RESULTS

Profile of mouse endometrial epithelial proteome during early pregnancy

Endometrial epithelial cells were collected from mice on Days 1 and 4 of pregnancy to examine protein expression using two-dimensional gel electrophoresis (2-DE), followed by colloidal Coomassie Brilliant Blue staining. Figure 1 shows a representative 2-DE reference map of the mouse endometrial epithelial proteome generated using pH 3–10 immobilized pH gradient strips and 10% SDS-polyacrylamide gels. Approximately 674 distinct protein spots were detected across samples collected on Days 1 and 4 of pregnancy.

Figure 1: Representative two-dimensional gels depicting the proteome profiles of mouse endometrial epithelial cells on Days 1 and 4 of pregnancy.

Quantitative changes in endometrial protein expression from Day 1 to Day 4 of pregnancy

To compare protein expression profiles between Days 1 and 4 of pregnancy, 2-DE gel images were analyzed using Melanie software (version 7). A total of 674 protein spots were consistently detected across all gels. Among these, 80 protein spots exhibited significant differential expression between the two time points (p < 0.05 and ≥2.0-fold change). The list of differentially expressed proteins is provided in Table 2. Of these, 42 proteins were upregulated on Day 4 (ratio <1), whereas 38 proteins were downregulated on Day 4 (ratio >1) relative to Day 1. Subsequent identification by MALDI-TOF/MS and MALDI-TOF/TOF analysis successfully characterized 52 of the 80 differentially expressed protein spots (Table 3). Importantly, this dataset represents a cell type–specific proteomic resource derived from isolated endometrial epithelial cells, providing a focused reference for molecular changes occurring at the embryo–maternal interface during the transition to uterine receptivity.

Spot No. | Day 1 volume (%) | Day 4 volume (%) | Ratio (Day 1/Day 4) | Regulation (Day 4 vs Day 1) | p-value
7 | 0.10 ± 0.04ᵇ | 0.52 ± 0.05ᵃ | 0.19 | Upregulated on Day 4 | 0.000283
11 | 0.76 ± 0.19ᵃ | 0.22 ± 0.18ᵇ | 3.45 | Downregulated on Day 4 | 0.023193
16 | 0.54 ± 0.12ᵃ | 0.27 ± 0.03ᵇ | 2.00 | Downregulated on Day 4 | 0.019474
23 | 1.21 ± 0.24ᵃ | 0.58 ± 0.13ᵇ | 2.08 | Downregulated on Day 4 | 0.016044
29 | 1.12 ± 0.24ᵃ | 0.43 ± 0.16ᵇ | 2.60 | Downregulated on Day 4 | 0.014381
33 | 0.10 ± 0.02ᵇ | 0.23 ± 0.07ᵃ | 0.43 | Upregulated on Day 4 | 0.040713
39 | 0.21 ± 0.02ᵃ | 0.10 ± 0.009ᵇ | 2.10 | Downregulated on Day 4 | 0.010155
46 | 0.32 ± 0.05ᵃ | 0.15 ± 0.08ᵇ | 2.13 | Downregulated on Day 4 | 0.000891
52 | 0.13 ± 0.02ᵃ | 0.06 ± 0.02ᵇ | 2.17 | Downregulated on Day 4 | 0.008505
55 | 0.19 ± 0.01ᵇ | 0.39 ± 0.04ᵃ | 0.49 | Upregulated on Day 4 | 0.025876
56 | 0.09 ± 0.04ᵇ | 0.28 ± 0.05ᵃ | 0.32 | Upregulated on Day 4 | 0.004535
57 | 0.09 ± 0.02ᵇ | 0.18 ± 0.04ᵃ | 0.50 | Upregulated on Day 4 | 0.005394
60 | 0.05 ± 0.01ᵇ | 0.12 ± 0.02ᵃ | 0.42 | Upregulated on Day 4 | 0.022701
67 | 0.05 ± 0.007ᵇ | 0.17 ± 0.04ᵃ | 0.29 | Upregulated on Day 4 | 0.015278
72 | 0.41 ± 0.04ᵃ | 0.20 ± 0.09ᵇ | 2.05 | Downregulated on Day 4 | 0.009048
76 | 0.12 ± 0.03ᵇ | 0.29 ± 0.06ᵃ | 0.41 | Upregulated on Day 4 | 0.032291
94 | 0.21 ± 0.05ᵃ | 0.07 ± 0.03ᵇ | 3.00 | Downregulated on Day 4 | 0.014791
100 | 0.08 ± 0.02ᵃ | 0.043 ± 0.01ᵇ | 1.86 | Downregulated on Day 4 | 0.011585
116 | 0.33 ± 0.12ᵃ | 0.05 ± 0.02ᵇ | 6.60 | Downregulated on Day 4 | 0.000400
137 | 0.08 ± 0.008ᵃ | 0.03 ± 0.01ᵇ | 2.67 | Downregulated on Day 4 | 0.000410
141 | 0.02 ± 0.007ᵇ | 0.10 ± 0.01ᵃ | 0.20 | Upregulated on Day 4 | 0.002816
144 | 0.05 ± 0.01ᵇ | 0.21 ± 0.02ᵃ | 0.20 | Upregulated on Day 4 | 0.002468
145 | 0.04 ± 0.009ᵇ | 0.28 ± 0.06ᵃ | 0.14 | Upregulated on Day 4 | 0.011013
154 | 0.16 ± 0.02ᵃ | 0.07 ± 0.008ᵇ | 2.29 | Downregulated on Day 4 | 0.001079
156 | 0.02 ± 0.02ᵇ | 0.09 ± 0.009ᵃ | 0.22 | Upregulated on Day 4 | 0.012501
160 | 0.24 ± 0.02ᵃ | 0.09 ± 0.02ᵇ | 2.67 | Downregulated on Day 4 | 0.023801
179 | 0.20 ± 0.04ᵃ | 0.09 ± 0.02ᵇ | 2.22 | Downregulated on Day 4 | 0.008831
197 | 0.10 ± 0.03ᵇ | 0.30 ± 0.09ᵃ | 0.33 | Upregulated on Day 4 | 0.005713
211 | 0.06 ± 0.01ᵇ | 0.15 ± 0.03ᵃ | 0.40 | Upregulated on Day 4 | 0.003560
222 | 0.16 ± 0.02ᵃ | 0.08 ± 0.02ᵇ | 2.00 | Downregulated on Day 4 | 0.017929
224 | 0.14 ± 0.02ᵇ | 0.28 ± 0.03ᵃ | 0.50 | Upregulated on Day 4 | 0.007739
229 | 0.15 ± 0.03ᵃ | 0.06 ± 0.03ᵇ | 2.50 | Downregulated on Day 4 | 0.000076
230 | 0.17 ± 0.03ᵃ | 0.07 ± 0.02ᵇ | 2.43 | Downregulated on Day 4 | 0.012954
236 | 0.13 ± 0.006ᵇ | 0.33 ± 0.02ᵃ | 0.39 | Upregulated on Day 4 | 0.001501
246 | 0.07 ± 0.02ᵇ | 0.21 ± 0.05ᵃ | 0.33 | Upregulated on Day 4 | 0.028508
249 | 0.06 ± 0.01ᵇ | 0.16 ± 0.02ᵃ | 0.38 | Upregulated on Day 4 | 0.012756
254 | 0.15 ± 0.04ᵃ | 0.07 ± 0.02ᵇ | 2.14 | Downregulated on Day 4 | 0.020577
264 | 0.43 ± 0.08ᵃ | 0.21 ± 0.03ᵇ | 2.05 | Downregulated on Day 4 | 0.001970
276 | 0.22 ± 0.02ᵃ | 0.11 ± 0.0ᵇ | 2.00 | Downregulated on Day 4 | 0.007829
278 | 0.10 ± 0.04ᵇ | 0.27 ± 0.05ᵃ | 0.37 | Upregulated on Day 4 | 0.005011
279 | 0.13 ± 0.06ᵇ | 0.32 ± 0.007ᵃ | 0.41 | Upregulated on Day 4 | 0.025550
284 | 0.13 ± 0.03ᵃ | 0.06 ± 0.03ᵇ | 2.20 | Downregulated on Day 4 | 0.000004
285 | 0.13 ± 0.02ᵇ | 0.51 ± 0.006ᵃ | 0.25 | Upregulated on Day 4 | 0.014231
287 | 0.25 ± 0.06ᵃ | 0.09 ± 0.02ᵇ | 2.78 | Downregulated on Day 4 | 0.028098
300 | 0.10 ± 0.02ᵃ | 0.04 ± 0.02ᵇ | 2.50 | Downregulated on Day 4 | 0.033515
302 | 0.18 ± 0.06ᵇ | 0.36 ± 0.08ᵃ | 0.50 | Upregulated on Day 4 | 0.016860
314 | 0.45 ± 0.07ᵃ | 0.17 ± 0.10ᵇ | 2.65 | Downregulated on Day 4 | 0.013880
315 | 0.42 ± 0.09ᵃ | 0.13 ± 0.08ᵇ | 3.23 | Downregulated on Day 4 | 0.015857
318 | 0.13 ± 0.05ᵃ | 0.02 ± 0.008ᵇ | 6.50 | Downregulated on Day 4 | 0.000283
341 | 0.10 ± 0.02ᵃ | 0.05 ± 0.01ᵇ | 2.00 | Downregulated on Day 4 | 0.021799

Spot No. | Protein identity | GenBank Accession | Gene symbol | Source species | Theoretical Mr/ pI | MALDI-MS PMFᵃ | MALDI-TOF/ TOF LIFTᵇ | Score/ threshold TOF | Score/ threshold TOF/TOF | Tolerance TOF (ppm)
7 | Glutathione S-transferase Mu | gi|6680121 | Gstm2 | Mus musculus | 25871/7.60 | 18/87(59) | 3(26,47,88) | 154/64 | 161/35 | 100
16 | Creatine kinase U-type, mitochondrial precursor | gi|6753428 | Ckmt1 | M. musculus | 47373/9.3 | 16/112(41) | 3(30,44,72) | 122/64 | 147/35 | 100
23 | Uncharacterized protein LOC433182 | gi|70794816 | na | M. musculus | 47453/6.4 | 17/100(46) | 2(54,88) | 116/64 | 142/35 | 100
29 | Keratin, type I cytoskeletal 19 | gi|6680606 | Krt19 | M. musculus | 44515/5.1 | 19/134(54) | 3(14,32,42) | 157/64 | 88/36 | 100
33 | Keratin, type II cytoskeletal 75 | gi|29789317 | Krt75 | M. musculus | 59932/9.1 | 14/125(23) | na | 65/64 | na | 100
39 | Stress-70 protein, mitochondrial | gi|162461907 | Hspa9 | M. musculus | 73701/5.7 | 36/118(54) | 4(28, 42, 58, 76) | 198/64 | 204/35 | 100
46 | Protein disulfide isomerase A3 precursor | gi|112293264 | Pdia3 | M. musculus | 57099/5.8 | 24/114(55) | 2(6,41) | 150/64 | 47/36 | 100
52 | 78 kDa glucose-regulated protein precursor | gi|254540166 | Hspa5 | M. musculus | 68570/5 | 26/124(38) | 2(56,61) | 123/64 | 117/35 | 100
55 | Protein disulfide isomerase precursor | gi|42415475 | Pdia1 | M. musculus | 57422/4.32 | 22/152(55) | 3(38,83,112) | 149/64 | 174/36 | 100
56 | Protein disulfide isomerase precursor | gi|42415475 | Pdia1 | M. musculus | 57422/4.62 | 13/50(36) | 3(25,42,53) | 117/64 | 120/35 | 100
60 | Hspd1 protein, partial | gi|76779273 | Hspa1 | M. musculus | 59559/8.88 | 17/120(24) | 2(10, 38) | 93/64 | 48/35 | 100
94 | 14-3-3 protein sigma | gi|3065927 | Sfn | M. musculus | 27803/4.6 | 14/80(52) | 3(11, 18, 32) | 96/64 | 49/35 | 100
116 | Sickle tail protein | gi|152061323 | Skt | M. musculus | 213874/8.3 | 37/125(23) | na | 96/64 | na | 100
137 | EndoA' cytokeratin (5' end put.); putative | gi|309215 | EndoA ' | M. musculus | 53210/5.3 | 20/125(36) | 3(23,29,76) | 86/64 | 128/34 | 100
144 | Vimentin | gi|55408 | Vim | M. musculus | 53746/4.9 | 23/58(39) | na | 140/64 | na | 100
145 | Reticulocalbin 3, EF-hand calcium binding domain, isoform CRA_a | gi|13529539 | Rcn3 | M. musculus | 39048/4.6 | 17/135(53) | 2(17,23) | 114/64 | 40/35 | 100
154 | mCG17595, isoform CRA_a | gi|148672065 | mCG17595, isoform CRA_a | M. musculus | 31866/4.8 | 65/43(21) | na | 65/64 | na | 100
156 | Lamin B2 | gi|228591 | Lmnb2 | M. musculus | 67476/5.3 | 143/107(37) | 3(16,26,38) | 143/64 | 80/36 | 100
160 | Glial fibrillary acidic protein | gi|51066 | Gfap | M. musculus | 48494/5.2 | na | 1(1) | na | 44/35 | 100
179 | Prelamin-A/C isoform A precursor | gi|162287370 | Lmna | M. musculus | 74478/6 | 223/93(53) | 2(28,68) | 223/64 | 96/34 | 100
197 | Prelamin-A/C isoform A precursor | gi|162287370 | Lmna | M. musculus | 74478/6.6 | 39/95(57) | 3(16, 32, 36) | 264/64 | 84/34 | 100
211 | Prelamin-A/C isoform A precursor | gi|162287370 | Lmna | M. musculus | 74478/6.6 | 30/83(47) | 1(1) | 184/64 | 38/35 | 100
222 | Spectrin alpha chain, non-erythrocytic 1 isoform 1 | gi|115496850 | Sptan1 | M. musculus | 286025/5.1 | 28/80(13) | na | 65/64 | na | 100
224 | Albumin | gi|26986064 | Alb | M. musculus | 20075/6 | 9/110(57) | 4(25,37,45,76) | 75/64 | 137/35 | 100
229 | ATP synthase beta-subunit | gi|2623222 | na | M. musculus | 56344/5 | 1/108(3) | 1(69) | na | 69/34 | 100
236 | Unnamed protein product | gi|26341396 | na | M. musculus | 67013/5.4 | 15/129(31) | 4(36,48,96,148) | 100/64 | 346/34 | 100
246 | Glutathione S-transferase Mu 7 | gi|113679874 | Gstm7 | M. musculus | 25864/6.4 | 17/61(58) | 1(46) | 125/64 | 46/35 | 100
249 | Adenylate kinase 2, mitochondrial isoform b | gi|34328230 | Ak2 | M. musculus | 32661/6 | 11/66(59) | 2(10, 37) | 86/64 | 47/34 | 100
276 | Arginase-1 | gi|7106255 | Arg1 | M. musculus | 34927/6.58 | 14/79(59) | 3(43, 57, 70) | 155/64 | 170/35 | 100
284 | Uncharacterized protein LOC433182 | gi|70794816 | na | M. musculus | 47453/6.4 | 22/135(60) | 3(44,80,87) | 104/64 | 211/35 | 100
287 | Uncharacterized protein LOC433182 | gi|70794816 | na | M. musculus | 47453/6.4 | 22/143(62) | 3(28,55,76) | 127/64 | 159/34 | 100
302 | Serine (or cysteine) peptidase inhibitor, clade H, member 1, isoform CRA_a | gi|148684430 | Serpinh1, Hsp47 | M. musculus | 45069/9.5 | 23/97(58) | 3(70,71,116) | 140/64 | 258/35 | 100
314 | Fructose-bisphosphate aldolase A isoform 2 | gi|6671539 | Aldoa | M. musculus | 39787/9.2 | 15/131(51) | 3(10,40,44) | 95/64 | 92/35 | 100
315 | 3-ketoacyl-CoA thiolase A, peroxisomal precursor | gi|18700004 | Acaa2 | M. musculus | 44382/9.7 | 16/101(49) | 2(12,63) | 103/64 | 75/35 | 100
318 | Unnamed protein product | gi|26328539 | na | M. musculus | 82105/10.3 | 17/99(22) | na | 78/64 | na | 100
341 | Heat shock 70 kDa protein 5 (glucose-regulated protein), isoform CRA_b | gi|148676670 | Hspa5 | M. musculus | 56314/4.9 | 14/76(34) | 2(9, 47) | 101/64 | 57/35 | 100
350 | Atp5b protein | gi|23272966 | Atp5b | M. musculus | 56632/5.1 | 4/114(29) | 4(8,61,74,98) | na | 241/35 | 100
357 | Vimentin | gi|2078001 | Vim | M. musculus | 51590/48 | 31/119(61) | 3(39,52,68) | 180/64 | 164/35 | 100
385 | Vimentin | gi|2078001 | Vim | M. musculus | 51590/4.8 | 26/109(54) | na | 143/64 | na | 100
424 | Zinc finger protein mfg2 | gi|199139 | Mfg2 | M. musculus | 48276/10.4 | 8/61(24) | na | 70/64 | na | 100
484 | Heat shock protein 65 | gi|51455 | Hsp65 | M. musculus | 61074/58 | 19/114/126(6) | 2(46,72) | 84/64 | 118/35 | 100
502 | Glutathione reductase 1 | gi|148703470 | Gsr | M. musculus | 46307/7.95 | 13/91(50) | na | 88/64 | na | 100
550 | Serum albumin precursor | gi|163310765 | Alb | M. musculus | 70700/5.7 | 14/104(29) | 4(12, 63, 78, 80) | 101/64 | 231/33 | 100
555 | Short-chain specific acyl-CoA dehydrogenase, mitochondrial precursor | gi|31982522 | Acads | M. musculus | 45146/9.4 | 15/111(40) | 3(7,16,17) | 75/64 | 41/35 | 100
563 | Peptidyl-prolyl cis-trans isomerase D | gi|13385854 | Ppid | M. musculus | 41116/7.8 | 12/129(34) | 2(21,50) | 74/64 | 71/36 | 100
573 | Acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase) | gi|20810027 | Acaa2 | M. musculus | 42288/9.3 | 20/123(65) | 3(51,55,90) | 122/64 | 196/36 | 100
597 | Peroxiredoxin-2 | gi|148747558 | Prdx2 | M. musculus | 23760/7.2 | 1/89(5) | 1(37) | na | 37/36 | 100
603 | Cathepsin B | gi|50597 | Ctsb | M. musculus | 3046/5.5 | 1/41(65) | 1(65) | na | 65/35 | 100
622 | Pyruvate carboxylase, mitochondrial isoform 1 | gi|251823980 | Pcx | M. musculus | 130491/6.3 | 32/110(31) | 3(16,32,67) | 130/64 | 115/35 | 100
633 | Sickle tail protein isoform b | gi|46358401 | Skt | M. musculus | 146438/9.8 | 21/74(20) | na | 75/64 | na | 100
664 | Vimentin V | gi|2078001 | Vim | M. musculus | 51590/4.8 | 25/104(48) | 3(5,43,54) | 129/64 | 104/35 | 100
673 | Catalase | gi|442441 | Cat | M. musculus | 59982/8.8 | 14/128(33) | 2(31,41) | 94/64 | 72/36 | 100

Functional classification of differentially expressed proteins

The 52 identified proteins were functionally annotated for subcellular localization, biological processes, and molecular functions using the AmiGO 2 Gene Ontology database, and the distribution of associated ontological categories is presented in Figure 2. Subcellular localization analysis revealed that the majority of proteins were localized to the cytoplasm (21%) and mitochondria (21%), followed by the membrane (15%), ER (12%), and nucleus (10%). Smaller proportions were detected in intracellular compartments (2%) and extracellular organelles (2%), whereas approximately 17% of the proteins could not be classified. Functional categorization based on biological processes demonstrated that these proteins were primarily associated with cellular metabolism (27%), biological regulation (13%), cellular development (11%), cellular localization (8%), and protein folding (8%). Additional roles included embryonic development (6%), cell adhesion (2%), cytoskeletal organization (2%), and reproductive system development (2%), while 21% of the proteins were not assigned to a specific biological process. Molecular function analysis indicated that most proteins were associated with protein binding (33%), catalytic activity (13%), antioxidant activity (11%), ion binding (6%), ATP binding (4%), ribosome binding (4%), DNA binding (4%), and RNA binding (4%), with approximately 21% remaining functionally uncharacterized. Collectively, these results highlight the diverse molecular pathways involved in endometrial remodeling during early pregnancy (Figure 2). This Gene Ontology analysis was performed for descriptive functional classification only and did not include statistical enrichment testing, multiple-testing correction, or comparison to a background gene set. Notably, several antioxidant proteins (Gstm2, Gstm7, Prdx2, and Cat) and ER stress-associated proteins (PDIA3 and HSPA5) exhibited coordinated expression changes, suggesting integrated regulation of oxidative stress and protein-folding stress responses during uterine receptivity.

Figure 2: Classification of differentially expressed proteins identified in this study. The identified proteins were annotated using the Gene Ontology database AmiGO 2 and categorized according to (A) subcellular localization, (B) biological processes, and (C) molecular functions.

Expression of vimentin and Gstm2 during early pregnancy

Immunofluorescence analysis was performed to determine the localization of vimentin and Gstm2 in uterine tissue sections collected on Days 1 and 4 of pregnancy (Figure 3). On Day 1, vimentin and Gstm2 signals were either absent or barely detectable in the luminal and glandular epithelia (Figure 3A). By contrast, on Day 4, low vimentin expression was observed in these epithelial regions, whereas Gstm2 exhibited strong immunoreactivity (Figure 3B). In addition, PCNA staining was more intense on Day 4 than on Day 1, indicating increased cellular proliferation during the implantation window (Figure 3A and B). To further validate these observations, reverse-transcription PCR and qPCR were performed to assess Vimentin and Gstm2 mRNA expression levels in isolated endometrial epithelial cells. Vimentin mRNA expression did not differ significantly between Day 1 and Day 4 (p > 0.05; Figures 4A and B). However, Gstm2 mRNA levels were significantly higher on Day 4 than on Day 1 (p < 0.05; Figures 4C and D). Although agarose gel electrophoresis (Figure 4A) demonstrated comparable Vimentin band intensities between the two time points, qPCR (Figure 4B) indicated a slight increase in Vimentin transcript levels on Day 4. The lack of statistical significance may be attributable to biological variability among replicates.

Figure 3: Immunolocalization of vimentin, Gstm2, and PCNA (green) in the mouse uterus on Days 1 (A) and 4 (B) of pregnancy. The negative control (NTC) was prepared by replacing the primary antibody with blocking buffer. Nuclei were counterstained with DAPI (blue). Merged fluorescent images of uterine sections were obtained through immunofluorescence staining. Scale bar: 100 μm. Immunofluorescent detection of vimentin, Gstm2, and PCNA was performed on uterine cross-sections to visualize epithelial and stromal compartments. Imaging was optimized for the epithelial regions; therefore, stromal vimentin-positive cells may appear faint. Gene expression analysis (quantitative real-time polymerase chain reaction) was performed on isolated epithelial cells to complement immunofluorescence findings.

Figure 4: Gene expression in mouse endometrial epithelial cells on Days 1 and 4 of pregnancy. (A and C) Representative agarose gel images showing the expression of Vimentin and Gstm2 following reverse-transcription PCR. (B and D) Relative expression levels of vimentin and Gstm2 determined by quantitative real-time polymerase chain reaction (qPCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference for normalization. Data are presented as the mean ± standard error of the mean (SEM) from three independent experiments. Asterisks (*) indicate significant differences in gene expression between Days 1 and 4 (p < 0.05). Note that the qPCR-derived relative transcript abundance may not be fully reflected by the band intensity observed in agarose gel visualization.