The embryonic capsule, which covers the equine blastocyst after it loses its zona pellucida, is composed of mucin-like glycoproteins. In the present study, we investigated both macroscopic and molecular changes in the capsule during development. The weight of the capsule increased from day 11\p=n-\12 of pregnancy and reached a maximum at about day 18, coinciding with the time during which the conceptus migrates extensively throughout the uterus. The sialic acid content of the capsule declined markedly from about day 16, the time of conceptus 'fixation' in the uterus, which suggests a unique developmentally regulated mechanism for the control of embryo mobility. These results lead us to propose that the capsule may have an anti-adhesion function in the developing conceptus, and that this effect could be regulated by the sugar side chains of the capsular glycoproteins. The glycosylation characteristics of the blastocyst coverings also underwent changes at about day 9 of pregnancy, which may be related to loss of the zona pellucida. An anti-capsule monoclonal antibody was raised and shown to recognize a tissue-specific antigen present only on the capsule and trophoblast. This antigen was present on the trophoblastic cells soon after the blastocyst is formed, reached a maximum concentration at about day 18, and was absent after day 22, coinciding with the disappearance of the capsule. Immunohistochemical studies indicate that the mucinlike capsular glycoproteins are secreted, at least in major part, by the trophoblast. The pattern of secretion of these glycoproteins and the coincidence of molecular changes with important events in early pregnancy strongly suggest that they play crucial roles in early embryonic development.
The equine embryonic capsule replaces the zona pellucida and envelopes the conceptus during the second and third weeks of pregnancy. Although this capsule was described more than 100 years ago, its molecular structure has not been characterized. Here we present evidence that the glycoprotein(s) of the equine capsule resembles those of the mucin glycoprotein family. The resistance of the capsule to chemical and enzymatic solubilization was confirmed, and, as in mucins, protein constituted only 35-40% of its total dry mass. Determination of the sugar composition of the capsule using colorimetric assays and high-performance anion-exchange chromatography also showed it to have mucin-like characteristics. Gal, GalNAc, sulfated sugars, and sialic acid make up a high proportion of the capsular carbohydrate, while GlcNAc, Glc, and Man are minor components. These findings were verified using lectin histochemical staining of frozen sections of conceptuses. The results of amino acid analysis were also consistent with the proposal that the capsular glycoproteins belong to the mucin family. Removal of the covalently bound carbohydrate by beta-elimination under reducing conditions demonstrated that the capsule is O-glycosylated mainly on threonine residues. Affinity chromatography on jacalin-agarose confirmed that, like mucins, the capsular glycoproteins are heavily O-glycosylated. SDS-PAGE analysis revealed a prominent 21-kDa band, specific to the capsule, in preparations solubilized by trypsin but not by other proteases. Characterization of its constituent glycoprotein(s) should be helpful in elucidating the role of the capsule (and analogous blastocyst coverings in other species) during early pregnancy.
State of latency, well known for several herpesviruses, has been proposed for equine herpesvirus-3 (EHV-3) and supported by epidemiological observations. No detailed assessment about reactivation, patterns of excretion and reexcretion has been formally reported. An experimental reactivation study by corticosteroid treatment in previously naturally infected horses was therefore carried out. Two polo mares with clinical and virologically confirmed history of equine coital exanthema were injected with dexamethasone and prednisolone on 3 successive days. Clinical signs, body temperature and clinical samples for virological and serological studies were obtained daily. Mares did not show any systemic clinical signs or hyperthermia. EHV-3 shedding, seroconversion and the presence of a small lesion were observed in one of the mares under study 2 weeks after corticosteroid treatment. The results demonstrate that this virus exhibits a latency-reactivation behaviour similar to that of other alpha herpesviruses. Reactivation of latency may have an important bearing on the appearance of clinical signs in mares and/or stallions during the breeding season without the actual evidence of transfer from mare to stallion or vice versa.
This research tested the hypothesis that immunological sensitization of mares by skin allografting, followed by the establishment of pregnancy using semen from the skin-graft donor, would give rise to secondary immune responses to the developing horse conceptus, resulting in an earlier demise of the fetally derived endometrial cups. Maiden mares received skin allografts from a stallion homozygous for Major Histocompatibility Complex (MHC) antigens and/or equivalent autografts and were subsequently mated to the skin-graft donor stallion during the next two breeding seasons. Mares that had been immunologically primed to the foreign MHC class I antigens of the skin-graft donor stallion developed strong secondary antibody responses early in their first pregnancies, whereas autografted mares made weak primary antibody responses in their first pregnancies and strong secondary responses in their second pregnancies. In contrast, histological examination of the endometrial cups after surgical pregnancy termination at Day 60 of gestation revealed no discernible differences between allografted and autografted mares, and there were no significant differences in the concentrations and/or duration of secretion of the endometrial cup-specific hormone, equine chorionic gonadotrophin (eCG), between allografted and autografted mares, nor in either group between first and second pregnancies. The vigorous antibody response observed in the pregnant allografted mares supported the first part of our hypothesis, providing evidence of systemic immunological priming. However, there was a lack of an equivalent heightened cellular response to the endometrial cups. These findings provided strong evidence for an asymmetric immune response to the conceptus, characterized by strong humoral immunity and a dampened cellular response.
The equine embryonic capsule, an acellular covering that envelops the conceptus during the second and third weeks of pregnancy, is composed of mucin-like glycoproteins. Its structure is consistent with a dual role during early pregnancy: protection of the conceptus, and communication between the embryo and the mother. Loss of sialic acid from the capsular glycoproteins at day 16 correlates with the time of "fixation," or loss of conceptus mobility throughout the uterine horns. This study investigated how the structure of the capsule is linked to the maintenance of pregnancy. Six pregnancies, confirmed by ultrasound, were terminated by prostaglandin injection on day 14, prior to the time of embryo fixation. These "defective" conceptuses were collected at day 17, and the structure and molecular properties of their capsules were compared to those of day 17 conceptuses collected from 5 normal pregnancies. Defective capsules were not significantly different from normal capsules in terms of dry weight, amino acid composition, and content of neutral and amino sugars. However, defective capsules failed to show the loss of sialic acid normally occurring around the time of embryo fixation. Analysis of the capsular mucins following trypsin digestion was carried out by radioactive labeling with 3H on sialyl-oligosaccharides and 125I on tyrosine residues, followed by fast protein liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Differences in the trypsin fragmentation patterns indicated increased susceptibility of the defective capsules to proteolysis. We conclude that there is a temporal association between desialylation of the equine capsule and embryonic survival, and that failure to desialylate alters the properties of the capsule.
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