Reproduction in Domestic Animals

Proteins in Early Equine Conceptuses 235pI 3 11p19p17p101 2 3 4 5 6 7 8p6pI 5.0 pI 5.8Fig. 4. Silver-stained 2D SDS–PAGE reducing gel of proteins inuterine flush collected from a control mare on day 16.5. Two bands inthe 6 kDa region were identified by MS ⁄ MS sequencing as forms ofuteroglobin. The major band at pI 4.9–5.0 and the minor band at pI5.8–6.0 both contained sequences of products of two uteroglobin genes(see Table 1)Fig. 3. Silver-stain (a) and immunoblot with antibody to a syntheticuteroglobin peptide (b) of 15% SDS-PAGE reducing gel of proteins inuterine flush samples collected on day 16.5 from control (lanes 1–4)and PGF 2a -treated (lanes 5–8) pregnant maresstronger signal in treated mares than in normal mares(Fig. 3b). This suggested that the form of uteroglobinthat increased after PGF 2a was different from theuteroglobin band in the normal pregnant uterus. By2D PAGE, the p6 bands migrated as a major form at pI4.9–5.0 and a minor form at pI 5.8–6.0 (Fig. 4), both ofwhich were identified as uteroglobin by MS ⁄ MSsequencing of in-gel trypsin digests (Table 1). Bothspots were composed of two forms, based on amino acidsequences predicted from two uteroglobin horse genomicsequences (GenBank 124072669). Two peptides ofmasses 932.547 (KTLVDFLPKN) and 1292.622(AVEPFKPDADMKA) were consistent with horsep6uteroglobin reference sequence NP_001075327 andsecretoglobin ⁄ CCSP of equine lung origin (AAW83215,AAW83216, AAW83215 and AAW83218). By comparison,two other sequenced peptides of masses 1362.712(KLMDKIAKSPLCA) and 1391.705 (KLMDKIVESPLCA) present in the major pI 4.9–5.0 spot indicatedthat two secretoglobins in the whole genome shotgunsequence of the horse (gi 124072669) were expressed.The increase in immunoreactive uteroglobin after luteolysiswas attributed to a selective increase in thesecretoglobin 1A1 form (NP_001075327) that includesthe peptide with tyrosine rather than alanine at position59 used to generate the antibody (Katavolos 2006).The minor spot (pI 5.8–6.0) was increased threefold(p = 0.025) at day 18 in the treated mares in comparisonwith control mares (N = 3 per group), as quantifiedby 2D-DIGE.DiscussionThese studies were conducted to identify potential proteinbiomarkers that might distinguish successful and unsuccessfulfixation of the equine conceptus, or that might helpto explain the molecular basis of these processes. For thisTable 1. Amino acid sequences of two forms of uteroglobin in uterine flush fluid from a day 15 normal pregnancySpot A,pISpot B,pIMeasuredmassPeptide sequence determinedby MALDI-TOF and MS ⁄ MSSimilarity with knownuteroglobin ⁄ CCSP sequences4.9–5.0 5.8–6.0 gi:20385506,gi:126352306gi:58978622, gi:58978613,gi:58978651, gi:58978641+ + 932.551 (K)TLVDFLPK(N) Yes Yes+ 1275.740 (K)TLVDFLPKNTK(D) Yes No+ + 1292.625 (A)VEPFKPDADMK(A) Yes Yes+ 1362.712 (K)LMDKIAKSPLCA(–) Yes No+ + 1391.705 (K)LMDKIVESPLCA(–) No Yes+ 1832.060 (K)TLVDFLPKNTKDSILK(L) Yes NoÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag