Wnt Signaling Pathway in Uterus of Normal and Seminal Vesicle Excised Mated Mice during Pre-implantation Window

Abstract

Introduction The importance of seminal vesicle secretion and uterine Wnt signaling for uterus preparation and embryo implantation has been described.

Materials and Methods In this study, we evaluated the gene expression of Wnt ligands (Wnt4 and Wnt5a) and their corresponding receptors (Fzd2 and Fzd6) using qRT-PCR and active β-catenin protein levels using western blotting in the uterine tissue of female mice mated with intact and seminal vesicle-excised (SVX) males during the pre-implantation window. We evaluated the association between these factors and implantation rates and embryo spacing.

Results mRNA expression of Wnt4 and Wnt5a and active β-catenin protein levels decreased from Day 1 to Day 4, but reached a peak on the fifth day of pregnancy. Fzd2 also reached its highest level on Day 5. Fzd6 expression showed a decreasing trend towards the day of implantation. Lack of seminal vesicle secretion decreased Wnt4 and Wnt5a expression on Days 1 and 5 and β-catenin levels on Day 5. There were almost no significant differences in expression levels of the Fzd2 and Fzd6 receptors between groups. There were positive and negative correlations, respectively, between implantation rates and embryo spacing and Wnt4, Wnt5a and active β-catenin in the control group, but such correlations were not observed in the SVX-mated mice.

Conclusions Significant changes occurred in the expression of several Wnt signaling members and there was a significant association between Wnt signaling and embryo implantation. Seminal vesicle secretion affects Wnt signaling in mice and consequently also affects murine embryo implantation.

Zusammenfassung

Einleitung Die Bedeutung von Bläschendrüsensekret und Wnt-Signale für die Vorbereitung der Gebärmutter auf die Implantation von Embryonen wurde bereits anderweitig beschrieben.

Material und Methoden Die Studie untersuchte die Genexpression der Wnt-Liganden Wnt 4 und Wnt 5a sowie deren Rezeptoren (Fzd2 und Fzd6) mithilfe von qRT-PCR und den aktiven β-Catenin-Proteinspiegel mithilfe von Westernblot im Gebärmuttergewebe von Mäusen während der Präimplantationszeit. Die Mäuseweibchen wurden mit Männchen verpaart, die entweder über intakte Samendrüsen verfügten oder die zuvor einer Exzision der Samendrüse (SVX) unterzogen worden waren. Die Assoziationen zwischen diesen Faktoren und den Implantationsraten bzw. dem Abstand zwischen den Embryonen wurde untersucht.

Ergebnisse Der mRNA-Expression von Wnt4 und Wnt5a und der aktive β-Catenin-Proteinspiegel sanken zwischen dem 1. und dem 4. Tag nach der Verpaarung, sie erreichten aber einen Spitzenweg am 5. Tag der Schwangerschaft. Die Expression von Fzd2 erreichte ebenfalls am 5. Tag ihren Höhepunkt. Hingegen zeigte die Expression von Fzd6 eine rückläufige Tendenz bis zum Tage der Implantation. Das Fehlen von Samenblasensekret führte zu einem Rückgang von Wnt4- und Wnt5a-Expression am 1. und 5. Tag und des β-Catenin-Spiegels am 5. Tag. Es gab keine signifikanten Unterschiede zwischen den beiden Gruppen hinsichtlich der Expression der Fzd2- und Fzd6-Rezeptoren. Es bestand jeweils eine positive bzw. negative Korrelation zwischen den Implantationsraten und den Abständen zwischen den Embryos und dem Wnt4-, Wnt5a- und β-Catenin-Spiegel in der Kontrollgruppe, aber diese Korrelation fand sich nicht bei den SVX-verpaarten Mäuseweibchen.

Schlussfolgerungen Die Expression verschiedener Wnt-Liganden hat sich signifikant verändert, und es gab ebenfalls eine signifikante Assoziation zwischen dem Wnt-Signalweg und der Implantation von Mäuseembryonen. Das Vorhandensein bzw. Fehlen von Samendrüsensekret beeinflusst den Wnt-Signalweg in Mäuseweibchen und wirkt sich daher auch auf die Implantation von Mäuseembryonen aus.

Key words

Wnt signaling - embryo implantation - seminal fluid - decidualization

Schlüsselwörter

Wnt-Signalwege - Embryoimplantation - Samenblasenflüssigkeit - Dezidualisierung

• References

• 1 Latifi Z, Fattahi A, Ranjbaran A. et al. Potential roles of metalloproteinases of endometrium-derived exosomes in embryo-maternal crosstalk during implantation. J Cell Physiol 2018; 233: 4530-4545
• 2 Zhang S, Lin H, Kong S. et al. Physiological and molecular determinants of embryo implantation. Mol Aspects Med 2013; 34: 939-980
• 3 Nejabati HR, Latifi Z, Ghasemnejad T. et al. Placental growth factor (PlGF) as an angiogenic/inflammatory switcher: lesson from early pregnancy losses. Gynecol Endocrinol 2017; 33: 668-674
• 4 Logan CY, Nusse R. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 2004; 20: 781-810
• 5 Brembeck FH, Rosário M, Birchmeier W. Balancing cell adhesion and Wnt signaling, the key role of beta-catenin. Curr Opin Genet Dev 2006; 16: 51-59
• 6 Xueling GE, Wang X. Role of Wnt canonical pathway in hematological malignancies. J Hematol Oncol 2010; 3: 33
• 7 Franco HL, Dai D, Lee KY. et al. WNT4 is a key regulator of normal postnatal uterine development and progesterone signaling during embryo implantation and decidualization in the mouse. FASEB J 2011; 25: 1176-1187
• 8 Sonderegger S, Pollheimer J, Knöfler M. Wnt signalling in implantation, decidualisation and placental differentiation–review. Placenta 2010; 31: 839-847
• 9 Zhang L, Xie Y, Dong M. et al. Wnt/beta-catenin signaling pathway regulates beta1, 4-galactosyltransferase l expression in the endometrium to affect the embryo implantation. Int J Clin Exp Pathol 2017; 10: 1756-1764
• 10 Hayashi K, Yoshioka S, Reardon SN. et al. WNTs in the neonatal mouse uterus: potential regulation of endometrial gland development. Biol Reprod 2011; 84: 308-319
• 11 Goad J, Ko Y-A, Kumar M. et al. Differential Wnt signaling activity limits epithelial gland development to the anti-mesometrial side of the mouse uterus. Dev Biol 2017; 423: 138-151
• 12 Hayashi K, Erikson DW, Tilford SA. et al. Wnt genes in the mouse uterus: potential regulation of implantation. Biol Reprod 2009; 80: 989-1000
• 13 Corda G, Sala A. Non-canonical WNT/PCP signalling in cancer: Fzd6 takes centre stage. Oncogenesis 2017; 6: e364
• 14 Lyons JP, Mueller UW, Ji H. et al. Wnt-4 activates the canonical beta-catenin-mediated Wnt pathway and binds Frizzled-6 CRD: functional implications of Wnt/beta-catenin activity in kidney epithelial cells. Exp Cell Res 2004; 298: 369-387
• 15 Daikoku T, Song H, Guo Y. et al. Uterine Msx-1 and Wnt4 signaling becomes aberrant in mice with the loss of leukemia inhibitory factor or Hoxa-10: evidence for a novel cytokine-homeobox-Wnt signaling in implantation. Mol Endocrinol 2004; 18: 1238-1250
• 16 Herington JL, Bi J, Martin JD. et al. Beta-catenin (CTNNB1) in the mouse uterus during decidualization and the potential role of two pathways in regulating its degradation. J Histochem Cytochem 2007; 55: 963-974
• 17 Tepekoy F, Akkoyunlu G, Demir R. The role of Wnt signaling members in the uterus and embryo during pre-implantation and implantation. J Assist Reprod Genet 2015; 32: 337-346
• 18 Endo M, Nishita M, Fujii M. et al. Insight into the role of Wnt5a-induced signaling in normal and cancer cells. Int Rev Cell Mol Biol 2015; 314: 117-148
• 19 Li C, Chen H, Hu L. et al. Ror2 modulates the canonical Wnt signaling in lung epithelial cells through cooperation with Fzd2. BMC Mol Biol 2008; 9: 11
• 20 Hou X, Tan Y, Li M. et al. Canonical Wnt signaling is critical to estrogen-mediated uterine growth. Mol Endocrinol 2004; 18: 3035-3049
• 21 Cha J, Bartos A, Park C. et al. Appropriate crypt formation in the uterus for embryo homing and implantation requires Wnt5a-ROR signaling. Cell Rep 2014; 8: 382-392
• 22 Matsuoka A, Kizuka F, Lee L. et al. Progesterone increases manganese superoxide dismutase expression via a cAMP-dependent signaling mediated by noncanonical Wnt5a pathway in human endometrial stromal cells. J Clin Endocrinol Metab 2010; 95: E291-E299
• 23 Robertson SA, Prins JR, Sharkey DJ. et al. Seminal fluid and the generation of regulatory T cells for embryo implantation. Am J Reprod Immunol 2013; 69: 315-330
• 24 Robertson SA, Guerin LR, Moldenhauer LM. et al. Activating T regulatory cells for tolerance in early pregnancy–the contribution of seminal fluid. J Reprod Immunol 2009; 83: 109-116
• 25 Robertson SA, Guerin LR, Bromfield JJ. et al. Seminal fluid drives expansion of the CD4+ CD25+ T regulatory cell pool and induces tolerance to paternal alloantigens in mice. Biol Reprod 2009; 80: 1036-1045
• 26 Sharkey DJ, Tremellen KP, Jasper MJ. et al. Seminal fluid induces leukocyte recruitment and cytokine and chemokine mRNA expression in the human cervix after coitus. J Immunol 2012; 188: 2445-2454
• 27 Shahnazi M, Nouri M, Mohaddes G. et al. Prostaglandin E pathway in uterine tissue during window of preimplantation in female mice mated with intact and seminal vesicle-excised male. Reprod Sci 2018; 25: 550-558 doi:10.1177/1933719117718272
• 28 Sharkey DJ, Tremellen KP, Briggs NE. et al. Seminal plasma pro-inflammatory cytokines interferon-γ (IFNG) and CXC motif chemokine ligand 8 (CXCL8) fluctuate over time within men. Hum Reprod 2017; 32: 1373-1381
• 29 Troedsson M. Uterine response to semen deposition in the mare. Proceedings of the Annual Meeting of the Society for Theriogenology; 1995 Sept 13 – 15, San Antonio, TX. 130-135
• 30 Sonomoto K, Yamaoka K, Oshita K. et al. Interleukin-1β induces differentiation of human mesenchymal stem cells into osteoblasts via the Wnt-5a/receptor tyrosine kinase-like orphan receptor 2 pathway. Arthritis Rheum 2012; 64: 3355-3363
• 31 Ge XP, Gan YH, Zhang CG. et al. Requirement of the NF-κB pathway for induction of Wnt-5A by interleukin-1β in condylar chondrocytes of the temporomandibular joint: functional crosstalk between the Wnt-5A and NF-κB signaling pathways. Osteoarthritis Cartilage 2011; 19: 111-117
• 32 Oguma K, Oshima H, Aoki M. et al. Activated macrophages promote Wnt signalling through tumour necrosis factor-alpha in gastric tumour cells. EMBO J 2008; 27: 1671-1681
• 33 Doncel GF, Anderson S, Zalenskaya I. Role of semen in modulating the female genital tract microenvironment–implications for HIV transmission. Am J Reprod Immunol 2014; 71: 564-574
• 34 North TE, Babu IR, Vedder LM. et al. PGE2-regulated wnt signaling and N-acetylcysteine are synergistically hepatoprotective in zebrafish acetaminophen injury. Proc Natl Acad Sci U S A 2010; 107: 17315-17320
• 35 Svala E, Thorfve AI, Ley C. et al. Effects of interleukin-6 and interleukin-1β on expression of growth differentiation factor-5 and Wnt signaling pathway genes in equine chondrocytes. Am J Vet Res 2014; 75: 132-140
• 36 Fattahi A, Darabi M, Farzadi L. et al. Effects of dietary omega-3 and-6 supplementations on phospholipid fatty acid composition in mice uterus during window of pre-implantation. Theriogenology 2018; 108: 97-102
• 37 Deb K, Reese J, Paria BC. Placenta and Trophoblast: Methodologies to study Implantation in Mice. In: Methods in molecular Medicine. Volume 121. Humana Press; 2006: 9-34
• 38 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001; 25: 402-408
• 39 Wang H, Dey SK. Roadmap to embryo implantation: clues from mouse models. Nat Rev Genet 2006; 7: 185-199
• 40 Mccormack J, Greenwald G. Progesterone and oestradiol-17β concentrations in the peripheral plasma during pregnancy in the mouse. J Endocrinol 1974; 62: 101-107
• 41 Nakamura T, Miyagawa S, Katsu Y. et al. Sequential changes in the expression of Wnt-and Notch-related genes in the vagina and uterus of ovariectomized mice after estrogen exposure. In Vivo 2012; 26: 899-906
• 42 Rider V, Talbott A, Bhusri A. et al. WINGLESS (WNT) signaling is a progesterone target for rat uterine stromal cell proliferation. J Endocrinol 2016; 229: 197-207
• 43 Robertson S. Seminal fluid signaling in the female reproductive tract: lessons from rodents and pigs. J Anim Sci 2007; 85: E36-E44
• 44 Robertson S, Mau V, Tremellen K. et al. Role of high molecular weight seminal vesicle proteins in eliciting the uterine inflammatory response to semen in mice. J Reprod Fertil 1996; 107: 265-277
• 45 Ramathal CY, Bagchi IC, Taylor RN. et al. Endometrial decidualization: of mice and men. Semin Reprod Med 2010; 28: 17-26
• 46 Chen Q, Zhang Y, Lu J. et al. Embryo-uterine cross-talk during implantation: the role of Wnt signaling. Mol Hum Reprod 2009; 15: 215-221
• 47 Mège RM, Gavard J, Lambert M. Regulation of cell-cell junctions by the cytoskeleton. Curr Opin Cell Biol 2006; 18: 541-548
• 48 Gama A, Paredes J, Gärtner F. et al. Expression of E-cadherin, P-cadherin and beta-catenin in canine malignant mammary tumours in relation to clinicopathological parameters, proliferation and survival. Vet J 2008; 177: 45-53
• 49 Zhang J-Y, Bai X-M, Zhang L. et al. PGE_2 promotes endometrial cancer cell growth through Wnt/β-catenin signaling pathway [J]. Acta Universitatis Medicinalis Nanjing (Natural Science) 2011; 31
• 50 Catalán V, Gómez-Ambrosi J, Rodríguez A. et al. Activation of noncanonical Wnt signaling through WNT5A in visceral adipose tissue of obese subjects is related to inflammation. J Clin Endocrinol Metab 2014; 99: E1407-E1417
• 51 Kiewisz J, Kaczmarek MM, Morawska E. et al. Estrus synchronization affects WNT signaling in the porcine reproductive tract and embryos. Theriogenology 2011; 76: 1684-1694
• 52 Ring L, Neth P, Weber C. et al. β-Catenin-dependent pathway activation by both promiscuous “canonical” WNT3a–, and specific “noncanonical” WNT4–and WNT5a–FZD receptor combinations with strong differences in LRP5 and LRP6 dependency. Cell Signal 2014; 26: 260-267
• 53 Topol L, Jiang X, Choi H. et al. Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation. J Cell Biol 2003; 162: 899-908