LPA3-mediated lysophosphatidic acid signalling in embryo implantation and spacing

Abstract

Every successful pregnancy requires proper embryo implantation. Low implantation rate is a major problem during infertility treatments using assisted reproductive technologies (ART) 1 . Here we report a new molecular influence on implantation through the lysophosphatidic acid (LPA) receptor LPA 3 2-4 . Targeted deletion of LPA 3 in mice resulted in significantly reduced litter size, which could be attributed to delayed implantation and altered embryo spacing. These two events led to delayed embryonic development, hypertrophic placentas shared by multiple embryos, and embryonic death. An enzyme demonstrated to influence implantation, cyclooxygenase-2 (COX-2) 5 , was downregulated in LPA 3 -deficient uteri during preimplantation. Down regulation of COX-2 led to reduced levels of prostaglandins that are critical for implantation 1 . Exogenous administration of the prostaglandins PGE 2 and cPGI into LPA 3 -deficient females rescued delayed implantation but did not rescue defects in embryo spacing. These data identify LPA 3 receptor-mediated signalling as a new influence on implantation and further indicate linkage between LPA signalling and prostaglandin biosynthesis.Multiple factors can adversely affect successful pregnancy. Two of these factors are failed synchronization between embryonic and endometrial development during implantation and occurrence of multiple gestations (especially monochorionic gestation), which can result in fetal demise 1,6-9 . These factors are particularly important for the clinical success and efficacy of ART. One molecular factor that has been previously implicated in female reproduction is the small, bioactive phospholipid LPA 10 . LPA has a range of influences that are mediated by at least four G protein-coupled receptors, LPA 1-4 2 . Deletion of LPA 1 and LPA 2 in mice revealed roles for these receptors in neural development, craniofacial formation, neuropathic pain, and altered cellular signalling, but without obvious effects on female reproduction 11-Correspondence and requests for materials should be addressed to J. Chun (e-mail:jchun@scripps.edu).. 6 These authors contributed equally to the work. Functional deletion of LPA 3 was achieved by replacing a fragment covering the untranslated region and the start codon in exon 2 with a neomycin-resistance gene in reverse orientation in R1 embryonic stem cells (supplementary Fig. S1). The LPA 3 -deficient mice were born with normal Mendelian frequency without sexual bias (supplementary Table S1), and appeared grossly normal (data not shown). However, LPA 3 -deficient females produced litter sizes of less than 50% compared to that from wild-type (WT) and LPA 3 heterozygote (Het) controls (supplementary Table S2), and showed a statistically significant prolongation of pregnancy (20.9±0.5 days, vs. 19.4±0.7 days in WT/Het controls, P<0.05). These phenotypes were independent of stud genotype, indicating defects in female reproduction. Supplementary InformationTowards determining whether LPA 3 deletion might directly affect the female...

Autotaxin–lysophosphatidic acid– LPA 3 signaling at the embryo‐epithelial boundary controls decidualization pathways

“…Anti‐ATX staining showed that ATX was predominantly expressed in the uteri and localized throughout the epithelial layer (Fig 7A). Administration of an ATX inhibitor (S15‐00826, Fig EV7A–E) into the uterine cavity of pregnant mice resulted in abnormal embryo spacing and implantation failure at 5.5 dpc (Fig 7B–D), as was observed in Lpar3 KO uteri (Ye et al , 2005; Hama et al , 2007). The expression levels of Hbegf , Ptgs2 , Bmp2 and Wnt4 were also reduced by the ATX inhibitor (Fig 7E and F).…”

“…Several tens of genes were up‐regulated 2 h after the T13 injection (Table 1). Among the genes, we focused on Hbegf and Ptgs2 (encoding COX‐2), because they are responsible for decidualization and knockout of these genes interfered with implantation (Lim et al , 1997; Song et al , 2002; Wang et al , 2004; Xie et al , 2007; Large et al , 2014), as was observed in Lpar3 KO mice (Ye et al , 2005). Both Hbegf and Ptgs2 were transiently induced, peaking at 2–9 h after the T13 injection (Fig 3A).…”

“…Studies of knock‐out (KO) mice and human genetic diseases of these LPA‐related genes have shown that LPA has various pathophysiological roles including angiogenesis (Yukiura et al , 2011), hair follicle formation (Inoue et al , 2011; Hayashi et al , 2015), bone development (Nishioka et al , 2016), and neural development (Yung et al , 2015). We previously showed that LPA 3 , which is highly expressed in the uterine epithelium during the peri‐implantation period (Ye et al , 2005), has a critical role in the early pregnancy. Lpar3 KO mice show many reproductive defects, including significantly reduced COX‐2 (a key enzyme for synthesis of prostaglandins), delayed implantation, aberrant embryo spacing, defects in placental formation and fetal development, and reduced litter size (Ye et al , 2005; Hama et al , 2007).…”

“…We previously showed that LPA 3 , which is highly expressed in the uterine epithelium during the peri‐implantation period (Ye et al , 2005), has a critical role in the early pregnancy. Lpar3 KO mice show many reproductive defects, including significantly reduced COX‐2 (a key enzyme for synthesis of prostaglandins), delayed implantation, aberrant embryo spacing, defects in placental formation and fetal development, and reduced litter size (Ye et al , 2005; Hama et al , 2007). However, the defects in Lpar3 KO were only partially recovered by administration of prostaglandins (Ye et al , 2005), suggesting that other unidentified factors should operate downstream of LPA 3 .…”

“…Lpar3 KO mice show many reproductive defects, including significantly reduced COX‐2 (a key enzyme for synthesis of prostaglandins), delayed implantation, aberrant embryo spacing, defects in placental formation and fetal development, and reduced litter size (Ye et al , 2005; Hama et al , 2007). However, the defects in Lpar3 KO were only partially recovered by administration of prostaglandins (Ye et al , 2005), suggesting that other unidentified factors should operate downstream of LPA 3 . In addition, it is almost unclear what kind of cellular events are affected in Lpar3 KO uteri.…”