Effects of bovine pregnancy-associated glycoproteins on gene transcription in bovine endometrial explants
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Pregnancy-associated glycoproteins (PAGs) are a complex gene family, whose members are expressed by trophoblasts of ruminants and related species. In cattle, the PAGs accumulate at the trophoblast-uterine interface and many can enter the maternal circulation. However, very little is known about the role they play in pregnancy although preliminary results suggest that PAGs at the placenta-uterine interface play roles involving matrix turnover and immune modulation. This study was designed to provide further insight into the biological roles of bovine PAGs by measuring changes in endometrial transcript abundance for some matrix metalloproteinases (MMPs) and chemokines/cytokines. PAGs for these experiments were purified from mid-gestation bovine placental extracts by affinity chromatography. Heifers were synchronized and bred by artificial insemination with high fertility semen (n = 14) or dead semen (n = 5). Heifers were slaughtered at day 18 post-insemination and the reproductive tracts were obtained and flushed to determine if a conceptus was present. Endometrial explants were collected and split between 4 groups: pregnant with and without 15 [mu]g/ml PAG (n = 10) and nonpregnant with and without 15 [mu]g/ml PAG (n = 9). Endometrial explants were cultured with or without added PAGs for up to 96 hours at 37 degreesC and 5 percent CO2 and samples were harvested at 24 hour intervals for extraction of RNA and fixation. This study focuses on the 48 and 72 hour collection points. Transcript abundance for target genes was analyzed in the endometrial tissue by quantitative PCR. The normalization control transcript was peptidylprolyl isomerase A (PPIA). After 48 and 72 hours, significant increases in CXCL1, CXCL2, and CXCL5 as well as MMP1, MMP3 and MMP13 were measured in the PAG-treated endometrium from both pregnant and non-pregnant animals (P<0.05). CCL11 was upregulated at both time points in the pregnant endometrium but only after 72 hours in the nonpregnant endometrium. There were also significant decreases in message for CCL2, CCL8d CCL16 in the PAG-treated groups from both pregnant and non-pregnant animals at each time point (P<0.05). Significant decreases in CXCL10, CXCL12, and Regakine message were seen only in PAG-treated endometrium from pregnant animals (P<0.05). Structural differences in the luminal and glandular epithelium were seen in the PAG-treated biopsies from both non-pregnant and pregnant heifers. These results suggest that PAGs are capable of inducing structural changes as well as changes in transcript abundance in bovine endometrial explants, which suggests that this model system might be useful to assess PAG function at the placenta-uterine interface.
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M.S.