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S. J. Kimber and S. Lindenberg

Summary. Ovariectomy and hormone replacement of mice were used to examine the hormonal control of expression on the uterine surface of a carbohydrate determinant (lacto-N-fucopentaose I, LNF I), involved in the initial interaction between the blastocyst and the endometrial epithelium at implantation. Pseudopregnant mice mated with sterile males were also used to elucidate the impact of embryonic signals on the expression of this antigen on the uterine surface. Two groups of fucosylated structures could be distinguished; one group was predominantly dependent on maternal oestrogen and progesterone, while the other group appeared to be less influenced by the hormonal milieu.

Keywords: implantation; uterus; hormones; carbohydrates; receptors; mouse

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S. Lindenberg, S. J. Kimber, and E. Kallin

Summary. Mouse blastocysts bound LNF I conjugated to BSA-FITC or HSA-FITC and binding was inhibited by LNF I-HSA and to some extent by free LNF I, suggesting that the trophectoderm carries receptors specific for LNF I-like structures previously shown to be involved in implantation.

Keywords: embryo; implantation; neoglycoprotein; receptor; trophoblast, mouse

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I. M. Illingworth and S. J. Kimber

The carbohydrate H-type-1 antigen has been implicated in attachment of the murine blastocyst to the endometrial epithelium. Monoclonal antibody 667/9E9 was used to investigate the steroidal dependency of expression of this antigen in the murine endometrial epithelium using intact or ovariectomized mice treated with oestrogen or the pure anti-oestrogen, ICI 182,780. The effects of this anti-oestrogen were also investigated in the endometrial epithelium from intact rats. In both ovariectomized, oestrogen-supplemented and intact mice after treatment with ICI 182,780, staining with monoclonal antibody 667/9E9 was abolished in the luminal epithelium on both the apical and lateral cell surfaces, whereas basal staining remained. Glandular staining in mice was not affected in the same manner. In intact rats, where H-type-1 antigen expression has been reported to be predominantly controlled by progesterone, the anti-oestrogen had little effect, in accordance with previous reports.

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S. J. Kimber, I. M. Illingworth, and S. R. Glasser

Monoclonal antibodies were used to examine the expression of a number of carbohydrate antigens in the rat endometrial epithelium from day 1 to day 8 of pregnancy and in ovariectomized rats supplemented with ovarian steroids. Carbohydrate antigens based on the Galβ1–GlcNAc backbone structure were expressed and some of these (Ley, Lex, B antigen) were present at all stages of pregnancy and independent of ovarian steroids. The H-type-2 antigen was stimulated by progesterone and expressed in the sensitized and receptive uterus, but was not detected after implantation or, in ovariectomized rats, in the refractory phase. The H-type-1 antigen, which is stimulated by oestrogen in the mouse, appeared to be stimulated by progesterone in rats. It was expressed throughout the period of pregnancy but maximal expression was found on days 4–5. The histo-blood group A antigen appeared in ovariectomized rats only after treatment with progesterone followed by three daily injections of progesterone and oestrogen, and in the corresponding postimplantation period of pregnant rats. Its appearance corresponded to the loss of detectable H-type-2 antigen. This study shows that the rat endometrial epithelium expresses some carbohydrate antigens not expressed in mice (A and antigen) or under completely different steroidal regulation (H-type-1). Moreover, the antigen was expressed on the endometrial epithelium adjacent to decidua on days 7 and 8 of pregnancy, but not in rats given ovarian steroids to mimic the sensitized, receptive or refractory phase. Differences in expression between glandular and luminal epithelia indicated differences in steroidal regulation, as found in mice.

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S. Lindenberg, K. Sundberg, S. J. Kimber, and A. Lundblad

Summary. Seven oligosaccharides isolated from human milk were tested for their effect in an in-vitro model of mouse blastocyst adhesion and trophoblast outgrowth on endometrial epithelial monolayers. One compound, lacto-N-fucopentaose I (LNF I), produced a significant reduction in the percentage of attached and outgrown blastocysts after co-culture for 72 h (P < 0·001). No significant effect of any other tested oligosaccharide was obtained.

Keywords: trophectoderm; uterine–epithelial cells; blastocyst; oligosaccharides; attachment; in vitro

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L Mohamet, J K Heath, and S J Kimber

Uteri of Lif null mice do not support embryo implantation. Since deletion of some genes often prevents the survival of null mice to adulthood, we have used a proven inhibitor of leukaemia inhibitory factor (LIF) signalling to identify the precise window of time during which LIF is required in vivo, and assessed the cellular expression of several LIF-associated targets. On day 4 of pregnancy, mice were injected with hLIF-05 (inhibitor) into the uterine lumen, with corresponding volumes of PBS (vehicle) injected into the contralateral horn. On days 5 and 6, the number of implantation sites was recorded and the uteri processed for immunohistochemistry. Blockade of LIF on day 4 reduced embryo implantation by 50% (P≤0.0001) and was effective maximally between 0930 and 1230 h. Antagonism of LIF signalling was evidenced by a lack of phosphorylated STAT3 in the luminal epithelium (LE). Amphiregulin was absent from the LE on day 4 evening and H-type-1 antigen expression was retained in the LE on day 5 in inhibited uteri. Interleukin-1α and oncostatin M expression were reduced in the stroma on day 6, following LIF inhibition. Unexpectedly, PTGS2 expression in stroma was unaffected by LIF inhibition in vivo, in contrast to Lif null mice. In summary, this suggests that LIF signalling is effective for implantation during a discrete time window on day 4 and antagonism of LIF signalling recapitulates many features exhibited in Lif null uteri. The data presented validates the use of antagonists to investigate tissue specific and temporal cytokine signalling in reproductive function.

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H Singh, L Nardo, S J Kimber, and J D Aplin

Our limited understanding of the processes underlying steroid hormonal control of human endometrial receptivity is largely due to the lack of a relevant model system. To overcome scarcity of material, we have developed a model in which mouse embryos attach to human Ishikawa cells, which express functional steroid hormone receptors. Blastocysts flushed from day 4 pregnant superovulated mice were transferred to confluent Ishikawa cell monolayers. After 48 h of co-culture, 85% of the blastocysts had attached loosely, but only 40% attached stably to the epithelial cell surface. In contrast, 95% of the embryos attached stably to tissue culture plastic. Thus, weak attachment of a majority of the embryos was followed by stronger adhesion of a smaller proportion. Seventeen percent of the transferred blastocysts modified the epithelial cell surface with loss of MUC1 at the attachment site, extending variably to adjacent epithelial cells. Initially, stable attachment occurred without disruption to the integrity of the epithelial monolayer, but at later stages after the embryo had spread laterally, displacement of subjacent cells was observed. A modest increase in stable attachment, but no changes to MUC1 clearance, was observed after assisted hatching. After 24 h priming of Ishikawa cells by 17β-oestradiol (OE2) followed by 72-h incubation with medroxyprogesterone acetate and OE2, stable attachment increased from 40 to 70%. Initial attachment is efficient either in the presence or in the absence of hormone; steroid treatment increased the incidence of stable attachment. Implantation failure is predicted to occur in this model when embryos fail to progress from initial to stable attachment.

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A H K El-Hashash and S J Kimber

Differentiation of trophoblast giant cells is an early event during the process of murine embryo implantation. However, differentiation of secondary trophoblast giant cells in the rodent is still only partially understood, probably because of the lack of suitable in vitro models and cell markers. In order to advance our understanding of trophoblast differentiation, suitable in vitro models and markers are required to study their development. The objectives of this study were to establish and characterise a serum-free in vitro model for murine secondary trophoblast cells. Secondary trophoblast giant cells growing in vitro and paraffin sections of day 8.5 postcoitum mouse embryos were processed for immunostaining to establish the expression of potential markers using antibodies to blood group antigens, E-cadherin, α7 integrins and activator protein-γ, as well as placental lactogen-II. Within 3 days in serum-free culture, ectoplacental cone-derived secondary trophoblast cells underwent simultaneous induction of both morphological and functional differentiation. Secondary trophoblasts grew in vitro as a monolayer of cells with giant nuclei and expressed B and Le-b/Le-y blood group antigens, α7 integrins and placental lactogen-II, as well as activator protein-γ. Transcripts for activator protein-γ and placental lactogen-II were detected in cultures by RT-PCR and for placental lactogen-II by in situ hybridisation. At later time-points apoptosis increased. A fibronectin substrate significantly increased secondary trophoblast cell numbers and surface area of outgrowth. The increase in cells with giant nuclei coincided with induction of placental lactogen-II expression. A relationship was found between the nuclear area of secondary trophoblast cells and expression of placental lactogen-II.

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C. J. P. Jones, S. J. Kimber, I. Illingworth, and J. D. Aplin

Biotinylated lectins from Sambucus nigra (SNA) and Maackia amurensis (MAA), which bind to α2,6-linked and α2,3-linked sialyl residues, respectively, were used as probes to study glycan terminal modifications associated with decidualization in the uterine stroma of pregnant rats and mice. Binding of lectins from Erythrina cristagalli (ECA), Phaseolus vulgaris (leukoagglutinin, L-PHA), Triticum vulgaris (WGA) and Bandeiraea simplicifolia (BSA-1B4) was also examined. Tissues from rats between day 5 and day 8 of gestation and mice between day 5 and day 7 of gestation were fixed in Bouin's solution and embedded in wax prior to lectin histochemistry. On day 7 in rats and day 6 in mice, there was a marked reduction in the binding of SNA in the subluminal decidua surrounding the implantation site. In rats, MAA binding to enlarged decidual cells around the implantation chamber was increased markedly, but there was no change in mice. In both species there was de novo binding of ECA in the SNA-negative area, suggesting that the loss of α2,6-linked sialyl residues unmasks terminal N-acetyl lactosamine. These findings are consistent with previous evidence of a close structural and functional similarity between the artificially induced deciduoma and true decidua of rats and show identical changes to the glycosylation patterns previously found in differentiating rat deciduoma. In both species, therefore, decidua exhibits regionally specific terminal glycosylation. However, the species-specific expression of α2,3-linked sialyl residues suggests distinct patterns of steroidally modulated sialyl transferase expression.

Open access

Adam J Watkins, Emma S Lucas, Stephanie Marfy-Smith, Nicola Bates, Susan J Kimber, and Tom P Fleming

Mammalian placentation is dependent upon the action of trophoblast cells at the time of implantation. Appropriate fetal growth, regulated by maternal nutrition and nutrient transport across the placenta, is a critical factor for adult offspring long-term health. We have demonstrated that a mouse maternal low-protein diet (LPD) fed exclusively during preimplantation development (Emb-LPD) increases offspring growth but programmes adult cardiovascular and metabolic disease. In this study, we investigate the impact of maternal nutrition on post-implantation trophoblast phenotype and fetal growth. Ectoplacental cone explants were isolated at day 8 of gestation from female mice fed either normal protein diet (NPD: 18% casein), LPD (9% casein) or Emb-LPD and cultured in vitro. We observed enhanced spreading and cell division within proliferative and secondary trophoblast giant cells (TGCs) emerging from explants isolated from LPD-fed females when compared with NPD and Emb-LPD explants after 24 and 48 h. Moreover, both LPD and Emb-LPD explants showed substantial expansion of TGC area during 24–48 h, not observed in NPD. No difference in invasive capacity was observed between treatments using Matrigel transwell migration assays. At day 17 of gestation, LPD- and Emb-LPD-fed conceptuses displayed smaller placentas and larger fetuses respectively, resulting in increased fetal:placental ratios in both groups compared with NPD conceptuses. Analysis of placental and yolk sac nutrient signalling within the mammalian target of rapamycin complex 1 pathway revealed similar levels of total and phosphorylated downstream targets across groups. These data demonstrate that early post-implantation embryos modify trophoblast phenotype to regulate fetal growth under conditions of poor maternal nutrition.