Reproduction in Domestic Animals

236 MA Hayes, BA Quinn, ND Keirstead, P Katavolos, RO Waelchli and KJ Betteridgecomparison, we used an experimental model of PGF 2a -induced luteolysis that leads to failure of fixation anddelayed loss of sialic acid from the embryonic capsule(Chu et al. 1997; Betteridge et al. 2006). These studiesfollow on from a previous demonstration that b 2 Massociated with the normal capsule is truncated to an8 kDa form (D9-b 2 M) around the time of fixation (Quinnet al. 2007). The present studies show that b 2 M exists asmultiple 10 kDa forms and one 8 kDa form that can beseparated by 2D-PAGE, and that luteolysis has only aminor influence on proteolysis or binding of b 2 M associatedwith the capsule.The most obvious luteolysis-associated alteration incapsule proteins was an increase in a 17 kDa secretoryphospholipase A2 (sPLA2) classified as type IIA on thebasis of amino acid and cDNA sequence homology of thecDNA sequence we obtained from pregnant endometrium.Previous reports have noted the presence of apartially characterized PLA2 in the normal pregnantuterus (Beier-Hellwig et al. 1995; Quinn et al. 2007) butits function there is unknown. PLA2s are a complexgroups of enzymes that cleave glycerophospholipids atthe sn-2 position and release a free fatty acid, most oftenarachidonic acid which is the cyclooxygenase substrateprecursor of various eicosanoids. PLA2s can thereby havediverse signalling roles that are especially important ininflammation and haemostasis, and in the regulation ofovarian function, pregnancy and parturition (Tithof et al.2007). Secretory PLA2-IIA has a high pI and binds toanionic phospholipid interface rather than phospholipidsin intact cell membranes (Beers et al. 2003; Birts et al.2007). Human sPLA2-IIA also has an ability to bind toheparin-sulphate proteoglycans (Birts et al. 2007). Whilethe functions of sPLA2-IIA are still unclear, the evidencesupports the view that they relate to its role as an innateimmune protein involved in the catabolism of cell debris(bacteria, apoptotic cells) in extracellular locations (Birtset al. 2007). Equine uterine sPLA2 characterized in ourstudies also has a high pI of 9.8, binds to the embryoniccapsule and increases when fixation has been blocked byadministration of PGF 2a . It is therefore plausible thatsPLA2 contributes to the imminent removal and degradationof the capsule or the conceptus.Uterocalin is a well characterized lipocalin secreted byendometrial glands in the luteal stage of the ovariancycle and in the early stages of normal pregnancy inequids (Stewart et al. 2000; Suire et al. 2001; Kennedy2005). Uterocalin is also a highly cationic protein(pI 9.4 similar to that of sPLA2) (Fig. 2) and thisproperty likely explains why it also binds to theembryonic capsule. Recent evidence suggests that uterocalinis involved in the transport of small lipophilic,mainly nutrient, substances across the glycan capsuleand into the yolk sac (Suire et al. 2001; Kennedy 2005;Quinn et al. 2007). Amounts in the uterine flush werelower after treatment with PGF 2a , consistent withprogesterone dependence (Suire et al. 2001). Theamounts present in the capsule were variable and bydays 16 and 18, some had been proteolytically convertedto smaller fragments of approximately 10 kDa (Fig. 2b).It appears likely that this alteration is part of the processof degradation of the capsule rather than a processrelevant to its function.Observations in the present study indicate that thereare at least two uteroglobin genes expressed in theequine uterus during early pregnancy. The form thatwas more consistent immunoreactively with equineuteroglobin (Mu¨ller-Scho¨ttle et al. 2002) was increasedsubstantially after luteolysis. Uteroglobin was firstidentified as a low molecular weight secreted uterineprotein in rabbits and is the founding member of thelarge secretoglobin superfamily of proteins that alsoincludes Clara cell secretory proteins (CCSP) (Beier2000; Mu¨ller-Scho¨ttle et al. 2002; Mukherjee et al.2007). These proteins are expressed in various tissues,mainly in the lung and uterus. Uteroglobin (secretoglobin1A1) is expressed in various mammals including thehorse (Mu¨ller-Scho¨ttle et al. 2002) and there are threeclosely similar genes in the horse genome, located onchromosome 12. Previous studies showed that theuteroglobin that is detected in immunoblots with anantibody to a peptide sequence of uteroglobin ⁄ secretoglobin1A1 is more abundant in the non-pregnant uterusthan during the fixation period of normal pregnancy(Quinn et al. 2007). Uteroglobin has a small lipophilicbinding pocket (von der Decken et al. 2005) and canbind prostaglandins such as PGF 2a (Mukherjee et al.2007) but the significance of these properties is unclear.Secretoglobins can bind various small lipophilic moleculesincluding retinoids and polychlorinated biphenyls(von der Decken et al. 2005; Mukherjee et al. 2007).CCSP has anti-inflammatory functions in the respiratorytract, where it might bind lipid mediators andsPLA2 (Mukherjee et al. 2007). Further studies arerequired to determine whether there is a relationshipbetween the increases in sPLA2 and uteroglobin inresponse to luteolysis.These studies have employed analytical methods witha level of sensitivity most suitable for demonstration ofchanges in the most abundant proteins. It is expectedthat there are also alterations in many cytokines andenzymes that are not apparent in these approaches.Moreover, it is still unclear whether the modifications inproteins and glycans that can be observed in the capsuleduring and after the period of fixation are functionallyimportant in interactions between the endometrium andconceptus. Some of these changes might be part of thenormal removal of the capsule. However, the increase insPLA2-type IIA in the capsule and a change in theexpression of uteroglobin genes might have a role in thepending demise of the conceptus in some circumstances.Alternatively, these changes might reflect the return tothe non-pregnant post-luteolysis condition.AcknowledgementsThis research was supported by the Natural Sciences and EngineeringResearch Council of Canada (NSERC); the Grayson Jockey ClubResearch Foundation Inc.; Equine Guelph; The E.P. Taylor researchFoundation; and the Ontario Ministry of Agriculture, Food and RuralAffairs (OMAFRA). Natalie Keirstead was supported by a Fellowshipfrom the Canadian Institutes of Health Research (CIHR). We thankDorothee Bienzle, Jeff Caswell, Gordon Kirby and John Lumsden foruse of analytical equipment funded by the Canadian Foundation forInnovation (CFI). We are grateful for technical advice of DavidHobson and Paul Huber in relation to DIGE methods, and for MSanalysis by Li Zhang at the Advanced Protein Technology Centre,Ó 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag