Reproduction in Domestic Animals

234 MA Hayes, BA Quinn, ND Keirstead, P Katavolos, RO Waelchli and KJ Betteridgewith trypsin fragment masses consistent with uterocalin.By day 18, most of the p10 form of b 2 M in the capsulehad been converted to the p8 form (D9-b 2 M) that lacksnine amino acids from the N-terminus (Quinn et al.2007).A band of 17 kDa (p17) was observed around thetime of fixation in normal capsules (Fig. 1), consistentwith previous studies (Quinn et al. 2007). This wasmarkedly increased in capsules from mares that weretreated with PGF 2a (Fig. 1). The p17 band of a day 18conceptus from a treated mare was tentatively identifiedas a secretory phospholipase A2 (sPLA2) based onhomology of two partial sequences (K)LLNYKFSYRand (K)YQYYNNK determined by MS ⁄ MS. Thesesequences were most similar to a rat sPLA2 (gi: 220858),the cDNA sequence of which was used to identify asingle cDNA sequence from an equine articular cartilagecDNA library (gi: 57706753). Primers designed from thissequence were used to amplify a complete cDNAsequence from cDNA prepared by RT-PCR of RNAextracted from endometrium from a normal pregnantmare (NM_001100113). This is 100% identical with acoding sequence in chromosome 2 of the horse genome(chr2:32592684–32595740). The predicted amino acidsequence included the two partial sequences we identifiedby MS ⁄ MS of the p17 band. Also, MALDI-TOFanalysis of trypsin digested p17 matched six peptidemasses of a theoretical digest of the predicted sPLA2protein from our cDNA sequence. The predictedsequence also had homology with an N-terminalsequence (XXLXFXKMIXLMTGKQAT) reported fora 17 kDa progesterone-dependent secreted phospholipasein the equine uterus (Beier-Hellwig et al. 1995).Polyclonal antibodies generated against a syntheticpeptide (KEATSSYGFYGC) of our sequence alsorecognized p17 in immunoblots of capsules from normaland treated mares (not shown).The p17 band was not detected in 2D-PAGE ofcontrol capsules (Fig. 2a,b). However, in capsules fromtreated mares, a single 17 kDa spot was present at pI 9.8on days 16 (Fig. 2c) and 18 (not shown).By 2D-PAGE of pooled days 13–15 control capsuleextracts, a 19 kDa uterocalin spot was observed at pI 9.5(Fig. 2a). The p19 spot was less prominent in control(Fig. 2b) and treated days 16 and 18 capsules (Fig. 2c),whereas a doublet of smaller spots observed at pI 9.5were consistent with uterocalin determined by MS ⁄ MSsequences (Fig. 2b).Before fixation (Fig. 2a), there were three prominent10 kDa bands and one 8 kDa band in the pI rangeof 5.0–6.5 that were identified as forms of b 2 M byMS ⁄ MS peptide sequences of in-gel trypsin digests.After fixation, the 10 kDa forms of b 2 M decreasedwhereas the 8 kDa form became more prominent(Fig. 2b).To evaluate the changes in uterine production ofsPLA2, we examined concentrated uterine flush fluidsamples from control and treated pregnancies. At day 16of gestation, flush samples from control mares containedvariable amounts of a 19 kDa band (Fig. 3a), consistentwith uterocalin by immunoblotting (Quinn et al. 2007)and by MALDI-TOF analysis of in-gel trypsin digests.By comparison, there was a less abundant band atpI 5 7 11p19p17p10p19p17p10p19p17p10Fig. 2. Silver-stained 2D SDS–PAGE reducing gel of proteins elutedfrom equine embryonic capsules. (a) Controls pooled from days 13.5 to15.5; (b) control from day 16; (c) treated from day 166 kDa (Fig. 3a), that migrated to the position ofimmunoreactive uteroglobin also found in previousstudies of uterine flush samples (Quinn et al. 2007).Uteroglobins migrate faster than expected for theircalculated molecular masses (Mu¨ller-Scho¨ttle et al.2002; Mukherjee et al. 2007). The p6 uteroglobin bandswere moderately increased in intensity in the silverstained gels of uterine flush fluids from mares that weretreated with the PGF 2a analogue compared with normalmares (Fig. 3a). However, in Western blots with polyclonalantibodies raised against the EPSKPDADMKAATTQLKTLV, uteroglobin bands gave a markedlyÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag