Expression of endometrial immune-related genes possibly functioning during early pregnancy in the mare

Abstract

Despite enormous efforts, biochemical and molecular mechanisms associated with equine reproduction, particularly processes of pregnancy establishment, have not been well characterized. Previously, PCR-selected suppression subtraction hybridization analysis was executed to identify unique molecules functioning in the equine endometrium during periods of pregnancy establishment, and granzyme B (GZMB) cDNA was found in the pregnant endometrial cDNA library. Because GZMB is produced from natural killer (NK) cells, endometrial expression of GZMB and immune-related transcripts were characterized in this study. The level of GZMB mRNA is higher in the pregnant endometrium than in non-pregnant ones. This expression was also confirmed through Western blot and immunohistochemical analyses. IL-2 mRNA declined as pregnancy progressed, while IL-15, IFNG and TGFB1 transcripts increased on day 19 and/or 25. Analyses of IL-4 and IL-12 mRNAs demonstrated the increase in these transcripts as pregnancy progressed. Increase in CCR5 and CCR4 mRNAs indicated that both Th1 and Th2 cells coexisted in the day 25 pregnant endometrium. Taken together, the endometrial expression of immune-related transcripts suggests that immunological responses are present even before the trophectoderm actually attaches to the uterine epithelial cells.

Fig. 1.

Expression of granzyme B (GZMB) mRNA and protein in the equine endometrium. A: Real-time PCR analysis of GZMB mRNA in the equine endometrium. Total RNA was extracted from equine endometrium in day 13 cyclic and in days 13, 19, and 25 pregnant animals, lymph node and spleen. Bars represent means ± SE. An asterisk indicates a significant difference (P<0.05) when compared with the value from the cyclic endometrium. B: Western blot analysis of GZMB in the cyclic and days 13, 19, and 25 pregnant endometrium, lymph node and spleen.

Fig. 2.

Immunohistochemical analysis of GZMB in the equine endometrium. Immunohistochemical localization of GZMB in the cyclic (a) and pregnant days 13 (b), 19 (c) and 25 (d) equine endometrium. Detection of GZMB with mouse anti-equine GZMB antibody [35]; the inset (panel a, upper right corner) was the negative control with normal mouse IgG. All slides were counterstained with hematoxylin. LE, luminal epithelium; GE, glandular epithelium; ST, subepithelial stroma. Bar=100 μm.

Fig. 3.

mRNA expression of immune-related genes involved in the activation of T lymphocyte and NK cells in the equine endometrium. Total RNA was extracted from the equine endometrium in day 13 cyclic and in days 13, 19 and 25 pregnant animals, and real-time PCR analysis was performed to determine the expression of IL-2, IL-15, IFNG, and TGFB1 mRNA. Bars represent means ± SE. An asterisk indicates a significant difference (P<0.05) when compared with the value from the cyclic endometrium.

Fig. 4.

mRNA expression of immune-related genes involved in the activation of T helper cells in the equine endometrium. Total RNA was extracted from the equine endometrium in day 13 cyclic and in days 13, 19 and 25 pregnant animals, and real-time PCR analysis was performed to determine the expression of IL-4, IL-12, CCR4 and CCR5 mRNA. Bars represent means ± SE. An asterisk indicates a significant difference (P<0.05) when compared with the value from the cyclic endometrium.

Fig. 5.

Possible model of immune-related gene expressions and their interactions during periods covering days 13, 19 and 25, while the equine conceptus goes through three stages of development; migration (day 13, Upper), fixation (day 19, Middle), and attachment (day 25, Lower) to the uterine endometrium. Intrauterine migration induces endometrial IL-15, resulting in NK cell recruitment. These uNK cells produce GZMB and induce other immune-related gene expressions at the phase of conceptus fixation. As the capsule subsides and conceptus attachment proceeds, Th1 and Th2 cells are recruited into the attachment area of the endometrium. These sequential gene expressions may be required for equine pregnancy to proceed.