The expression of mucin genes and the presence of mucin gene products in the equine endometrium Eva Maischberger a,1 , Carolyn A. Cummins a , Eamonn Fitzpatrick a , Mary E. Gallagher a , Sheila Worrall a , Karine Rousseau b , David J. Thornton b , Wim G. Meijer c , Raúl Miranda-CasoLuengo c , Vivienne E. Duggan a , Stephen D. Carrington a , Jane A. Irwin a,⇑ , Colm J. Reid a a School of Veterinary Medicine, Veterinary Sciences Centre, University College Dublin, Belfield, Dublin 4, Ireland b University of Manchester, Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, The Michael Smith Building, Manchester M13 9PT, United Kingdom c School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland article info Article history: Received 17 July 2012 Accepted 17 March 2013 Keywords: Equine mucin genes Equine female reproductive tract Oestrous cycle Reverse transcriptase polymerase chain reaction In situ hybridisation abstract In the equine reproductive tract, little is known about mucin gene expression and the role of mucins in barrier function and host-cell interaction. The aims of the study were to identify equine orthologs of mammalian mucin genes using available equine sequence data, to profile expression of equine ortholo- gous mucin genes in the endometrium using reverse transcriptase polymerase chain reaction (RT-PCR), to determine spatial expression patterns of mucin genes using in situ hybridisation, and to confirm the pres- ence of mucin gene products using Western blotting and equine-specific mucin antibodies during oestrus and dioestrus. While the mucin gene expression pattern in equine endometrium is similar to that of other mammals, several mucins appear to be uniquely expressed in this tissue (eqMUC3B, 7, 18, and 20) and one is hormonally regulated (eqMUC3B). Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction The lining epithelium of the equine uterus is comprised of mu- cus-secreting and ciliated cells. The mucus-secreting cells are found both in the surface endometrium and also within endome- trial glands, located in the underlying lamina propria. The secre- tions of these cells lubricate and hydrate the mucosal surface, and protects it against infection (Causey, 2007). The endometrium also plays an important role during embryo implantation and development, and acts as a habitat for commensal bacteria when the cervix is open (Lagow et al., 1999; Rutllant et al., 2005). Mucus forms a semi-adhesive, enzyme-resistant barrier against pathogens and toxins (Lagow et al., 1999; Hattrup and Gendler, 2008; Thorn- ton et al., 2008). In acute and sub-acute endometritis, mucus pro- duction by the endometrium increases, and changes in the production, elasticity or viscosity of this mucus may interfere with mucociliary clearance and hence fertility (Causey, 2007; LeBlanc, 2010). Consequently, the characterisation of equine endometrial mucin genes will provide an understanding of the composition of mucus in normal, healthy mares, and the basis for further studies dealing with the role of mucus in reproductive diseases. The dry matter of mucus is composed largely of mucin glyco- proteins. Due to their high content of O-linked oligosaccharides (Lagow et al., 1999; Hebbar et al., 2005), they bind substantial amounts of water upon secretion to generate a hydrated biopoly- meric mucus gel (Andersch-Bjorkman et al., 2007). This gel is a dy- namic and responsive part of the mucosal barrier. Mucin core proteins contain domains comprised of variable numbers of tan- dem repeat (VNTR) sequences that contain proline and are rich in serine and/or threonine (Lagow et al., 1999; Rose and Voynow, 2006; Andersch-Bjorkman et al., 2007), which are linked to O-gly- cans. These heavily glycosylated domains are flanked by unique, non-repetitive NH 2 - and COOH-terminal domains, which are un- ique in sequence and length for each mucin (Hebbar et al., 2005; Rose and Voynow, 2006; Hattrup and Gendler, 2008). Mucins occur as secreted gel-forming mucins, secreted non-gel- forming mucins, and membrane-bound mucins (Linden et al., 2008). The combination and relative amount of individual mucins in mucus are cell and tissue-specific. Their localisation within the epithelium also differs (Thornton et al., 2008). The cysteine-rich re- gions of the N- and C-termini of the large secreted gel-forming mucins MUC2, MUC5AC, MUC5B, MUC6, and MUC19 allow poly- merisation through inter-molecular disulphide bonds, accounting for their gel-forming ability (Gipson et al., 1997; Lagow et al., 1999; Rose and Voynow, 2006; Linden et al., 2008). Membrane- bound mucins consist of two subunits connected via sodium dodecyl sulphate (SDS)-labile bonds (Hattrup and Gendler, 2008). 0034-5288/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2013.03.012 ⇑ Corresponding author. Tel.: +353 1 7166216; fax: +353 1 7166237. E-mail address: jane.irwin@ucd.ie (J.A. Irwin). 1 Present address: University of Zurich, Vetsuisse Faculty, Clinic for Equine Internal Medicine, Winterthurerstrasse 260, 8057 Zurich, Switzerland. Research in Veterinary Science 95 (2013) 169–175 Contents lists available at SciVerse ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc