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, Integrin-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. Reproduction (2019) 158 47–59 Introduction secretion of progesterone (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 estradiol 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 cellular differentiation 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