Shuo Xiao

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.

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.

Temporal expression pattern of progesterone receptor in the uterine luminal epithelium suggests its requirement during early events of implantation

OBJECTIVE To determine the precise timing of progesterone receptor (PR) disappearing from the uterine luminal epithelium (LE) to help understand the significance of the dynamic PR expression in the LE during embryo implantation. DESIGN Experimental rodent models. SETTING University research laboratories. ANIMAL(S) Mice and hamsters. INTERVENTION(S) Pseudopregnancy and artificial decidualization. MAIN OUTCOME MEASURE(S) Blue dye injection for detecting embryo attachment; immunohistochemistry, immunofluorescence, and in situ hybridization for detecting gene expression. RESULT(S) Progesterone receptor remained expressed in the LE up to 6 hours after the initial detection of blue dye reaction in mice (day 3, 22:00 hours), but disappeared first from LE cells at the implantation site and subsequently from the entire LE layer by day 4, 06:00 hours, when uterine stromal decidualization had become obvious. Progesterone receptor remained highly expressed in the LE of day 4 at 11:00 hours in pseudopregnant mice, but it disappeared from the entire LE layer by day 4 at 06:00 hours in artificially decidualized pseudopregnant mice. CONCLUSION(S) Progesterone receptor disappears from the LE after implantation has initiated and before the histologic decidualization manifests, suggesting an active role of continued PR expression in the LE for the initial implantation process. The disappearance of PR expression in the LE is regulated by uterine factor(s) produced upon embryo attachment.

Altered Spatiotemporal Expression of Collagen Types I, III, IV, and VI in Lpar3-Deficient Peri-Implantation Mouse Uterus

Lpar3 is upregulated in the preimplantation uterus, and deletion of Lpar3 leads to delayed uterine receptivity in mice. Microarray analysis revealed that there was higher expression of Col3a1 and Col6a3 in the Preimplantation Day 3.5 Lpar3−/− uterus compared to Day 3.5 wild-type (WT) uterus. Since extracellular matrix (ECM) remodeling is indispensable during embryo implantation, and dynamic spatiotemporal alteration of specific collagen types is part of this process, this study aimed to characterize the expression of four main uterine collagen types: fibril-forming collagen (COL) I and COL III, basement membrane COL IV, and microfibrillar COL VI in the peri-implantation WT and Lpar3−/− uterus. An observed delay of COL III and COL VI clearance in the Lpar3−/− uterus may be associated with higher preimplantation expression of Col3a1 and Col6a3. There was also delayed clearance of COL I and delayed deposition of COL IV in the decidual zone in the Lpar3−/− uterus. These changes were different from the effects of 17beta-estradiol and progesterone on uterine collagen expression in ovariectomized WT uterus, indicating that the altered collagen expression in Lpar3−/− uterus is unlikely to be a result of alterations in ovarian hormones. Decreased expression of several genes encoding matrix-degrading metallo- and serine proteinases was observed in the Lpar3−/− uterus. These results demonstrate that pathways downstream of LPA3 are involved in the dynamic remodeling of ECM in the peri-implantation uterus.

Broad Gap Junction Blocker Carbenoxolone Disrupts Uterine Preparation for Embryo Implantation in Mice

ABSTRACT Gap junctions have an important role in cell-to-cell communication, a process obviously required for embryo implantation. Uterine luminal epithelium (LE) is the first contact for an implanting embryo and is critical for the establishment of uterine receptivity. Microarray analysis of the LE from peri-implantation mouse uterus showed low-level expression of 19 gap junction proteins in preimplantation LE and upregulation of gap junction protein, beta 2 (GJB2, connexin 26, Cx26) in postimplantation LE. Time course study using in situ hybridization and immunofluorescence revealed upregulation of GJB2 in the LE surrounding the implantation site before decidualization. Similar dynamic expression of GJB2 was observed in the LE of artificially decidualized mice but not pseudopregnant mice. To determine the potential function of uterine gap junctions in embryo implantation, carbenoxolone (CBX), a broad gap junction blocker, was injected i.p. (100 mg/kg) or via local uterine fat pad (10 mg/kg) into pregnant mice on Gestation Day 3 at 1800 h, a few hours before embryo attachment to the LE. These CBX treatments disrupted embryo implantation, suggesting local effects of CBX in the uterus. However, i.p. injection of glycyrrhizic acid (100 mg/kg), which shares similar structure and multiple properties with CBX but is ineffective in blocking gap junctions, did not affect embryo implantation. Carbenoxolone also inhibited oil-induced artificial decidualization, concomitant with suppressed molecular changes and ultrastructural transformations associated with uterine preparation for embryo implantation, underscoring the adverse effect of CBX on uterine preparation for embryo implantation. These data demonstrate that uterine gap junctions are important for embryo implantation.

Differential gene expression profiling of mouse uterine luminal epithelium during periimplantation.

Uterine luminal epithelium (LE) is critical for establishing uterine receptivity. Microarray analysis of gestation day 3.5 (D3.5, preimplantation) and D4.5 (postimplantation) LE from natural pregnant mice identified 382 upregulated and 245 downregulated genes in the D4.5 LE. Gene Ontology annotation grouped 186 upregulated and 103 downregulated genes into 22 and 15 enriched subcategories, respectively, in regulating DNA-dependent transcription, metabolism, cell morphology, ion transport, immune response, apoptosis, signal transduction, and so on. Signaling pathway analysis revealed 99 genes in 21 significantly changed signaling pathways, with 14 of these pathways involved in metabolism. In situ hybridization confirmed the temporal expression of 12 previously uncharacterized genes, including Atp6v0a4, Atp6v0d2, F3, Ggh, Tmprss11d, Tmprss13, Anpep, Fxyd4, Naip5, Npl, Nudt19, and Tpm1 in the periimplantation uterus. This study provides a comprehensive picture of the differentially expressed genes in the periimplantation LE to help understand the molecular mechanism of LE transformation upon establishment of uterine receptivity.

Perinatal Bisphenol A Exposure in C57B6/129svj Male Mice: Potential Altered Cytokine/Chemokine Production in Adulthood

Pregnant mice (n = 3) were exposed to BPA by intraperitoneal injection, from gestation day 9.5 until end of lactation. Male offspring were evaluated for cytokine production at 20 wk-of-age. One pregnant control mouse produced no males, precluding statistical analysis. However, recurring shifts in cytokines were suggested in the adult BPA offspring. Serum showed a numeric increase in 16 of 21 basal cytokine levels. ConA-stimulated splenocytes showed a numeric increase in 17 of 21 cytokines, and LPS-stimulated splenocytes an increase in 18 of 21 cytokines. The cytokine profile was one of TH1 up-regulation more than TH2, and with skewing toward TH17 responses.

Uterine luminal epithelium–specific proline-rich acidic protein 1 (PRAP1) as a marker for successful embryo implantation

Proline-rich acidic protein 1 mRNA is highly expressed in the uterine luminal epithelium (LE) of day 0.5 mouse uterus, disappears in the preimplantation day 3.5 uterus, and reappears abundantly in the LE after embryo implantation has occurred or upon artificial decidualization. In ovariectomized uterus, Prap1 is down-regulated by P, transiently down-regulated by E(2) treatment for 6 hours, but dramatically induced by E(2) treatment for 3 days.

Postweaning dietary genistein exposure advances puberty without significantly affecting early pregnancy in C57BL/6J female mice

An epidemiological study indicates higher plasma level of genistein in girls with earlier puberty. This study tests the hypothesis in C57BL/6J mice that postweaning (peripubertal) dietary genistein exposure could result in earlier puberty in females assessed by vaginal opening, estrous cyclicity, corpus luteum and mammary gland development. Newly weaned female mice were fed with 0, 5, 100, or 500 ppm genistein diets. Decreased age at vaginal opening, increased length on estrus stage, and accelerated mammary gland development were detected in 100 and 500 ppm genistein-treated groups. Increased presence of corpus luteum was found in 5 ppm genistein-treated group at 6 weeks old only. Increased expression of epithelial-specific genes but not that of ERα or ERβ was detected in 500 ppm genistein-treated mammary glands at 5 weeks old. No significant adverse effect on embryo implantation was observed. These data demonstrate causal effect of dietary genistein on earlier puberty in female mice.

Progesterone receptor-mediated up-regulation of transthyretin in preimplantation mouse uterus

Transthyretin (TTR), a carrier for thyroxine and retinol, has its messenger RNA (mRNA) expressed in the glandular endometrial epithelium and its protein detected in the glandular endometrial epithelium and the uterine lumen. TTR mRNA is dramatically up-regulated in the preimplantation mouse uterus as well as the P-treated ovariectomized mouse uterus, and in both situations the up-regulation of TTR is blocked by treatment with the P receptor antagonist RU486.