Characterization of the Th Profile of the Bovine Endometrium during the Oestrous Cycle and Early Pregnancy

Despite extensive research in the area of cow fertility, the extent to which the maternal immune system is modulated during pregnancy in cattle remains unclear. Therefore, the objective of the current study was to characterize the presence and response profile of B, T-helper (LTh), T- cytotoxic (LTc), gamma delta-T (γδT) and natural killer (NK) lymphocytes in terms of cell number, distribution and cytokine expression in bovine endometrial tissue to pregnancy. Endometrial tissue samples were collected from beef heifers on Days 5, 7, 13 and 16 of the estrous cycle or pregnancy. Samples were analysed by immunofluorescence to identify the presence and abundance of B-B7 (B-cells), CD4 (LTh), CD8 (LTc), γδT cell receptor (TCR) and CD335/NKp46 (NK cells) -positive immune cells. Quantitative real time PCR (QPCR) was carried out to analyse mRNA relative abundance of FOXP3 (a marker of regulatory T (Treg) cells) and a panel of immune factors, including MHC-I, LIF, Interleukins 1, 2, 6, 8, 10, 11,12A, IFNa and IFNG. Results indicate that B-B7+ cells are quite populous in bovine endometrial tissue, CD4+ and CD8+ -cells are present in moderate numbers and γδTCR+ and CD335+ cells are present in low numbers. Pregnancy affected the total number and distribution pattern of the NK cell population, with the most significant variation observed on Day 16 of pregnancy. Neither B lymphocytes nor T lymphocyte subsets were regulated temporally during the oestrous cycle or by pregnancy prior to implantation. mRNA transcript abundance of the immune factors LIF, IL1b, IL8 and IL12A, IFNa and IFNG, expression was regulated temporally during the estrous cycle and LIF, IL1b, IL-10, IL11, IL12A were also temporally regulated during pregnancy. In conclusion, the endometrial immune profile of the oestrous cycle favours a Th2 environment in anticipation of pregnancy and the presence of an embryo acts to fine tune this environment.Science Foundation Irelan

Characterization of the Th Profile of the Bovine Endometrium during the Oestrous Cycle and Early Pregnancy

<div><p>Despite extensive research in the area of cow fertility, the extent to which the maternal immune system is modulated during pregnancy in cattle remains unclear. Therefore, the objective of the current study was to characterize the presence and response profile of B, T-helper (LTh), T- cytotoxic (LTc), gamma delta-T (γδT) and natural killer (NK) lymphocytes in terms of cell number, distribution and cytokine expression in bovine endometrial tissue to pregnancy. Endometrial tissue samples were collected from beef heifers on Days 5, 7, 13 and 16 of the estrous cycle or pregnancy. Samples were analysed by immunofluorescence to identify the presence and abundance of B-B7 (B-cells), CD4 (LTh), CD8 (LTc), γδT cell receptor (TCR) and CD335/NKp46 (NK cells) -positive immune cells. Quantitative real time PCR (QPCR) was carried out to analyse mRNA relative abundance of FOXP3 (a marker of regulatory T (Treg) cells) and a panel of immune factors, including MHC-I, LIF, Interleukins 1, 2, 6, 8, 10, 11,12A, IFNa and IFNG. Results indicate that B-B7+ cells are quite populous in bovine endometrial tissue, CD4+ and CD8+ -cells are present in moderate numbers and γδTCR+ and CD335+ cells are present in low numbers. Pregnancy affected the total number and distribution pattern of the NK cell population, with the most significant variation observed on Day 16 of pregnancy. Neither B lymphocytes nor T lymphocyte subsets were regulated temporally during the oestrous cycle or by pregnancy prior to implantation. mRNA transcript abundance of the immune factors LIF, IL1b, IL8 and IL12A, IFNa and IFNG, expression was regulated temporally during the estrous cycle and LIF, IL1b, IL-10, IL11, IL12A were also temporally regulated during pregnancy. In conclusion, the endometrial immune profile of the oestrous cycle favours a Th2 environment in anticipation of pregnancy and the presence of an embryo acts to fine tune this environment. </p> </div

Immunofluorescent labeling of endometrial tissue for NK cells.

<p>(a-h) Representative images of immunofluorescent CD335⁺ NK cell receptor labeling (red) in cells (nuclei labeled with DAPI [blue]) of the endometrial SS from cyclic (C) and pregnant (P) heifers on Days 13 and of the deep stroma (DS) from C and P heifers on day 16 (original magnification ×40). B) Line graphs detailing the total number of NK cells in cyclic (red line) and pregnant (blue line) endometrial tissue based on the sum of immunofluorescent labeled cells per 2.25 mm² field of view, in all areas (total CD335⁺) ) and specifically in the luminal epithelium (LE), shallow stroma (SS) and in the deep stroma (DS). Values plotted are least squared means and standard errors across across 5 replicate animals per timepoint and per status.</p

A) Immunofluorescent labeling of endometrial tissue for CD4⁺ and CD8+ T cells.

<p>(a-j) Representative images of immunofluorescent CD4⁺ cells labeling (green) and CD8⁺ cells labeling (red) and nuclei labeled with DAPI [blue]), in the endometrial shallow stroma (SS) ; deep stroma (DS) and myometrium (M) in cyclic at day 7 (a-d) ; 13 (e-h) and 16 (i-l) of estrus cycle and at day 5 (m-p) ; 13 (q-t) and 16 (u-x) of pregnancy. (original magnification ×40). B) Line graphs detailing the total number of endometrial CD4+  and CD8+ cells. CD4+ (a-e) and CD8+ (f-j) -cell numbers in cyclic (red line) and pregnant (blue line) endometrial tissue are based on the sum of immunofluorescent labeled cells per 2.25 mm² field of view, in all areas (total CD4+ or CD8+) and specifically in the luminal epithelium (LE), SS, DS and M. Values plotted are least squared means and standard errors across 5 replicate animals per timepoint and per status.</p

Cytokine mRNA expression profiles.

<p>mRNA abundance of selected cytokines in cyclic (red line) and pregnant (blue line) endometrial tissue during the pre- and peri-implantation period. Values plotted are least squared means and standard errors across across 5 replicate animals per timepoint and per status. Asterisks indicate significant difference between pregnant and cyclic tissue at a particular time point: * = <i>P</i> < 0.05.</p

Immunofluorescent labeling of endometrial tissue for γδT-cells.

<p>(a-f) Representative images of immunofluorescent γδT cell receptor (WC1) labeling (green) in cells (nuclei labeled with DAPI [blue]) of the endometrial deep stroma (DS) from cyclic (C) Day 5 and pregnant (P) Day 7 and 16 -heifers (original magnification ×40). (g-i) Line graphs detailing the total number of γδT-cells in cyclic (red line) and pregnant (blue line) endometrial tissue based on the sum of immunofluorescent labeled cells per 2.25 mm² field of view, in all areas (total γδT-cells⁺) and specifically in the SS and DS. Values plotted are least squared means and standard errors across 5 replicate animals per timepoint and per status.</p