Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Mar 2;12(3):388.
doi: 10.3390/biom12030388.

Heat Stress Impairs Maternal Endometrial Integrity and Results in Embryo Implantation Failure by Regulating Transport-Related Gene Expression in Tongcheng Pigs

Weisi Lian  1 , Dengying Gao  1 , Cheng Huang  1 , Qiqi Zhong  1 , Renwu Hua  1 , Minggang Lei  1   2   3
Affiliations

Heat Stress Impairs Maternal Endometrial Integrity and Results in Embryo Implantation Failure by Regulating Transport-Related Gene Expression in Tongcheng Pigs

Weisi Lian et al. Biomolecules. .

Abstract

Heat stress (HS) poses a significant threat to production and survival in the global swine industry. However, the molecular regulatory effects of heat stress on maternal endometrial cells are poorly understood in pigs during early embryo implantation. In this study, we systematically examined morphological changes in the endometrium and the corresponding regulation mechanism in response to HS by combining scanning electron microscopy (SEM), hematoxylin/eosin (H&E) staining, western blot, and RNA-seq analyses. Our results showed that HS led to porcine endometrium damage and endometrial thinness during embryo implantation. The expression levels of cell adhesion-related proteins, including N-cadherin and E-cadherin, in the uterus were significantly lower in the heat stress group (39 ± 1 °C, n = 3) than in the control group (28 ± 1 °C, n = 3). A total of 338 up-regulated genes and 378 down-regulated genes were identified in porcine endometrium under HS. The down-regulated genes were found to be mainly enriched in the pathways related to the microtubule complex, immune system process, and metalloendopeptidase activity, whereas the up-regulated genes were mainly involved in calcium ion binding, the extracellular region, and molecular function regulation. S100A9 was found to be one of the most significant differentially expressed genes (DEGs) in the endometrium under HS, and this gene could promote proliferation of endometrial cells and inhibit their apoptosis. Meanwhile, HS caused endometrial epithelial cell (EEC) damage and inhibited its proliferation. Overall, our results demonstrated that HS induced uterine morphological change and tissue damage by regulating the expression of genes associated with calcium ions and amino acid transport. These findings may provide novel molecular insights into endometrial damage under HS during embryo implantation.

Keywords: S100A9; cell adhesion; heat stress; porcine endometrial cells; tight junction; transport activity.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effects of heat stress on the endometrium during embryo implantation. (A,B) Image of luminal epithelium at day 4 post heat stress (H/E staining) (scale bar, A 100 μm; B 50 μm). (C) Luminal epithelial thickness in the HS-treated group and the control group. *** p < 0.001. The data were expressed as means ± SD (n = 5). (D–F) Scanning electron microscopy (SEM) images of uteri under heat stress. Uteri collected on day 14 of pregnancy were treated with heat stress (scale bar, 20 μm). (E–G) High magnification (scale bar, 10 μm).
Figure 2
Figure 2
(A) Western blot analysis of effects of heat stress on expression levels of E-cadherin, N-cadherin, occludin, and claudin-1 proteins. (B) Densitometry analysis of proteins related to epithelial cell integrity. * p < 0.05.
Figure 3
Figure 3
Differentially expressed genes (DEGs) analysis. (A) Principal component analysis (PCA) and heatmap analysis of the control and HS group samples. (B) Volcano map of DEGs in the control group and the HS group. (C) Hierarchical cluster analysis of DEGs.
Figure 4
Figure 4
Gene ontology (GO) enrichment analysis of DEGs in the control group and the HS group during embryo implantation. (A) Up-regulated DEGs. (B) Down-regulated DEGs. Kyoto encyclopedia of genes and genomes (KEGG) analysis of DEGs in the control group and the HS group during embryo implantation. (C) Up-regulated DEGs. (D) Down-regulated DEGs.
Figure 5
Figure 5
Cluster analysis of DEGs related to four major signaling pathways. (A) Embryo implantation. (B) Calcium binding. (C) Transport. (D) Transmembrane receptor. The red color presents the up-regulated, and blue color indicates the down-regulated genes.
Figure 6
Figure 6
Protein–protein interaction analysis of up-regulated (A) and down-regulated (B) genes in the endometrium during embryo implantation in the HS treatment group and the control group. (C) Validation of the RNA-seq results by qRT-PCR. The data are presented as the means ± standard deviation (SD). * p < 0.05; ** p < 0.01; and *** p < 0.001.
Figure 7
Figure 7
Impact of heat stress on porcine endometrial epithelial cells. (A) Morphological changes of EECs after heat stress. (B) Viability of EECs under HS. (C) Impact of HS on expression level of genes associated with cell proliferation (PCNA). (D) Impact of HS on expression level of genes associated with embryo implantation and cell adhesion (MUC1 and ZO-1). * p < 0.05 and *** p < 0.001. ab means in the same bar without a common letter differ at p < 0.05.
Figure 8
Figure 8
Effects of S100A9 on proliferation and apoptosis of EECs. (A) Impact of HS on expression of S100A9. (B) Relative expression of S100A9 transfected with pcDAN3.1(+)-S100A9, pcDNA3.1(+), siS100A9, and NC at 24 h post transfection in EECs. (C,D) Flow cytometry analysis of cell cycle distribution. (E,F) Apoptosis by flow cytometry. * p < 0.05, ** p < 0.01 and *** p < 0.001. G1, first gap; S, synthesis; G2, second gap; NC, negative control.
Figure 9
Figure 9
Model of heat-stress effects on endometria in pigs during embryo implantation.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Lindenberg S.J. Experimental studies on the initial trophoblast endometrial interaction. Dan. Med. Bull. 1991;38:371–380. - PubMed
    1. Zhang S., Lin H., Kong S., Wang S., Wang H., Wang H., Armant D.R. Physiological and molecular determinants of embryo implantation. Mol. Asp. Med. 2013;34:939–980. doi: 10.1016/j.mam.2012.12.011. - DOI - PMC - PubMed
    1. Kim S.-M., Kim J.-S.J.D. A review of mechanisms of implantation. Dev. Reprod. 2017;21:351. doi: 10.12717/DR.2017.21.4.351. - DOI - PMC - PubMed
    1. Hu W., Liang Y.-X., Luo J.-M., Gu X.-W., Chen Z.-C., Fu T., Zhu Y.-Y., Lin S., Diao H.-L., Jia B.J.C.D., et al. Nucleolar stress regulation of endometrial receptivity in mouse models and human cell lines. Cell Death Dis. 2019;10:1–16. doi: 10.1038/s41419-019-2071-6. - DOI - PMC - PubMed
    1. Zheng H.T., Zhang H.Y., Chen S.T., Li M.Y., Fu T., Yang Z. The detrimental effects of stress-induced glucocorticoid exposure on mouse uterine receptivity and decidualization. FASEB J. 2020;34:14200–14216. doi: 10.1096/fj.201902911RR. - DOI - PubMed

Publication types