Modeling Trophoblast Differentiation using Equine Chorionic Girdle Vesicles

de Mestre, A.M.; Bacon, S.J.; Costa, C.C.; Leadbeater, J.C.; Noronha, L.E.; Stewart, F.; Antczak, D.F.

doi:10.1016/j.placenta.2007.10.005

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Volume 29, Issue 2 , Pages 158-169, February 2008

Modeling Trophoblast Differentiation using Equine Chorionic Girdle Vesicles

A.M. de Mestre
- Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
- Corresponding author. Department of Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London, NW1 0TU, UK. Tel.: +44 207 121 1906; fax: +44 207 7388 2342.
S.J. Bacon
- Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
- Current address: Department of Biological Sciences, Mount Holyoke College, South Hadley, MA, USA.
C.C. Costa
- Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
J.C. Leadbeater
- Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
L.E. Noronha
- Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
F. Stewart
- Thoroughbred Breeders’ Association Equine Fertility Unit, Mertoun Paddocks, Woodditton Road, Newmarket, CB8 9BH, UK
- Deceased.
D.F. Antczak
- Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA

Accepted 17 October 2007. published online 03 December 2007.

Abstract

The chorionic girdle of the equine conceptus is comprised of specialized trophoblast cells which, at day 36–38 of equine pregnancy, gain an invasive phenotype and invade the endometrium to form endometrial cups. Studies of equine endometrial cups remain difficult to perform because of the invasive techniques required to obtain cup tissue and because sampling requires termination of the pregnancy. In this study we developed a system to model trophoblast differentiation and trophoblast–immune interactions in vitro and in vivo. We utilized a method of culturing chorionic girdle pieces in serum-free medium to promote spontaneous formation of vesicle structures enriched for terminally differentiated binucleate cells that secreted equine chorionic gonadotrophin (eCG). Immunohistochemical staining and scanning electron microscopy showed that the cells of the vesicles closely resembled the outer layers of chorionic girdle immediately prior to invasion. Chorionic girdle vesicles were harvested after 72h in culture and ectopically transplanted via injection into the vulvar mucosa of recipient mares. At 7, 14, 21 and 28days after transplantation, biopsies of the injection sites were obtained. Immunohistochemical labeling of cryostat sections of the biopsies with a panel of monoclonal antibodies to horse trophoblast molecules demonstrated survival, differentiation, and presence of trophoblast cells for at least 21days. Serial sections of the biopsies labeled with antibodies to the equine lymphocyte surface markers CD4 and CD8, together with lymphocyte microcytotoxicity assays, revealed that the recipients mounted both cellular and humoral antibody immune responses to the transplanted trophoblast cells. This new method for culturing equine chorionic girdle trophoblast cells, and for transplanting trophoblast vesicles to ectopic sites, should allow identification of key aspects of trophoblast differentiation and the interactions that occur between invasive trophoblast and the maternal immune system.