Xiaoqin Ye

LPA3-mediated lysophosphatidic acid signalling in embryo implantation and spacing

Every successful pregnancy requires proper embryo implantation. Low implantation rate is a major problem during infertility treatments using assisted reproductive technologies. Here we report a newly discovered molecular influence on implantation through the lysophosphatidic acid (LPA) receptor LPA3 (refs 2–4). Targeted deletion of LPA3 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 (COX2) (ref. 5), was downregulated in LPA3-deficient uteri during pre-implantation. Downregulation of COX2 led to reduced levels of prostaglandins E2 and I2 (PGE2 and PGI2), which are critical for implantation. Exogenous administration of PGE2 or carbaprostacyclin (a stable analogue of PGI2) into LPA3-deficient female mice rescued delayed implantation but did not rescue defects in embryo spacing. These data identify LPA3 receptor-mediated signalling as having an influence on implantation, and further indicate linkage between LPA signalling and prostaglandin biosynthesis.

Characterization of lpa2 (Edg4) and lpa1/lpa2 (Edg2/Edg4) Lysophosphatidic Acid Receptor Knockout Mice: Signaling Deficits without Obvious Phenotypic Abnormality Attributable to lpa2

ABSTRACT Lysophosphatidic acid (LPA), a bioactive lipid produced by several cell types including postmitotic neurons and activated platelets, is thought to be involved in various biological processes, including brain development. Three cognate G protein-coupled receptors encoded by lpa1 /lp A1/Edg-2/Gpcr26, lpa2 /lp A2/Edg-4, and lpa3 /lp A3/Edg-7 mediate the cellular effects of LPA. We have previously shown that deletion of lpa1 in mice results in craniofacial dysmorphism, semilethality due to defective suckling behavior, and generation of a small fraction of pups with frontal hematoma. To further investigate the role of these receptors and LPA signaling in the organism, we deleted lpa2 in mice. Homozygous knockout (lpa2 (−/−)) mice were born at the expected frequency and displayed no obvious phenotypic abnormalities. Intercrosses allowed generation of lpa1 (−/−) lpa2 (−/−) double knockout mice, which displayed no additional phenotypic abnormalities relative to lpa1 (−/−) mice except for an increased incidence of perinatal frontal hematoma. Histological analyses of lpa1 (−/−) lpa2 (−/−) embryonic cerebral cortices did not reveal obvious differences in the proliferating cell population. However, many LPA-induced responses, including phospholipase C activation, Ca2+ mobilization, adenylyl cyclase activation, proliferation, JNK activation, Akt activation, and stress fiber formation, were absent or severely reduced in embryonic fibroblasts derived from lpa1 (−/−) lpa2 (−/−) mice. Except for adenylyl cyclase activation [which was nearly abolished in lpa1 (−/−) fibroblasts], these responses were only partially affected in lpa1 (−/−) and lpa2 (−/−) fibroblasts. Thus, although LPA2 is not essential for normal mouse development, it does act redundantly with LPA1 to mediate most LPA responses in fibroblasts.

Embryo Spacing and Implantation Timing Are Differentially Regulated by LPA3-Mediated Lysophosphatidic Acid Signaling in Mice

Abstract In polytocous animals, blastocysts are evenly distributed along each uterine horn and implant. The molecular mechanisms underlying these precise events remain elusive. We recently showed that lysophosphatidic acid (LPA) has critical roles in the establishment of early pregnancy by affecting embryo spacing and subsequent implantation through its receptor, LPA3. Targeted deletion of Lpa3 in mice resulted in delayed implantation and embryo crowding, which is associated with a dramatic decrease in the prostaglandins and prostaglandin-endoperoxide synthase 2 expression levels. Exogenous administration of prostaglandins rescued the delayed implantation but did not rescue the defects in embryo spacing, suggesting the role of prostaglandins in implantation downstream of LPA3 signaling. In the present study, to know how LPA3 signaling regulates the embryo spacing, we determined the time course distribution of blastocysts during the preimplantation period. In wild-type (WT) uteri, blastocysts were distributed evenly along the uterine horns at Embryonic Day 3.8 (E3.8), whereas in the Lpa3-deficient uteri, they were clustered in the vicinity of the cervix, suggesting that the mislocalization and resulting crowding of the embryos are the cause of the delayed implantation. However, embryos transferred singly into E2.5 pseudopregnant Lpa3-deficient uterine horns still showed delayed implantation but on-time implantation in WT uteri, indicating that embryo spacing and implantation timing are two segregated events. We also found that an LPA3-specific agonist induced rapid uterine contraction in WT mice but not in Lpa3-deficient mice. Because the uterine contraction is critical for embryo spacing, our results suggest that LPA3 signaling controls embryo spacing via uterine contraction around E3.5.

Lysophospholipid signaling in the function and pathology of the reproductive system

BACKGROUND Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are two prominent signaling lysophospholipids (LPs) exerting their functions through a group of G protein-coupled receptors (GPCRs). This review covers current knowledge of the LP signaling in the function and pathology of the reproductive system. METHODS PubMed was searched up to May 2008 for papers on lysophospholipids/LPA/S1P/LPC/SPC in combination with each part of the reproductive system, such as testis/ovary/uterus. RESULTS LPA and SIP are found in significant amounts in serum and other biological fluids. To date, 10 LP receptors have been identified, including LPA(1-5) and S1P(1-5). In vitro and in vivo studies from the past three decades have demonstrated or suggested the physiological functions of LP signaling in reproduction, such as spermatogenesis, male sexual function, ovarian function, fertilization, early embryo development, embryo spacing, implantation, decidualization, pregnancy maintenance and parturition, as well as pathological roles in ovary, cervix, mammary gland and prostate cancers. CONCLUSIONS Receptor knock-out and other studies indicate tissue-specific and receptor-specific functions of LP signaling in reproduction. More comprehensive studies are required to define mechanisms of LP signaling and explore the potential use as a therapeutic target.

Preimplantation exposure to bisphenol A (BPA) affects embryo transport, preimplantation embryo development, and uterine receptivity in mice

To investigate the effects of bisphenol A (BPA) on embryo and uterine factors in embryo implantation, timed pregnant C57BL6 females were treated subcutaneously with 0, 0.025, 0.5, 10, 40, and 100mg/kg/day BPA from gestation days 0.5-3.5. In 100mg/kg/day BPA-treated females, no implantation sites were detected on day 4.5 but retention of embryos in the oviduct and delayed embryo development were detected on day 3.5. When untreated healthy embryos were transferred to pseudopregnant females treated with 100mg/kg/day BPA, no implantation sites were detected on day 4.5. In 40 mg/kg/day BPA-treated females, delayed implantation and increased perinatal lethality of their offspring were observed. Implantation seemed normal in the rest BPA-treated groups or the female offspring from 40 mg/kg/day BPA-treated group. These data demonstrate the adverse effects of high doses of BPA on processes critical for embryo implantation: embryo transport, preimplantation embryo development, and establishment of uterine receptivity.

Lysophosphatidic acid in neural signaling

The physiological and pathological importance of lysophosphatidic acid (LPA) in the nervous system is underscored by its presence, as well as the expression of its receptors in neural tissues. In fact, LPA produces responses in a broad range of cell types related to the function of the nervous system. These cell types include neural cell lines, neural progenitors, primary neurons, oligodendrocytes, Schwann cells, astrocytes, microglia, and brain endothelial cells. LPA-induced cell type-specific effects include changes in cell morphology, promotion of cell proliferation and cell survival, induction of cell death, changes in ion conductance and Ca2+ mobilization, induction of pain transmission, and stimulation of vasoconstriction. These effects are mediated through a number of G protein-coupled LPA receptors that activate various downstream signaling cascades. This review provides a current summary of LPA-induced effects in neural cells in vitro or in vivo in combination with our current understanding of the signaling pathways responsible for these effects.

Lysophosphatidic acid (LPA) signaling in vertebrate reproduction.

Lysophosphatidic acid (LPA) is a cell membrane phospholipid metabolite that can act as an extracellular signal. Its effects are mediated through at least five G protein-coupled receptors, LPA(1-5), and probably others as well. Studies in multiple species including LPAR-deficient mice and humans have identified or implicated important roles for receptor-mediated LPA signaling in multiple aspects of vertebrate reproduction. These include ovarian function, spermatogenesis, fertilization, early embryo development, embryo implantation, embryo spacing, decidualization, pregnancy maintenance and parturition. LPA signaling can also have pathological consequences, influencing aspects of endometriosis and ovarian cancer. Here we review recent progress in LPA signaling research relevant to female and male reproduction.

Age-Dependent Loss of Sperm Production in Mice via Impaired Lysophosphatidic Acid Signaling

Abstract Approximately half of all infertility cases can be attributed to male reproductive dysfunction for which low sperm count is a major contributing factor. The current study identified receptor-mediated lysophosphatidic acid (LPA) signaling as a new molecular component influencing male fertility. LPA is a small signaling phospholipid, the effects of which are mediated through at least five G protein-coupled receptors, named LPA 1–5. LPA1/2/3, but not LPA4/5, show high expression in mouse testis. Mice deficient in LPA1/2/3 showed a testosterone-independent reduction of mating activity and sperm production, with an increased prevalence of azoospermia in aging animals. A significant increase of germ cell apoptosis also was observed in testes. Germ cell apoptosis led to a reduction in germ cell proliferation. These data demonstrate a novel in vivo function for LPA signaling as a germ cell survival factor during spermatogenesis.

Signaling Mechanisms Responsible for Lysophosphatidic Acid-induced Urokinase Plasminogen Activator Expression in Ovarian Cancer Cells

Lysophosphatidic acid (LPA) enhances urokinase plasminogen activator (uPA) expression in ovarian cancer cells; however, the molecular mechanisms responsible for this event have not been investigated. In this study, we used the invasive ovarian cancer SK-OV-3 cell line to explore the signaling molecules and pathways essential for LPA-induced uPA up-regulation. With the aid of specific inhibitors and dominant negative forms of signaling molecules, we determined that the Gi-associated pathway mediates this LPA-induced event. Moreover, constitutively active H-Ras and Raf-1-activating H-Ras mutant enhance uPA expression, whereas dominant negative H-Ras and Raf-1 block LPA-induced uPA up-regulation, suggesting that the Ras-Raf pathway works downstream of Gi to mediate this LPA-induced process. Surprisingly, dominant negative MEK1 or Erk2 displays only marginal inhibitory effect on LPA-induced uPA up-regulation, suggesting that a signaling pathway distinct from Raf-MEK1/2-Erk is the prominent pathway responsible for this process. In this report, we demonstrate that LPA activates NF-κB in a Ras-Raf-dependent manner and that blocking NF-κB activation with either non-phosphorylable IκB or dominant negative IκB kinase abolished LPA-induced uPA up-regulation and uPA promoter activation. Furthermore, introducing mutations to knock out the NF-κB binding site of the uPA promoter results in over 80% reduction in LPA-induced uPA promoter activation, whereas this activity is largely intact with the promoter containing mutations in the AP1 binding sites. Thus these results suggest that the Gi-Ras-Raf-NF-κB signaling cascade is responsible for LPA-induced uPA up-regulation in ovarian cancer cells.

Postweaning exposure to dietary zearalenone, a mycotoxin, promotes premature onset of puberty and disrupts early pregnancy events in female mice.

Zearalenone (ZEA) is a mycotoxin commonly found in contaminated livestock feed and human food with levels in the range of ppb and low ppm. It was hypothesized that ZEA, an endocrine disruptor, could affect puberty and early pregnancy. To test this hypothesis, newly weaned (3 weeks old) C57BL/6J female mice were exposed to 0, 0.002, 4, 10, and 40 ppm ZEA and 0.05 ppm diethylstilbestrol (positive control) in phytoestrogen-free AIN-93G diet. Females exposed to 10 and 40 ppm ZEA diets showed earlier onset of vaginal opening. Those treated with 40 ppm ZEA diet also had earlier first copulation plug and irregular estrous cyclicity. At 8 weeks old, all females were mated with untreated stud males on AIN-93G diet during mating. Treatment resumed upon identification of a vaginal plug on gestation day 0.5 (D0.5). Embryo implantation was assessed on D4.5. Exposure to 40 ppm ZEA diet resulted in reduced percentage of plugged mice with implantation sites, distended uterine appearance, and retained expression of progesterone receptor in D4.5 uterine epithelium. To determine the exposure timing and mechanisms of disrupted embryo implantation, four groups of females were fed with 0 or 40 ppm ZEA diets during premating (weaning to mating) and postmating (D0.5-D4.5), respectively. Premating exposure to 40 ppm ZEA diet reduced fertilization rate, whereas postmating exposure to 40 ppm ZEA diet delayed embryo transport and preimplantation embryo development, which subsequently affected embryo implantation. These data demonstrate that postweaning exposure to dietary ZEA can promote premature onset of puberty and disrupt early pregnancy events.