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INVESTIGATION OF BOVINE CONCEPTUS GALECTINS AND THE EFFECT OF BOVINE CONCEPTUS SECRETORY PROTEINS ON THE ENDOMETRIAL EPITHELIAL TRANSCRIPTOME Heather L. Baldwin Pregnancy loss in cattle is greatest in the first 30 days of gestation. Near d15 of gestation, bovine conceptuses elongate and express LGALS1, an immunosuppressive glycan-binding protein believed to be essential for implantation. However, other members of the bovine galectin family, including LGALS3, LGALS4, LGALS7, LGALS8, LGALS9 and a galectin-13-like gene (LGALS13L), also may be expressed by the elongating conceptus. Therefore, galectin expression in elongating, d16 bovine conceptuses and intercaruncular endometrium as well as elongating, d14 ovine conceptuses and intercaruncular endometrium was assessed by RT-qPCR. Angus heifer and Texel ewes underwent estrous cycle synchronization and either inseminated or not bred. On d16 (heifers) or d14 (ewes) after synchronized estrus (d0), reproductive tracts of pregnant (heifers, n=4; ewes, n=3) and cyclic (heifers, n=4; ewes, n=3) animals were removed. Conceptuses and intercaruncular endometrial samples were then collected. Conceptuses and intercaruncular endometrium samples were individually snap frozen and stored at -80°C until RT-qPCR. Bovine and ovine conceptus gene expression was normalized to control genes GAPDH and YWHAZ, and endometrial gene expression was normalized to control genes H3F3A and RNF11. A general linear model procedure (PROC GLM) in SAS was used to test for differences. For the conceptus data, alpha was set at 0.05, and a Tukey’s test was included in the analysis. Conceptus data are presented as non-transformed means ± standard deviation (SD) of the mean. Endometrial data are presented as non-transformed least square means (LSM) ± standard error of the mean (SEM) and P ≤ 0.05 was considered significant. In bovine conceptuses, expression of LGALS3 (24.47 ± 6.41) and LGALS1 (21.80 ± 2.07) was similar, yet, greater than all other galectins measured (α = 0.05). Expression of LGALS8 (1.73 ± 0.27) was 12-fold less than LGALS1, however, LGALS8 expression was greater than LGALS9 (0.77 ± 0.77), LGALS13L (0.30 ± 0.10), LGALS4 (0.08 ± 0.01) and LGALS7 (0.02 ± 0.01) (α = 0.05). In bovine intercaruncular endometrium, expression of LGALS1, LGALS8, and LGALS9 were greatest. Compared to cyclic heifers, endometrial expression of LGALS7 was less (5.80 ± 2.35 and 2.62 ± 2.35; cyclic and pregnant, respectively; P ≤ 0.05), while expression of LGALS9 (9.26 ± 2.25 and 24.56 ± 2.25; cyclic and pregnant, respectively; P ≤ 0.001) and LGALS3 were greater (2.62 ± 0.52 and 4.40 ± 0.52; cyclic and pregnant, respectively; P ≤ 0.05) during pregnancy. In sheep conceptuses, expression of LGALS3 (4.30 ± 0.78), LGALS1 (1.90 ± 0.25) and LGALS8 (1.68 ± 0.25) were similar. Compared to all other galectins measured, LGALS4 (0.20 ± 0.10) had the lowest level of expression (α = 0.05). In ovine intercaruncular endometrium, expression of LGALS3, LGALS8, and LGALS15 were greatest. Compared to cyclic ewes, expression of LGALS7 was greater in pregnant ewes (0.81 ± 0.24 and 2.25 ± 0.24; cyclic and pregnant, respectively; P ≤ 0.05) while LGALS4 (3.10 ± 0.38 and 1.78 ± 0.38; cyclic and pregnant, respectively; P = 0.06) tended to be less during pregnancy and LGALS15 tended to be greater during pregnancy (104.30 ± 234.20 and 591.35 ± 234.20, P = 0.07; cyclic and pregnant, respectively). In conclusion, elongating bovine and ovine conceptuses express various members of the galectin gene family, and select galectin genes are differentially expressed in the endometrium during pregnancy. The galectins may have important functions during early pregnancy in ruminants. Interferon-tau (IFNT), secreted by the elongating bovine conceptus, is the maternal recognition of pregnancy signal that suppresses the endometrial luteolytic mechanism by modifying expression of genes in specific cell types within the endometrium. The conceptus also secretes galectin proteins, specifically galectin-3. This study assessed the effect of IFNT (Experiment 1) and LGALS3 (Experiment 2) on bovine endometrial epithelial and fibroblast cell transcriptomes when the mid-luteal phase endometrial cells were cultured in a 3D environment. In Experiment one, epithelial cells, cultured in Transwell inserts above the fibroblast cells, were treated with RPMI media alone (control; n=4) or treatment media (RPMI with 5% FBS and 1% ABAM) containing 100 ng of recombinant ovine IFNT (n=3) for 6 h. In Experiment two, cells were treated with either RPMI media alone (Control; n=5), pregnant ovine uterine flush fluid, theoretically containing INFT, diluted with treatment media 1:1 (n=4), or treatment media (RPMI with 5% FBS and 1% ABAM) containing either 100 or 1000 ng of recombinant mouse LGALS3 (n=5) for 6 h. Total RNA was extracted from both cell types and the transcriptomes were assayed by RNA Sequencing and RT-qPCR. Interferon-tau regulated 673 and 83 genes (differentially expressed genes or DEGs) in epithelial and fibroblast cells, respectively (P ≤ 0.001; FDR P ≤ 0.05). Many of the DEGs identified in the epithelial (205) and fibroblast cells (64) were previously reported as IFNT and bovine conceptus-induced DEGs in intact bovine endometrium, including well-known ISGs such as ISG15, MX1, MX2, OAS1, and OAS2. Surprisingly, the ZC3HAV1 gene, which was recently found to be up-regulated in intact bovine endometrium in response to the conceptus, was only up-regulated in fibroblast cells despite applying IFNT to the apical surface of the epithelial cells in 3D culture. The RNA-Seq analysis of the bovine endometrial cells treated with mouse LGALS3 protein resulted in the identification of a single gene, RPL23A, that was upregulated in epithelial cells treated with 100 ng of LGALS3 (P ≤ 0.001; FDR P ≤ 0.05). This study supports the idea that the 3D culture of bovine endometrial cells may be utilized to study early conceptus-maternal crosstalk and identify endometrial cell type-specific responses to the conceptus.

Investigation of bovine conceptus galectins and the effect of bovine conceptus secretory proteins on the endometrial epithelial transcriptome