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Intrauterine administration of peripheral blood mononuclear cells (PBMCs) prior to bovine embryo transfer (ET) was previously shown to improve the pregnancy rate. To better understand how PBMCs improve the pregnancy rate, we examined gene expression in the cells from uterine lumen and evaluated the morphology of bovine pre-attachment embryos in utero following intrauterine administration of PBMCs. On day 3 of the estrous cycle (day 0 = estrous), bovine PBMCs were isolated and suspended in RPMI 1640, and were incubated for 24 hr. The cultured PBMCs were administered non-surgically to the uterine horn ipsilateral to the corpus luteum on day 4 of the estrous cycle (PBMC group). On day 9, endometrial-luminal lymphoid cells from uterine lumen ipsilateral to the corpus luteum were collected by uterine flushing. Transcripts for macrophage-colony stimulating factor in the lymphoid cells were more abundant in the PBMC group than in the control group (P < 0.05). On day 7 (of the separate experiments), five blastocysts were each transferred to the luminal area, to which PBMCs had been administered on day 4. These embryos were allowed to develop in utero until day 15 of gestation, when embryos were non-surgically retrieved from the uterus. The average length of trophoblasts recovered from the PBMC group was significantly longer than that of the control group (51.6 ± 7.8 vs. 27.4 ± 6.0 mm, P < 0.05). Our results strongly suggest that intrauterine administration of PBMCs improves endometrial environment, which promotes early development of pre-attachment conceptuses.

Abstract. The sex ratio of mammals has previously been shown to be affected by maternal stress. In our previous study, the proportion of female embryos collected from superovulated and artificially inseminated Holstein heifers that were frequently placed in stanchions and subjected to transrectal examinations of the ovaries during the follicular phase tended to be higher than the expected 50%. The goal of the present study was to test the validity of this observation using a greater number of heifers. Superovulated heifers were artificially inseminated at 56 and 72 h after PGF2α treatment using a single batch of frozen semen. Frequent capture (FC), transrectal examination and/or blood sampling were performed at 4-h intervals from 36 to 76 h after PGF2α treatment (n=13). Nine heifers were used as the Control (non-treatment). Seven-day-embryos were recovered by uterine flushing. Male and female embryos were separated using the loop-mediated isothermal amplification procedure. The proportion of female transferable embryos in the FC group (67.8%, 78/115) was significantly higher than that in the Control group (51.2%, 43/84, P<0.05). The peak concentration of plasma cortisol during the follicular phase following superovulatory treatment was 20.6 ng/ml in the FC group. These results suggest that subjecting heifers to stress during the follicular phase following superovulatory treatment may increase the female sex ratio of embryos.

We have identified members of the Xenopus cortical granule lectin (xCGL) family as candidate target glycoproteins of Xenopus galectin-VIIa (xgalectin-VIIa) in Xenopus embryos. In addition to the original xCGL, we also identified a novel member of the xCGL family, xCGL2. Expression of the mRNAs of xCGL and xCGL2, as well as that of xgalectin-VIIa, was observed throughout early embryogenesis. Two and three potential N-glycosylation sites were deduced from the amino acid sequences of xCGL and xCGL2, respectively, and xgalectin-VIIa recognizes N-glycans linked to a common site in xCGL and xCGL2 and also recognizes N-glycans linked to a site specific to xCGL2. However, interaction between xgalectin-Ia and xCGLs was not detectable. We also obtained consistent results on surface plasmon resonance analysis involving xCGLs as ligands and xgalectins as analytes. The Kd value of the interaction between xgalectin-VIIa and xCGLs was calculated to be 35.9 nM. The structures of the N-glycans of xCGLs, which were recognized by xgalectin-VIIa, were analyzed by the two-dimensional sugar map method, and three kinds of N-acetyllactosamine type, biantennary N-glycans were identified as the major neutral N-glycans. The binding specificity of oligosaccharides for xgalectin-VIIa was analyzed by frontal affinity chromatography (FAC). The oligosaccharide specificity pattern of xgalectin-VIIa was similar to that of the human homolog galectin-3, and it was also shown that the N-acetyllactosamine type, biantennary N-glycans exhibit high affinity for xgalectin-VIIa (Kd = 11 microM). These results suggest that xgalectin-VIIa interacts with xCGLs through binding to N-acetyllactosamine type N-glycans and that this interaction might make it possible to organize a lectin network involving members of different lectin families.

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