Protein expression profiles in Meishan and Duroc sows during mid-gestation reveal differences affecting uterine capacity, endometrial receptivity, and the maternal– fetal Interface

- Protein expression profiles in Meishan and Duroc sows during mid-gestation reveal differences affecting uterine capacity,.
- Background: Embryonic mortality is a major concern in the commercial swine industry and primarily occurs early in gestation, but also during mid-gestation.
- Results: In this study, protein expression patterns in endometrial tissue from Meishan and Duroc sows were examined during mid-gestation.
- After statistical analysis, 70 and 114 differentially expressed proteins (DEPs) were identified in Meishan and Duroc sows, respectively.
- Between Meishan and Duroc sows, 114 DEPs were detected at day 49, and 98 DEPs were detected at day 72.
- Functional enrichment analysis revealed differences in protein expression patterns in the two breeds.
- Our results suggest that the differences in expression affect uterine capacity, endometrial matrix remodeling, and maternal-embryo cross-talk, and may be major factors influencing the differences in embryonic loss between Meishan and Duroc sows during mid-gestation..
- Conclusions: Our data showed differential protein expression pattern in endometrium between Meishan and Duroc sows and provides insight into the development process of endometrium.
- Keywords: Protein expression, iTRAQ, Endometrium, Meishan and Duroc pigs.
- 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0.
- Full list of author information is available at the end of the article.
- Embryonic mortality accounts for over 30% of the overall mortality in swine herds and remains a challenge to the commercial swine industry [6].
- Comparisons between the Meishan and other pig breeds indicate that litter size is determined mainly by the recipient females [16, 17].
- The larger litter size of Meishan pigs partly results from the changes in the uterine milieu as well as a higher uterine capacity [15].
- Evidence from these stud- ies urged us to study the molecular basis of fetal loss during mid-gestation through comparing Meishan and European sows..
- for of the total, but till now very few molecular genetic data were collected on sows at this stage of pregnancy for investigating embryonic mortality..
- Here, in an effort to identify the molecular mecha- nisms involved in fetal loss during mid-gestation, we used iTRAQ (isobaric tags for relative and absolute quantification) to globally characterize differentially expressed proteins from endometrial tissues of Meishan and Duroc sows..
- All animal procedures used in this study strictly followed protocols approved by Animal Welfare Committee in the State Key Laboratory for Agro-biotechnology at China Agricultural University (Approval number XK257).
- During the fourth pregnancy, on days 49 and 72 of gestation, three Meishan and three Duroc sows were rendered uncon- scious by electrical stunning and then immediately bled by cutting the throat.
- The analysis (one MS scan followed by 20 MS/MS scans) was applied to the top 20 precursor ions above a threshold ion count of 1E4 in the MS survey scan with 30.0 s dynamic exclusion.
- MS/MS data were processed using the Mascot search engine (v.2.3.0) and tandem mass spectra were compared to entries in the Uniprot Sus scrofa database (21,047 sequences).
- Gene Ontology (GO) annota- tion of the proteome was implemented using the UniProt-GOA database (http://www.ebi.ac.uk/GOA/)..
- A two-tailed Fisher’s exact test was employed to test for enrichment of the differentially expressed proteins relative to all identified proteins..
- of the total, genomic studies in sows at this stage of pregnancy have not been done.
- Comparisons between the Meishan and other breeds indicate that.
- We therefore used iTRAQ to compare protein expression profiles in endometrial tissue from Meishan and Duroc sows on days 49 and 72 of pregnancy to identify proteins that are potentially involved in prolificacy differences..
- A total of 14,629 and 16, 565 unique peptides were identified in the two replicas with a minimum confidence level of 99%, representing 3672 and 4012 proteins, respectively.
- In the biological process category, most proteins are involved in cellular process (GO:0009987), single organism process (GO:0044699), metabolic process (GO and single organism cellular process (GO:.
- Differentially expressed pro- teins (DEPs) were identified based on the geometrical mean of the fold change value calculated for each protein in the two replicates.
- Five differentially expressed proteins (GLA, CRYAB, CTSB, ASAH1, and DPP4) were randomly selected and quantitated by western blot to test the reliability of the iTRAQ analysis (Fig.
- Differential protein expression during pregnancy in Meishan and Duroc pigs.
- MSD72 vs.
- Heatmap comparing average fold change in expression of the five genes as measured by western blot and iTRAQ (f).
- Functional enrichment analysis results are summarized for each DEP in Additional file 2: Table S2.
- Two proteins, CNN1 and TRIM29, were identified in the in- creased DEPs from MSD72 vs MSD49 and DUD72 vs DUD49.
- DPP4 and ANXA10 were identified in the de- creased DEPs from MSD72 vs MSD49 and DUD72 vs DUD49.
- Three proteins, PODN, ASAH1 and CPS1, ex- hibited differential reverse expression patterns between Meishan and Duroc pigs during mid-pregnancy..
- Functional clustering of DEPs at days 49 and days 72 To characterize the differences in endometrium protein profiles between Meishan and Duroc pigs, proteins from each developmental stage were compared, and then the DEPs were subjected to functional enrichment analysis..
- 3 Correlation and functional enrichment analysis.
- At day 72, 98 DEPs (56 increased and 42 decreased) were identified between the two breeds (Additional file 4:.
- Figure 4c and d show the results of the func- tional enrichment analyses for increased and decreased DEPs.
- To analyze the expression patterns of the two breeds in more detail, the DEPs obtained from analyses of MSD72 vs.
- 4 Functional enrichment analysis for DEPs between Meishan and Duroc sows at days 49 and days 72.
- 5 and Additional file 3: Table S3).
- We also identified six increased proteins (KRT18, ACTC1, DES, SYNM, CRYAB, and PRPH) involved in intermediate filament cytoskeleton organization and contractile fiber formation in the MSD72 vs.
- In the pig endometrium, intermediate filaments are involved in mechanisms re- sponsible for elongation of conceptuses, and for adaptive responses of the uterine wall to the increasing mechan- ical forces due to fetal growth and accumulation of fetal fluids [33].
- DUD49 are compared in the bottom panel.
- The color shading in the DEP boxes is proportional to the DEP fold change values, which were obtained from MSD49 vs.
- proteins that are expressed at different levels in Meishan and Duroc sows might be responsible for enlarging uter- ine size and retarding fetal growth rate, accounting for the differences in embryonic loss between the two breeds..
- The difference in endometrial structural modifica- tion is the main reason for the functional difference be- tween Meishan and Yorkshire uteri .
- DUD49 (LAMB2, TGFBI, HAPLN1, COL3A1, CLU and FBN1) that clustered in the extracellular matrix (Additional file 2:.
- These proteins are components of the extracellu- lar matrix [42, 43], and are significantly increased in the endometrium at the time of implantation, suggesting a role in blastocyst attachment [44].
- CTSB, CTSC, and CTSZ are involved in the degradation of extra- cellular matrix proteins including laminin, fibrilin, and col- lagen [45].
- In contrast, no significant differences in the levels of these proteins were observed in MSD72 vs..
- DUD72 (Additional file 4:.
- These proteases inhibitors were all enriched in the GO term serine proteases inhibitor (Additional file 4: Table S4).
- data suggest that different proteases and protease inhibi- tors control the remodeling of the endometrium extracel- lular matrix during mid-gestation in Meishan and Duroc sows..
- Maternal-embryo cross-talk is first de- tected in the implantation window [50] and continues throughout the pregnancy process.
- In this study, enriched GO terms such as extracellular vesicular exo- some and membrane-bounded vesicle were associated with many DEPs detected in the proteomic comparison between Meishan and Duroc sows during pregnancy..
- It is very interesting that ASAH1 was increased in the former comparison group, but decreased in the latter comparison group (Additional file 1: Table S1 and Fig.
- DPP4 levels decreased at day 72 relative to day 49 in both Meishan and Duroc sows, but the decrease was more pronounced in Duroc sows (Additional file 1: Table S1 and Fig.
- Together, these data suggest that the level of maternal-embryo cross-talk dif- fers between Duroc and Meishan sows in the middle of pregnancy.
- We hypothesize that this factor might contrib- ute to the differences in prolificacy between Meishan and Duroc sows.
- This study shows differential protein expres- sion pattern in endometrium between Meishan and Duroc sows during mid-gestation, and might provide evidence for elucidating the differences in embryonic loss between two breeds.
- Signifi- cant results in the study may be due to biological vari- ation, the expression patterns are only specific to the investigated individuals and may not a characteristic of the study population.
- More samples will be needed for further identification of the differential proteins and its function analysis.
- In addition, the size and weight of the fetuses are also related to embryo loss according to our data and previous publications [24, 25], which should also be included for further understanding of the genetic mechanism of embryo loss..
- In summary, we found differences in the protein expres- sion pattern in endometrium tissue derived from Meishan and Duroc sows during mid-gestation and pro- vided insight into the development process of endomet- rium.
- Additional file 1: Table S1.
- Additional file 2: Table S2.
- List of DEPs and functional enrichment analysis results for DEPs in Duroc sows..
- Additional file 3: Table S3.
- Additional file 4: Table S4.
- Additional file 5: Table S5.
- Additional file 6: Figure S1.
- Additional file 7: Figure S2.
- Additional file 8: Figure S3.
- Experimental procedures were approved by the Animal Welfare Committee in the State Key Laboratory for Agro-biotechnology at China Agricultural Uni- versity (Approval number XK257) and this study was carried out in strict ac- cordance with the guidelines and regulations established by this committee..
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- Transcriptome analysis revealed the embryo-induced gene expression patterns in the endometrium from Meishan and Yorkshire pigs.
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