93 SEX-SPECIFIC DEVELOPMENTAL PROGRAMMING OF THE BOVINE EMBRYO BY COLONY STIMULATING FACTOR 2 (CSF2)

Abstract

Competence of the bovine embryo to establish pregnancy can be enhanced by treatment with colony stimulating factor 2 (CSF2) from Days 5 to 7 post-insemination. The mechanism is unknown but could involve developmental reprogramming. Here, we questioned whether treatment with CSF2 from Days 5 to 7 alters growth, interferon-τ (IFNT) secretion, and the methylome of extra-embryonic membranes (EEM) at Day 15. In vitro-produced embryos were treated with either 0 or 10 ng mL^–1 bovine CSF2 from Days 5 to 7. Expanded blastocysts were transferred into synchronized recipients. On Day 15, conceptuses were recovered by flushing the uterus and sex determined by PCR (n = 4 males and 4 females per treatment). Statistical analysis of IFNT and length was performed using analysis of variance with the general linear models procedure of SAS (SAS Institute Inc., Cary, NC, USA). There was a sex × treatment interaction for conceptus length (P < 0.003) and IFNT in uterine flushing (P < 0.05) (as determined by antiviral activity). CSF2 increased length in males (least squares means ± s.e.m.: 93 ± 19 mm v. 20 ± 19 mm) and decreased length in females (22 ± 19 mm v. 103 ± 19). Similarly, CSF2 increased IFNT in males (45 483 ± 22 615 IU mL^–1 v. –2536.27 ± 13 385 IU mL^–1) and decreased IFNT in females (–14 012 ± 13 642 IU mL^–1 v. 35 404 ± 7514.91 IU mL^–1). The EmbryoGENE DNA Methylation Array (Edmonton, AB, Canada) was used to assess CSF2 effects at 418 805 positions across the genome in a subset of EEM (n = 2 for vehicle and 4 for CSF2 in males; n = 3 for vehicle and n = 3 for CSF2 in females). CSF2 caused hypermethylation for 9842 probes in males and 6227 probes in females and hypomethylation for 9322 probes in males and 3292 probes in females. An analysis was conducted to evaluate if clusters of differentially-methylated probes were non-randomly distributed spatially in 5 Mb regions of individual chromosomes using a 500-kb moving window. Statistical significance was determined using chi-squared tests for each 500-kb window. Differential methylation was not uniformly distributed but rather there were regions or hyper- and hypomethylation that varied with sex. The most common situation was where a region was differentially methylated in one sex but not the other. In some cases, a region was differentially methylated in opposite directions for males and females. For example, a region of BTA1 between positions 67 Mb–77.5 Mb contained 1632 probes, of which 59 probes were hypo-methylated in males and 35 were hypermethylated in females. In conclusion, changes in developmental programming of the bovine embryo caused by CSF2 occur in a sex-specific manner. This result suggests a possible mechanism by which environmental effects on the female affect male embryos differently than female embryos.

Support was provided by U.S. Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) grant 2011-67015-30688.