Proceedings of the 5th International Symposium on EQUINE ...

Equine Embryo TransferPRODUCTION OF CAPSULAR MATERIAL BY EQUINETROPHOBLAST TRANSPLANTED INTOIMMUNODEFICIENT MICEA. Albihn, J. Samper*, J. G. Oriol, B. A. Croy and K. J. BetteridgeDepartment <strong>of</strong> Biomedical Sciences, Ontario Veterinary College; *The Equine Research Centre,University <strong>of</strong> Guelph, Guelph, Ontario, N1G 2W1, CanadaThe equine embryonic capsule is presumedessential to normal embryonic development(Betteridge 1989; Stout et al. 1997). It also seemsto impede <strong>the</strong> successful freezing <strong>of</strong> equineembryos (Bruyas 1997). The capsule is consideredto be produced largely by <strong>the</strong> trophoblast (Oriol etal. 1993) but, because production <strong>of</strong> this mucinlikeglycoprotein by equine embryos has not so farbeen demonstrated in vitro (Betteridge 1989;McKinnon et al. 1989), this assumption is difficultto prove. The present study was <strong>the</strong>refore designedto test <strong>the</strong> hypo<strong>the</strong>sis that capsular material isproduced by <strong>the</strong> trophoblast, independently <strong>of</strong>maternal contributions. To do so, xenogeneictransplantation <strong>of</strong> equine endometrium and/ortrophoblast into mice with severe combinedimmunodeficiency (SCID; scid/scid orscid/scid.bg/bg mice; Croy and Chapeau 1990)was used as an ‘in vivo culture system’.To develop <strong>the</strong> procedures (Experiment 1),endometrial biopsy samples from dioestrous mareswere partly infiltrated with India ink, <strong>the</strong>n used toprepare multiple 1 mm 3 grafts. These weresurgically transplanted into various sites in 18mice for 4, 8 or 16 days. The results <strong>of</strong> Experiment1 are summarised in Table 1.Overall, 23/52 (44%) grafts were recovered inhistological sections, proportionately more from<strong>the</strong> ovarian fat pad or kidney capsule than from <strong>the</strong>uterine lumen. Histomorphology was wellmaintained for up to 16 days and ink staininghelped recovery (78% recovery for ink-stainedversus 26% for non-stained grafts). Technicaldifficulties ra<strong>the</strong>r than graft rejection mostprobably accounted for <strong>the</strong> failure to find somegrafts at <strong>the</strong> time <strong>of</strong> euthanasia. Thus,xenotransplantation into SCID mice was shown tobe a useful culture system for grafted equineendometrium.In Experiments 2 and 3, endometrial biopsysamples and conceptuses from 5 mares 13–15 daysafter ovulation were used to prepare grafts <strong>of</strong>endometrium (E), trophoblast (T) and capsule (C)for transplantation into 59 mice. At this stage <strong>of</strong>development, ‘trophoblast’ would have comprisedtrophectoderm, endoderm and possibly mesoderm.Grafts recovered at <strong>the</strong> time <strong>of</strong> euthanasia wereexamined for <strong>the</strong> presence <strong>of</strong> capsule-like materialei<strong>the</strong>r histochemically (Experiment 2, 20 mice) orimmunohistochemically (Experiment 3, 39 mice).PAS staining was used in Experiment 2, a mousemonoclonal antibody against equine capsule(MAb OC-1; Oriol et al. 1993) in Experiment 3.The overall graft recovery rate in Experiment2 was 22/49 (45%; 11/28 single grafts, and 11/21when E and T were co-engrafted). Capsule-likeextracellular glycoprotein at <strong>the</strong> graft site wasidentified by PAS staining <strong>of</strong> histologicalsections as summarised in Table 2. Strong PASpositivereactions (5–7 mm thick) were found inTABLE 1: Graft recovery rates in Experiment 1Graft recovery ratesNo. mice Ink infiltration Kidney Uterine lumen Ovarian fatpad Total %12 - 3/11 3/12 3/11 9/34 26%6 + 6/6 2/6 6/6 14/1878%189/17 5/189/17 23/52 44%60