highly regulated membrane transformation is a hallmark event for uterine during the window of implantation in mouse and receptivity acquisition in various species including the human (Lee et al. 2010). To locate whether uterine TPPP human (Murphy 2004). Here, our study demonstrated and ovarian hormones were correlated, the delayed that depolymerization of MTs via nocodazole leads to implantation experiment was done in ovariectomized LE modification and distorted pinopodes morphology mice. Our findings indicated that E2 upregulates in mouse uterus on D5 of pregnancy. The expression TPPP, whereas P4 could adversely affect its regulation. of receptivity markers (LIF, HoxA10, Integrin β-3, IHH Nocodazole disrupts MTs by binding to β-tubulin and WNT4) was also found to be decreased in MT and preventing the formation of one of the two inter- depolymerized group. Further, nocodazole treatment chain disulfide linkages, thus inhibiting MT dynamics prevented the blastocyst attachment and the adhesion (Mitchison & Kirschner 1984). Hence, we used reaction to mouse EECs. Although the contribution of nocodazole to assess the effect of MT depolymerization embryonic MTs in successful implantation cannot be in peri-implantation events. The reduction in number ruled out, a significant contribution is made by EECs of blastocysts on D5 was observed in mice receiving which basically undergoes the acquisition of receptive nocodazole treatment at pre-implantation phase in utero. state and is required for successful blastocyst adhesion In spatiotemporal studies, the increased expression of and attachment. TPPP and α/β-tubulin was seen in LE cells and stromal In pregnant mouse uterus, WNT4 expression is cells in peri-implantation period (D5) which was found increased in the stroma surrounding the blastocyst with to be suppressed in nocodazole-treated mice uteri, the onset of attachment reaction at midnight of D4, and which indicated the involvement of MT polymerization further enhanced on D5 and beyond upto D7 (Franco at the time of implantation. Steroid receptors ER and PR et al. 2011). The reduction in uterine glands may take have fundamental role in maintaining and regulating charge for the impaired implantation in Wnt4-deficient the embryo implantation (Bulun 2017). Studies targeting female mice (Li et al. 2007). Previous reports confirmed PR-A or PR-B showed specific roles of each PR isoform that WNT regulates MT dynamics through canonical in mediating P4 activities on the murine uterus (Patel or non-canonical WNT signaling pathway in monkey

Reproduction (2019) 158 47–59 https://rep.bioscientifica.com

Downloaded from Bioscientifica.com at 09/26/2021 07:57:48AM via free access MT depolymerization inhibits implantation 57 kidney and mouse neuronal cells (Ciani et al. 2004, of PGE2 and PGF2α were observed in EECs under the Salinas 2007). In the current study, we observed that MT condition of MT depolymerization. CaMKIIα knockdown depolymerization caused inhibition in the receptivity and cyclosporine A treatment also suppressed the marker WNT4, which is the upstream target of WNT secretory levels of PGE2 and PGF2α in the EECs. signaling. which led to suppressed WNT4 and Fz-2 The suppressed levels of PGE2 and PGF2α might be interaction as evident by co-IP experiments. responsible for the failure of uterine receptivity as well In the non-canonical WNT pathway, binding of as implantation. Overall, these findings proposed that WNTs to Fz receptors increases intracellular calcium MT depolymerization inhibits WNT4/CaMKIIα signaling levels and activates PKC in a DVL-dependent manner during peri-implantation stage in mice. Although the (Sheldahl et al. 2003). Ca+2 homeostasis is the essential specific role of these microtubule-dependent calcineurin phenomenon during blastocyst implantation and the signaling remains to be validated and investigated further, maintenance of optimum Ca+2 levels is important at these findings do support our notion on the importance of maternal–fetal interface (Suzuki et al. 2008). In our cytoskeletal proteins in the process of implantation. study, mouse EECs after nocodazole or cyclosporine In conclusion, MT depolymerization suppressed the A (calcineurin inhibitor) treatment or CaMKIIα siRNA WNT4/CaMKIIα signaling, decreased prostaglandins knockdown showed an increase in cytoplasmic Ca+2 PGE2 and PGF2α in EECs subsequently leading to level. Also, MT depolymerization notably reduced the implantation failure in mice. The findings of this study expression of calcium-dependent proteins calmodulin, substantiate the importance of MT polymerization and CaMKIIα and calcineurin during peri-implantation associated proteins upholding the complex mechanism phase and also in EECs in mouse. of embryo implantation. Future studies on such proteins To explore the WNT4 downstream cascades, we in human clinical samples might aid in the understanding functionally knocked down the CaMKIIα and found of biological mechanisms involved in female fertility and an inhibition in the expression of calcineurin, NFAT will also provide cues for development of newer strategies and NF-κB. Further, in the presence of cyclosporine for treatment of endometrium-based infertility in women. A, attenuation of transcription factor NFAT along with suppression of NF-κB, was observed. Besides, NF-κB- luc transcriptional activation was significantly inhibited Declaration of interest in EECs when treated with nocodazole or transfected The authors declare that there is no conflict of interest that with CaMKIIα siRNA or cyclosporine A. Previous reports could be perceived as prejudicing the impartiality of the show that calcineurin, NFAT and NF-κB are expressed research reported. in rodents, in human endometrium and first-trimester human trophoblast (Ponce et al. 2009, Abraham et al. 2012, Celik et al. 2013, Wang et al. 2013). The elevations Funding 2+ of decidual markers induced by Ca have been suggested This work was financially supported by CSIR network project to be mediated partially through the calcineurin/NFAT BSC0101. This is CSIR-CDRI communication number 9830. pathway (Maldonado-Perez et al. 2007, Macdonald et al. 2011, Abraham et al. 2012). Thus, our results indicated that MT depolymerization suppresses implantation by Author contribution statement inhibiting CaMKIIα-mediated calcineurin/NFAT/NF-κB A D conceptualized the study. A D and V S designed and signaling. However, the suppression of WNT4 can also executed the experiments. V S, J B K, P P, R K and P K A affect canonical pathway; hence, it will be interesting to performed the experiments. K M analyzed TEM. A D and V evaluate further the canonical signaling molecules which S analyzed the entire data and drafted the manuscript. All may also be important in the regulation of receptivity authors have approved the final version of the manuscript. markers in the uterus. Defective endometrial PG synthesis has been linked with repeated implantation failure in patients undergoing Acknowledgments in vitro fertilization (Achache et al. 2010). Interaction The authors thank Dr Kavita Singh and Ms Garima Pant, SAIF- of PGE2 with the EP1 receptor mobilizes intracellular facility, CSIR-CDRI for help in confocal microscopy and TEM, calcium and PGF receptor activation is coupled to respectively. V S is the recipient of Senior Research Fellowship 2+ phospholipase C-IP3 pathway and Ca mobilization from Indian Council of Medical Research, New Delhi and is (Harks et al. 2003, Sales & Jabbour 2003). PGE2 and PhD scholar of AcSIR, CSIR-CDRI campus, Lucknow. PGF2α concentrations are also increased in the human endometrial fluid during the window of implantation References (Vilella et al. 2013). Furthermore, in rodents and bovines, PGE and PGF2 have been reported to play an important Abraham F, Sacerdoti F, De León R, Gentile T & Canellada A 2012 2 α Angiotensin II activates the calcineurin/NFAT signaling pathway and role in blastocyst implantation (Matsumoto et al. 2001, induces cyclooxygenase-2 expression in rat endometrial stromal cells. Parent et al. 2003). In our study, the decreased levels PLoS ONE 7 e37750. (https://doi.org/10.1371/journal.pone.0037750) https://rep.bioscientifica.com Reproduction (2019) 158 47–59

Downloaded from Bioscientifica.com at 09/26/2021 07:57:48AM via free access 58 V Shukla and others

Achache H, Tsafrir A, Prus D, Reich R & Revel A 2010 Defective oscillations in monolayers of gap junctionally coupled NRK fibroblasts. endometrial prostaglandin synthesis identified in patients with repeated Pflugers Archiv 447 78–86. (https://doi.org/10.1007/s00424-003-1126-8) implantation failure undergoing in vitro fertilization. Fertility and Sterility Huang J, Qin H, Yang Y, Chen X, Zhang J, Laird S, Wang CC, Chan TF 94 1271–1278. (https://doi.org/10.1016/j.fertnstert.2009.07.1668) & Li TC 2017 A comparison of transcriptomic profiles in endometrium Angers S & Moon RT 2009 Proximal events in Wnt signal transduction. during window of implantation between women with unexplained Nature Reviews: Molecular 10 468–477. (https://doi. recurrent implantation failure and recurrent miscarriage. Reproduction org/10.1038/nrm2717) 153 749–758. (https://doi.org/10.1530/REP-16-0574) Bazer FW, Spencer TE, Johnson GA, Burghardt RC & Wu G 2009 Isshiki K, Hirase T, Matsuda S, Miyamoto K, Tsuji A & Yuasa K 2015 Comparative aspects of implantation. Reproduction 138 195–209. Death-associated protein kinase 2 mediates nocodazole-induced (https://doi.org/10.1530/REP-09-0158) apoptosis through interaction with tubulin. Biochemical and Biophysical Bhutada S, Basak T, Savardekar L, Katkam RR, Jadhav G, Metkari SM, Research Communications 468 113–118. (https://doi.org/10.1016/j. Chaudhari UK, Kumari D, Kholkute SD, Sengupta S et al. 2014 High bbrc.2015.10.151) mobility group box 1 (HMGB1) protein in human uterine fluid and its Kaczyński P, Baryla M, Goryszewska E, Bauersachs S & WacŁawik A 2018 relevance in implantation. Human Reproduction 29 763–780. (https:// Prostaglandin F2α promotes embryo implantation and development in doi.org/10.1093/humrep/det461) the pig. Reproduction 156 405–419. (https://doi.org/10.1530/REP-18- Bulun SE 2017 Steroids, cytokines, and implantation. Endocrinology 158 0225) 1575–1576. (https://doi.org/10.1210/en.2017-00407) Kathuria M, Bhattacharjee A, Sashidhara KV, Singh SP & Mitra K Celik O, Celik E, Turkcuoglu I, Yilmaz E, Ulas M, Simsek Y, Karaer A, 2014 Induction of mitochondrial dysfunction and oxidative stress in Celik N, Aydin NE, Ozerol I et al. 2013 Surgical removal of endometrioma Leishmaniadonovani by orally active clerodanediterpene. Antimicrobial decreases the NF-kB1 (p50/105) and NF-kB p65 (Rel A) expression in the Agents and Chemotherapy 58 5916–5928. (https://doi.org/10.1128/ eutopic endometrium during the implantation window. Reproductive AAC.02459-14) Sciences 20 762–770. (https://doi.org/10.1177/1933719112466307) Kaushal JB, Sankhwar P, Kumari S, Popli P, Shukla V, Hussain MK, Cha J, Sun X & Dey SK 2012 Mechanisms of implantation: strategies for Hajela K & Dwivedi A 2017 The regulation of Hh/Gli1 signaling cascade successful pregnancy. Nature Medicine 18 1754–1767. (https://doi. involves GSK3β-mediated mechanism in estrogen-derived endometrial org/10.1038/nm.3012) hyperplasia. Scientific Reports 7 6557. (https://doi.org/10.1038/s41598- Chen W, White MA & Cobb MH 2002 Stimulus-specific requirements 017-06370-1) for MAP3 kinases in activating the JNK pathway. Journal of Biological Kaushal JB, Popli P, Sankhwar P, Shukla V & Dwivedi A 2018 Sonic Chemistry 277 49105–49110. (https://doi.org/10.1074/jbc.M204934200) hedgehog protects endometrial hyperplasial cells against oxidative stress Choi HJ, Fukui M & Zhu BT 2011 Role of cyclin B1/Cdc2 up-regulation via suppressing mitochondrial fission protein dynamin-like GTPase in the development of mitotic prometaphase arrest in human breast (Drp1). Free Radical Biology and Medicine 129 582–599. (https://doi. cancer cells treated with nocodazole. PLoS ONE 6 e24312. (https://doi. org/10.1016/j.freeradbiomed.2018.10.427) org/10.1371/journal.pone.0024312) Lagos-Cabré R & Moreno RD 2008 Mitotic, but not meiotic, oriented cell Ciani L, Krylova O, Smalley MJ, Dale TC & Salinas PC 2004 A divergent divisions in rat spermatogenesis. Reproduction 135 471–478. (https:// canonical WNT-signaling pathway regulates microtubule dynamics: doi.org/10.1530/REP-07-0389) dishevelled signals locally to stabilize microtubules. Journal of Cell Large MJ & DeMayo FJ 2012 The regulation of embryo implantation Biology 164 243–253. (https://doi.org/10.1083/jcb.200309096) and endometrial decidualization by progesterone receptor signaling. Diaz-Gimeno P, Horcajadas JA, Martinez-Conejero JA, Esteban FJ, Molecular and Cellular Endocrinology 358 155–165. (https://doi. Alama P, Pellicer A & Simon C 2011 A genomic diagnostic tool for org/10.1016/j.mce.2011.07.027) human endometrial receptivity based on the transcriptomic signature. Lee DK, Kurihara I, Jeong JW, Lydon JP, DeMayo FJ, Tsai MJ & Tsai SY 2010 Fertility and Sterility 95 50.e1–60.e1. (https://doi.org/10.1016/j. Suppression of ERalpha activity by COUP-TFII is essential for successful fertnstert.2010.04.063) implantation and decidualization. Molecular Endocrinology 24 Domínguez F, Garrido-Gómez T, López JA, Camafeita E, Quiñonero A, 930–940. (https://doi.org/10.1210/me.2009-0531) Pellicer A & Simón C 2009 Proteomic analysis of the human Li Q, Kannan A, Wang W, DeMayo FJ, Taylor RN, Bagchi MK & Bagchi IC receptive versus non-receptive endometrium using differential in-gel 2007 Bone morphogenetic protein 2 functions via a conserved signaling electrophoresis and MALDI-MS unveils stathmin 1 and annexin A2 as pathway involving Wnt4 to regulate uterine decidualization in the mouse differentially regulated. Human Reproduction 24 2607–2617. (https:// and the human. Journal of Biological Chemistry 282 31725–31732. doi.org/10.1093/humrep/dep230) (https://doi.org/10.1074/jbc.M704723200) Franco HL, Dai D, Lee KY, Rubel CA, Roop D, Boerboom D, Jeong JW, Liang XH, Deng WB, Li M, Zhao ZA, Wang TS, Feng XH, Cao YJ, Lydon JP, Bagchi IC, Bagchi MK et al. 2011 WNT4 is a key regulator Duan EK & Yang ZM 2014 Egr1 protein acts downstream of estrogen- of normal postnatal uterine development and progesterone signaling leukemia inhibitory factor (LIF)-STAT3 pathway and plays a role during during embryo implantation and decidualization in the mouse. FASEB implantation through targeting Wnt4. Journal of Biological Chemistry Journal 25 1176–1187. (https://doi.org/10.1096/fj.10-175349) 289 23534–23545. (https://doi.org/10.1074/jbc.M114.588897) Fujimoto GI, Saldana LR & Gaskin F 1976 Uterine tubulin production during Lindsay LA, Dowland SN & Murphy CR 2016 Uterine focal adhesions early pregnancy in the rabbit. Endocrine Research Communications 3 retained at implantation after rat ovarian hyperstimulation. Reproduction 219–229. (https://doi.org/10.3109/07435807609056902) 152 753–763. (https://doi.org/10.1530/REP-16-0331) Green CJ, Fraser ST & Day ML 2015 -like growth factor 1 increases Lopez BJ & Valentine MT 2015 Molecular control of stress transmission apical in blastocysts to increase blastocyst attachment to in the microtubule cytoskeleton. Biochimica and Biophysica Acta 1853 endometrial epithelial cells in vitro. Human Reproduction 30 284–298. 3015–3024. (https://doi.org/10.1016/j.bbamcr.2015.07.016) (https://doi.org/10.1093/humrep/deu309) Macdonald LJ, Sales KJ, Grant V, Brown P, Jabbour HN & Catalano RD 2011 Guo X, Zhang X, Li Y, Guo Y, Wang J, Li Y, Shen B, Sun D & Zhang J 2012 Prokineticin 1 induces Dickkopf 1 expression and regulates cell proliferation Nocodazole increases the ERK activity to enhance MKP-1 expression and decidualization in the human endometrium. Molecular Human which inhibits p38 activation induced by TNF-α. Molecular and Cellular Reproduction 17 626–636. (https://doi.org/10.1093/molehr/gar031) Biochemistry 364 373–380. (https://doi.org/10.1007/s11010-012-1239-5) Maldonado-Perez D, Evans J, Denison F, Millar RP & Jabbour HN Gupta K, Sirohi VK, Kumari S, Shukla V, Manohar M, Popli P & Dwivedi A 2007 Potential roles of the prokineticins in reproduction. Trends in 2018 Sorcin is involved during embryo implantation via activating Endocrinology and Metabolism 18 66–72. (https://doi.org/10.1016/j. VEGF/PI3K/Akt pathway in mice. Journal of Molecular Endocrinology 60 tem.2006.12.002) 119–132. (https://doi.org/10.1530/JME-17-0153) Manohar M, Khan H, Sirohi VK, Das V, Agarwal A, Pandey A, Siddiqui WA Hantak AM, Bagchi IC & Bagchi MK 2014 Role of uterine stromal-epithelial & Dwivedi A 2014a Alteration in endometrial proteins during early- and crosstalk in embryo implantation. International Journal of Developmental mid-secretory phases of the cycle in women with unexplained infertility. Biology 58 139–146. (https://doi.org/10.1387/ijdb.130348mb) PLoS ONE 9 e111687. (https://doi.org/10.1371/journal.pone.0111687) Harks EG, Scheenen WJ, Peters PH, van Zoelen EJ & Theuvenet AP Manohar M, Khan H, Shukla V, Das V, Agarwal A, Pandey A & Siddiqui WA 2003 Prostaglandin F2α induces unsynchronized intracellular calcium 2014b Proteomic identification and analysis of human endometrial

Reproduction (2019) 158 47–59 https://rep.bioscientifica.com

Downloaded from Bioscientifica.com at 09/26/2021 07:57:48AM via free access MT depolymerization inhibits implantation 59

proteins associated with unexplained infertility. Journal of Proteomics stromal cells and impairs decidualization. Journal of Endocrinology 240 and Bioinformatics 07 359–366. (https://doi.org/10.4172/jpb.1000340) 417–429. (https://doi.org/10.1530/JOE-18-0459) Matsumoto H, Ma WG, Smalley W, Trzaskos J, Breyer RM & Dey SK 2001 Signoretto E, Honisch S, Briglia M, Faggio C, Castagna M & Lang F Diversification of cyclooxygenase-2-derived prostaglandins in ovulation 2016 Nocodazole Induced suicidal death of human erythrocytes. and implantation. Biology of Reproduction 64 1557–1565. (https://doi. Cellular Physiology and Biochemistry 38 379–392. (https://doi. org/10.1095/biolreprod64.5.1557) org/10.1159/000438638) Maurya VK, Jha RK, Kumar V, Joshi A, Chadchan S, Mohan JJ & Laloraya M Suzuki Y, Kovacs CS, Takanaga H, Peng JB, Landowski CP & Hediger MA 2013 Transforming growth factor-beta 1 (TGF-B1) liberation from its latent 2008 Calcium channel TRPV6 is involved in murine maternal-fetal complex during embryo implantation and its regulation by estradiol calcium transport. Journal of Bone and Mineral Research 23 1249–1256. in mouse. Biology of Reproduction 89 84. (https://doi.org/10.1095/ (https://doi.org/10.1359/jbmr.080314) biolreprod.112.106542) Tamura K, Hara T, Yoshie M, Irie S, Sobel A & Kogo H 2003 Enhanced Mitchison T & Kirschner M 1984 Dynamic instability of microtubule expression of uterine stathmin during the process of implantation and growth. Nature 312 237–242. (https://doi.org/10.1038/312237a0) decidualization in rats. Endocrinology 144 1464–1473. (https://doi. Montazeri M, Sanchez-Lopez JA, Caballero I, Maslehat Lay N, Elliott S, org/10.1210/en.2002-220834) Lopez-Martin S, Yáñez-Mó M & Fazeli A 2015 Activation of Toll- Tsai CY, Chou CK ,Yang CW, Lai YC, Liang CC, Chen CM & Tsai TF like receptor 3 reduces actin polymerization and adhesion molecule 2008 Hurp deficiency in mice leads to female infertility caused by an expression in endometrial cells, a potential mechanism for viral-induced implantation defect. Journal of Biological Chemistry 283 26302–26306. implantation failure. Human Reproduction 30 893–905. (https://doi. (https://doi.org/10.1074/jbc.C800117200) org/10.1093/humrep/deu359) Vilella F, Ramirez L, Berlanga O, Martinez S, Alama P, Meseguer M, Murphy CR 2004 Uterine receptivity and the plasma membrane Pellicer A & Simón C 2013 PGE2 and PGF2α concentrations in human transformation. Cell Research 14 259–267. (https://doi.org/10.1038/ endometrial fluid as biomarkers for embryonic implantation. Journal sj.cr.7290227) of Clinical Endocrinology and Metabolism 98 4123–4132. (https://doi. Parent J, Villeneuve C & Fortier MA 2003 Evaluation of the contribution of org/10.1210/jc.2013-2205) cyclooxygenase 1 and cyclooxygenase 2 to the production of PGE2 and Wang H & Dey SK 2006 Roadmap to embryo implantation: clues from PGF2 alpha in epithelial cells from bovine endometrium. Reproduction mouse models. Nature Reviews: Genetics 7 185–199. (https://doi. 126 539–547. (https://doi.org/10.1530/rep.0.1260539) org/10.1038/nrg1808) Patel B, Elguero S, Thakore S, Dahoud W, Bedaiwy M & Mesiano S Wang SC, Tang ChL, Piao HL, Zhu R, Sun Ch, Tao Y, Fu Q, Li DJ & Du MR 2015 Role of nuclear progesterone receptor isoforms in uterine 2013 Cyclosporine A promotes in vitro migration of human first-trimester pathophysiology. Human Reproduction Update 21 155–173. (https:// trophoblasts via MAPK/ERK1/2-mediated NF-κB and Ca2+/calcineurin/ doi.org/10.1093/humupd/dmu056) NFAT signaling. Placenta 34 374–380. (https://doi.org/10.1016/j. Paule SG, Airey LM, Li Y, Stephens AN & Nie G 2010 Proteomic approach placenta.2013.01.009) identifies alterations in cytoskeletal remodelling proteins during Wang Y, Zhu L, Kuokkanen S & Pollard JW 2015 Activation of protein decidualization of human endometrial stromal cells. Journal of Proteome synthesis in mouse uterine epithelial cells by estradiol-17β is mediated Research 9 5739–5747. (https://doi.org/10.1021/pr100525a) by a PKC-ERK1/2-mTOR signaling pathway. PNAS 112 E1382–E1391. Ponce C, Torres M, Galleguillos C, Sovino H, Boric MA, Fuentes A (https://doi.org/10.1073/pnas.1418973112) & Johnson MC 2009 Nuclear factor κB pathway and interleukin-6 Watanabe S, Shioi G, Furuta Y & Goshima G 2016 Intra-spindle are affected in eutopic endometrium of women with endometriosis. microtubule assembly regulates clustering of microtubule-organizing Reproduction 137 727–737. (https://doi.org/10.1530/REP-08-0407) centers during early mouse development. Cell Reports 15 54–60. (https:// Popli P, Sirohi VK, Manohar M, Shukla V, Kaushal JB, Gupta K & Dwivedi A doi.org/10.1016/j.celrep.2016.02.087) 2015 Regulation of cyclooxygenase-2 expression in rat oviductal Wu GJ, Simerly C, Zoran SS, Funte LR & Schatten G 1996 Microtubule epithelial cells: evidence for involvement of GPR30/Src kinase-mediated and chromatin dynamics during fertilization and early development EGFR signaling. Journal of Steroid Biochemistry and in rhesus monkeys, and regulation by intracellular calcium ions. 154 130–141. (https://doi.org/10.1016/j.jsbmb.2015.07.019) Biology of Reproduction 55 260–270. (https://doi.org/10.1095/ Sales KJ & Jabbour HN 2003 Cyclooxygenase enzymes and prostaglandins biolreprod55.2.260) in pathology of the endometrium. Reproduction 126 559–567. (https:// Yan LY, Huang JC, Zhu ZY, Lei ZL, Shi LH, Nan CL, Zhao ZJ, OuYang YC, doi.org/10.1530/rep.0.1260559) Song XF, Sun QY et al. 2006 NuMA distribution and microtubule Salinas PC 2007 Modulation of the microtubule cytoskeleton: a role for a configuration in rabbit oocytes and cloned embryos. Reproduction 132 divergent canonical Wnt pathway. Trends in Cell Biology 17 333–342. 869–876. (https://doi.org/10.1530/rep.1.01224) (https://doi.org/10.1016/j.tcb.2007.07.003) Zhang H, Shi X, Zhang QJ, Hampong M, Paddon H, Wahyuningsih D & Sawyer HR, Abel AH jr, Mcclellan MC, Schmitz M. & Niswender GD 1979 Pelech S 2002 Nocodazole-induced p53-dependent c-Jun N-terminal Secretory granules and progesterone secretion by ovine corpus lutea in kinase activation reduces apoptosis in human colon carcinoma HCT116 vitro. Endocrinology 104 476–486. cells. Journal of Biological Chemistry 277 43648–43658. (https://doi. Sheldahl LC, Slusarski DC, Pandur P, Miller JR, Kühl M & Moon RT org/10.1074/jbc.M203214200) 2003 Dishevelled activates Ca2+ flux, PKC, and CamKII in vertebrate Zhang S, Ong CN & Shen HM 2004 Critical roles of intracellular thiols embryos. Journal of Cell Biology 161 769–777. (https://doi.org/10.1083/ and calcium in parthenolide-induced apoptosis in human colorectal jcb.200211094) cancer cells. Cancer Letters 208 143–153. (https://doi.org/10.1016/j. Shi X, Zhang H, Paddon H, Lee G, Cao X & Pelech S 2006 Phosphorylation canlet.2003.11.028) of STAT3 serine-727 by cyclin-dependent kinase 1 is critical for Zou T, Ouyang L, Chen L, Dong W, Qiao H, Liu Y & Qi Y 2008 The nocodazole-induced mitotic arrest. Biochemistry 45 5857–5867. role of microtubule-associated protein 1S in SOCS3 regulation of IL-6 (https://doi.org/10.1021/bi052490j) signaling. FEBS Letters 582 4015–4022. (https://doi.org/10.1016/j. Shukla V, Chandra V, Sankhwar P, Popli P, Kaushal JB, Sirohi VK & febslet.2008.10.055) Dwivedi A 2015 Phytoestrogen genistein inhibits EGFR/PI3K/NF-kB activation and induces apoptosis in human endometrial hyperplasial cells. RSC Advances 5 56075–56085. (https://doi.org/10.1039/ C5RA06167A) Shukla V, Popli P, Kaushal JB, Gupta K & Dwivedi A 2018 Uterine TPPP3 plays important role in embryo implantation via modulation of β- Received 4 December 2018 catenin. Biology of Reproduction 99 982–999. (https://doi.org/10.1093/ First decision 3 January 2019 biolre/ioy136) Shukla V, Kaushal JB, Sankhwar P, Manohar M & Dwivedi A 2019 Inhibition Revised manuscript received 28 March 2019 of TPPP3 attenuates β-catenin/NF-κB/COX-2 signaling in endometrial Accepted 4 April 2019

https://rep.bioscientifica.com Reproduction (2019) 158 47–59

Downloaded from Bioscientifica.com at 09/26/2021 07:57:48AM via free access