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Altered expression of chemokines and their receptors at porcine maternal-fetal interface during early and mid-gestational fetal loss

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Abstract

Chemokines play a significant role in pregnancy, especially during embryonic attachment and placental development. During early pregnancy, immune cells are recruited extensively to the endometrium in several species including pigs. However, this recruitment is solely mediated by the presence of the conceptus in pigs making it a unique feature compared with other species (humans, primates and mice). To understand the biological significance of chemokine expression and immune cell recruitment in the context of fetal loss, we investigate a well-characterized porcine fetal loss model during the window of early pregnancy at gestational day (gd) 20 and mid-pregnancy (gd50). These periods coincide with 25–40 % of conceptus loss. Using targeted quantitative polymerase chain reaction and Western blot approaches, we screened a specific set of chemokines. Comparisons were made with endometrial lymphocytes (ENDO LY), endometrium and chorioallantoic membranes (CAM) associated with spontaneously arresting and healthy conceptus attachment sites (CAS). mRNA expression studies revealed an increased expression of CXCR3 and CCR5 in ENDO LY and of CXCL10, CXCR3, CCL5 and CCR5 in the endometrium associated with arresting CAS at gd20. DARC was decreased in the endometrium at gd50. CCL1 was increased in CAM associated with arresting CAS at gd50. Some of these differences were also noted at the protein level (CXCL10, CXCR3, CCL5 and CCR5) in the endometrium and CAM. CD45+ immunohistochemistry demonstrated a significantly higher localization in ENDO LY in the endometrium associated with healthy versus arresting counterparts. Most of these differences were observed in early pregnancy and might contribute towards a shift in immune cell functions.

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Abbreviations

CAM:

Chorioallantoic membrane

CAS:

Conceptus attachment sites

ENDO LY:

Endometrial lymphocytes

LCM:

Laser capture microdissection

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Acknowledgments

The authors thank Dr. Stephen C. Pang for access to the real-time PCR facilities at the Department of Biomedical and Molecular Sciences, Queen’s University. The support from technical staff at the Arkell Swine Research Station and the Meat Wing of the Animal and Poultry Science at the University of Guelph is gratefully acknowledged. We also thank Allison Felker at Queen’s University for technical assistance with the immunofluorescence technique.

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Authors and Affiliations

  1. Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada, K7L 3N6

    Mallikarjun Bidarimath, Kasra Khalaj, Madhuri Koti & Chandrakant Tayade

  2. Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada, N1G 2W1

    Kasra Khalaj, Rami T. Kridli, Jocelyn M. Wessels & Chandrakant Tayade

  3. Department of Animal Production, Faculty of Agriculture, Jordan University of Science and Technology, Irbid, 22110, Jordan

    Rami T. Kridli

  4. Department of Obstetrics and Gynecology, McMaster University, Hamilton, ON, Canada, L8S 4L8

    Jocelyn M. Wessels

Authors
  1. Mallikarjun Bidarimath
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  2. Kasra Khalaj
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  3. Rami T. Kridli
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  4. Jocelyn M. Wessels
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  5. Madhuri Koti
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  6. Chandrakant Tayade
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Corresponding author

Correspondence to Chandrakant Tayade.

Additional information

Generous funding support from the NSERC-CRD, Ontario Pork, OMAFRA and Bioniche Life Sciences (C.T.) and via the Queen’s Graduate Award, Queen’s University (M.B.) is greatly appreciated.

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Supplemental Figure 1

Protein expression of chemokines and their receptors in chorioallantoic membrane at gd20. Chemokines (CCL5, CCR5, CXCL10, CXCR3 and CCL1) that were significantly different between healthy and arresting conceptus attachment sites (CAS) at gd20 were quantified. Relative quantification was performed by using ACTB as the control protein. Each healthy and arresting group consisted in n = 12 per group and n = 6 sample type (n = 6 HCAM, n = 6 ACAM). Densitometry analysis was performed to give a ratio of chemokine proteins to ACTB by using Image J. No significant differences (P < 0.05) were evident in the expression of selected chemokines and their receptors at the protein level in the ACAM compared with HCAM at gd20 (IDV individual densitometry values). Densitometric values are presented as means ± SEM. *Statistical significance at P < 0.05. (GIF 114 kb)

High Resolution Image (TIF 2343 kb)

Supplemental Figure 2

Protein expression of chemokines and their receptors in chorioallantoic membrane at gd50. Chemokines (CCL5, CCR5, CXCL10, CXCR3 and CCL1) that were significantly different between healthy and arresting conceptus attachment sites (CAS) were quantified in chorioallantoic membranes collected at gd50. Relative quantification was performed by using ACTB as the reference protein. Each healthy and arresting group consisted in n = 12 per group and n = 6 sample type (n = 6 HCAM, n = 6 ACAM). Densitometry analysis was performed to give a ratio of chemokine to ACTB by using Image J. Relative expression of chemokine proteins (CCL5, CCR5, CXCL10, CXCR3 and CCL1) between HCAM and ACAM did not differ significantly at gd50 (IDV individual densitometry values). Densitometric values are presented as means ± SEM. *Statistical significance at P < 0.05. (GIF 114 kb)

High Resolution Image (TIF 1836 kb)

Supplemental Figure 3

Immunolocalization of Foxp3+ cells in the porcine endometrium at gd20. Left and right columns represent the distribution of Foxp3+ cells in the endometrium associated with healthy (HE) and arresting (AE) CAS, respectively. Nuclei are stained with 4,6-diamidino-2-phenylindole (DAPI, blue in a, b), Foxp3+ is stained with Anti-Mouse/Rat Foxp3 PE (DsRed, red in c, d) and the merged images (e, f) demonstrate their localization in the endometrium. Magnification ×400. Bar 400 μm. (GIF 131 kb)

High Resolution Image (TIF 2377 kb)

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Bidarimath, M., Khalaj, K., Kridli, R.T. et al. Altered expression of chemokines and their receptors at porcine maternal-fetal interface during early and mid-gestational fetal loss. Cell Tissue Res 366, 747–761 (2016). https://doi.org/10.1007/s00441-016-2470-2

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