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  • Abstract: placenta x
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  • Abstract: decidualization x
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The distribution and intensity of reaction for adenosine-5′-monophosphatase, adenosine-5′-triphosphatase, inosine-triphosphatase, thiamine-pyrophosphatase, uridine diphosphatase, β-glycerophosphatase and glucose-6-phosphatase were studied in relation to the implanting rat embryo and developing decidua at 3½, 4½, 5, 5½, 6, 6½, 7½, 8½ and 9½ days of pregnancy. The distribution of glycogen was also studied from 4½ days onwards, using the pas procedure in combination with diastase and/or dimedone blockade.

Glycogen was demonstrated in the stroma antimesometrial and lateral to the embryo, accumulating at 6 to 6½ days and gradually being compressed laterally by the expanding decidua, the lateral part of which also contained some glycogen.

All of the enzymes studied accumulated to a greater or lesser degree in the stroma initially and then in the antimesometrial decidua, where their accumulation was paralleled by a falling off of glycogen concentration.

Significant differences in distribution, particularly in the mesometrial region, were noted, adenosine-5′-monophosphatase, uridine-diphosphatase, and glucose-6-phosphatase accumulating in the cells of the glycogen wings, while the remaining enzymes did not. Adenosine-5′-triphosphatase, thiamine pyrophosphatase, and inosine-triphosphatase accumulated in the cells lining the sinusoids of this region.

Adenosine-5′-triphosphatase was the only enzyme studied to show any alteration between 3½ and 4½ days, and it is suggested that the observed antimesometrial accumulation of this enzyme may be a reflection of the oestrogen surge which occurs at this time.

An attempt is made to correlate the presence and alteration in distribution of these enzymes, and of glycogen, with what is known of their physiological functions with respect to glycogen metabolism, energy production, transport of substances across the cell membrane, and nutrition of the embryo.

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Y. Tsunoda and D. G. Whittingham

Summary. The effects of purified antibody (IgG) to mechanically isolated or detergent-solubilized zonae pellucidae on the development and implantation of mouse embryos were studied in vitro by culture and in utero by transfer of treated embryos. The proportion of 8-celled embryos treated with zona antibody that developed into blastocysts and attached to the culture dish in vitro were not significantly different from those obtained after treatment of embryos with IgG from normal rabbit serum and antiserum raised against mouse liver and kidney. Pregnancy rate, mean number of implantations and live fetuses, and fetal sex ratio after transfer of zona antibody treated embryos were not significantly different from the values obtained after transfer of embryos treated with control serum IgG. It was concluded that treatment with zona antibody did not inhibit the development and implantation of mouse embryos in vitro or in vivo.

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P. Carthew, Maureen J. Wood and Carol Kirby

Summary. Preimplantation embryos collected from mice in the acute phase of Sendai virus infection were not infected. Transfer of embryos from infected donors did not transmit the virus to the recipient foster mothers or to their progeny. The pregnancies were normal with no differences in implantation rate or number of live births when compared with control transfers of embryos collected from non-infected donors.

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Jing Xiong, Pan Zeng, Xue Cheng, Sen Miao, Le Wu, Sheng Zhou, Ping Wu and Duyun Ye

Embryo implantation involves a complex regulatory network of steroid hormones, inflammatory cytokines, and immune cells. Lipoxin A4 (LXA4), a biologically active eicosanoid with specific anti-inflammatory and pro-resolving properties, was recently found to be a novel modulator of estrogen receptor α (ERα). In this study, we investigated the potential role of LXA4 in implantation. We found that LXA4 blocked embryo implantation in mice and significantly reduced the expression of inflammatory mediators associated with uterine receptivity and embryo implantation, including corticotropin-releasing factor (CRF), cyclooxygenase 2-derived prostaglandin I2 and prostaglandin E2, leukemia inhibitory factor, and interleukin 6, but this effect was independent of LXA4 receptor. Subsequent investigation revealed enhanced ERα activity in the uteri of LXA4-treated mice during the peri-implantation period. ERα and phosphorylated ERα were significantly increased following LXA4 treatment. Finally, it was demonstrated that the inhibitory effect of LXA4 on embryo implantation was mediated through ERα. In the presence of the ERα antagonist ICI 182 780, LXA4 failed to block embryo implantation. LXA4 also failed to inhibit CRF expression. These results suggested that LXA4 blocks embryo implantation by controlling ERα activity, and this effect appeared to be related to the suppression of the inflammatory microenvironment necessary for implantation.

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Kazuhiro Tamura, Mikihiro Yoshie, Hirotaka Nishi, Yumi Osakabe, Keiichi Isaka, Takahiko Hara and Hiroshi Kogo

The cytosolic phosphoprotein stathmin is upregulated at the site of embryo implantation in the rodents. However, stathmin expression in the human uterus has not yet been investigated. The distribution of uterine and placental stathmin was analyzed by immunohistochemistry, while stathmin mRNA expression was detected in endometrial tissues by the reverse transcriptase-PCR. Cultured endometrial stromal cells were used to investigate whether stathmin plays a role in decidualization. Stathmin is expressed specifically in the glandular epithelium and the stromal cells of human endometrial tissue. It is also expressed by cytotrophoblasts and extravillous trophoblasts, but not by syncytiotrophoblasts or decidual tissues during the first trimester of pregnancy. When stromal cells isolated from normal endometrial tissues were cultured and stimulated to decidualize by progesterone (P4) plus estrogen or dibutyryl cyclic 3′,5′-AMP, their total and phosphorylated stathmin levels decreased. Knocking down stathmin expression in the cultured stromal cells using small interfering RNA, before the cells were exposed to the decidualizing agents, significantly suppressed decidualization, as indicated by the decreased expression of IGF-binding protein-1 and prolactin. Stathmin is differently expressed in human endometrial and placental cells and may participate in the decidualization of endometrial stromal cells.

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Paola I Ingaramo, Jorgelina Varayoud, María M Milesi, Marlise Guerrero Schimpf, Mónica Muñoz-de-Toro and Enrique H Luque

In this study, we investigated whether neonatal exposure to a glyphosate-based herbicide (GBH) alters the reproductive performance and the molecular mechanisms involved in the decidualization process in adult rats. Newborn female rats received vehicle or 2 mg/kg/day of a GBH on postnatal days (PND) 1, 3, 5 and 7. On PND90, the rats were mated to evaluate (i) the reproductive performance on gestational day (GD) 19 and (ii) the ovarian steroid levels, uterine morphology, endometrial cell proliferation, apoptosis and cell cycle regulators, and endocrine pathways that regulate uterine decidualization (steroid receptors/COUP-TFII/Bmp2/Hoxa10) at the implantation sites (IS) on GD9. The GBH-exposed group showed a significant increase in the number of resorption sites on GD19, associated with an altered decidualization response. In fact, on GD9, the GBH-treated rats showed morphological changes at the IS, associated with a decreased expression of estrogen and progesterone receptors, a downregulation of COUP-TFII (Nr2f2) and Bmp2 mRNA and an increased expression of HOXA10 and the proliferation marker Ki67(Mki67) at the IS. We concluded that alterations in endometrial decidualization might be the mechanism of GBH-induced post-implantation embryo loss.

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Sylvia Sanches Cortezzi, Elaine Cristina Cabral, Marcello Garcia Trevisan, Christina Ramires Ferreira, Amanda Souza Setti, Daniela Paes de Almeida Ferreira Braga, Rita de Cássia Sávio Figueira, Assumpto Iaconelli Jr, Marcos Nogueira Eberlin and Edson Borges Jr

This study has evaluated the performance of a multivariate statistical model to predict embryo implantation potential by processing data from the chemical fingerprinting of culture medium samples used for human embryo culture. The culture medium for 113 embryos from 55 patients undergoing ICSI was collected after embryo transfer. The samples were split into positive (n=29) and negative (n=84) implantation groups according their implantation outcomes (100% or 0% implantation). The samples were individually diluted and analyzed by electrospray ionization mass spectrometry (ESI-MS). The m/z ratios and relative abundances of the major ions in each spectrum were considered for partial least square discriminant analysis. Data were divided into two subsets (calibration and validation), and the models were evaluated and applied to the validation set. A total of 5987 ions were observed in the groups. The multivariate statistical model described more than 82% of the data variability. Samples of the positive group were correctly identified with 100% probability and negative samples with 70%. The culture media used for embryos that were positive or negative for successful implantation showed specific biochemical signatures that could be detected in a fast, simple, and noninvasive way by ESI-MS. To our knowledge, this is the first report that uses MS fingerprinting to predict human embryo implantation potential. This biochemical profile could help the selection of the most viable embryo, improving single-embryo transfer and thus eliminating the risk and undesirable outcomes of multiple pregnancies.

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RT Lambert, CJ Ashworth, L Beattie, FE Gebbie, JS Hutchinson, DJ Kyle and PA Racey

The roe deer blastocyst is in diapause between August and December, after which time it expands and elongates rapidly before implantation. Blood samples were taken from 30 animals to define temporal changes in reproductively important hormones to investigate the physiological cues present at embryo reactivation. In 15 of these animals, changes in uterine and conceptus protein synthesis and secretion, and luteal progesterone release during diapause and reactivation, were assessed after culture of these tissues in vitro. Oestradiol concentrations remained low during diapause (1.07 +/- 0.4 pg ml(-1)) and expansion (1.2 +/- 0.4 pg ml(-1)) but increased by 30 times at trophoblast elongation (49.17 +/- 0.37 pg ml(-1)). Prolactin remained at basal concentrations (4.69 +/- 0.86 ng ml(-1)) and increased after implantation (12.34 +/- 2.71 ng ml(-1)). Peripheral progesterone concentrations and luteal progesterone release remained constant throughout diapause, reactivation and implantation (peripheral progesterone: 3.82 +/- 1.97 ng ml(-1); luteal progesterone: 6.72 +/- 0.81 ng mg(-1) protein). Incorporation of a radiolabel into conceptus secretory proteins increased by four times at expansion compared with diapause, whereas incorporation into endometrial secretions remained constant. At elongation, incorporation into endometrial secretions increased two times and conceptus secretions increased 32 times. Two-dimensional electrophoresis and fluorography showed that the profile of endometrial secretory proteins was constant until implantation when qualitative changes were evident. Although a role for an endocrine maternal trigger of reactivation from diapause cannot be dismissed, these data provide no supporting evidence and indicate that the conceptus itself may drive reactivation.

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J Sengupta, P G L Lalitkumar, A R Najwa, D S Charnock-Jones, A L Evans, A M Sharkey, S K Smith and D Ghosh

Maternal endometrial vascular endothelial growth factor (VEGF) is considered important in blastocyst implantation. However, there is no direct evidence to support this conjecture in the primate. In the present study, we have examined this hypothesis by testing whether immunoneutralization of VEGF during the peri-implantation stage of gestation affects embryo implantation in the rhesus monkey. Adult female animals (n = 36) during mated ovulatory cycles were randomly assigned to one of the experimental groups treated subcutaneously with either isotype-matched mouse immunoglobulin (group 1: control, n = 8) or monoclonal mouse antibody against VEGF-A (anti-VEGF Mab; group 2: 10 mg on day 5 after ovulation, n = 8; group 3: 20 mg on day 5 after ovulation, n = 8; group 4: 10 mg on day 10 after ovulation, n = 4; group 5: 10 mg on days 5 and 10 after ovulation, n = 8). Anti-VEGF Mab-treated animals in groups 2–4 did not show any marked inhibition in pregnancy establishment. On pooled analysis, however, anti-VEGF Mab administration in groups 2–5 (n = 28) resulted in a significant (P < 0.04) decline in the number of viable term pregnancy when compared with control animals. The observed difference was explained by the fact that 10 mg anti-VEGF Mab given to each animal on days 5 and 10 after ovulation in group 5 (n = 8) inhibited pregnancy establishment significantly (P < 0.02) when compared with control group 1. There was no significant change in serum concentrations of estradiol-17β, progesterone, and free VEGF among groups. Furthermore, animals treated with anti-VEGF Mab (n = 8) as in group 5 revealed marked decrease in immunoreactive VEGF, fms-like tyrosine kinase-1, and kinase-insert domain region in trophoblast cells associated with shallow uterine invasion on day 13 of gestation when compared with samples from control group animals (n = 8). Thus, VEGF action is required for successful blastocyst implantation in the rhesus monkey.

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Manabu Ozawa, Qi-En Yang and Alan D Ealy

The overall aim of this work was to examine the expression profiles for fibroblast growth factor receptors (FGFRs) and describe their biological importance during bovine pre- and peri-implantation conceptus development. FGFR1 and FGFR2 mRNAs were detected at 1-, 2-, 8-cell, morula and blastocyst stages whereas FGFR3 and FGFR4 mRNAs were detected after the 8-cell stage but not earlier. The abundance of FGFR1, FGFR3, and FGFR4 mRNAs increased at the morula and blastocyst stages. Immunofluorescence microscopy detected FGFR2 and FGFR4 exclusively in trophoblast cells whereas FGFR1 and FGFR3 were detected in both trophoblast cells and inner cell mass in blastocysts. Neither transcripts for FGF10 nor its receptor (FGFR2b) were temporally related to interferon τ (IFNT) transcript profile during peri- and postimplantation bovine conceptus development. A series of studies used a chemical inhibitor of FGFR kinase function (PD173074) to examine FGFR activation requirements during bovine embryo development. Exposing embryos to the inhibitor (1 μM) beginning on day 5 post-fertilization did not alter the percentage of embryos that developed into blastocysts or blastocyst cell numbers. The inhibitor did not alter the abundance of CDX2 mRNA but decreased (P<0.05) the relative abundance of IFNT mRNA in blastocysts. Exposing blastocysts to the inhibitor from days 8 to 11 post-fertilization reduced (P<0.05) the percentage of blastocysts that formed outgrowths after transfer to Matrigel-coated plates. In conclusion, each FGFR was detected in bovine embryos, and FGFR activation is needed to maximize IFNT expression and permit outgrowth formation.