The mammalian embryo displays sexual dimorphism in the preimplantation period. Moreover, competence of the embryo to develop is dependent on the sire from which the embryo is derived and can be modified by embryokines produced by the endometrium such as colony stimulating factor 2 (CSF2). The preimplantation period is characterized by large changes in epigenetic modifications of DNA and histones. It is possible, therefore, that effects of sex, sire, and embryo regulatory molecules are mediated by changes in epigenetic modifications. Here it was tested whether global levels of two histone modifications in the trophectoderm of the bovine blastocyst were affected by sex, sire, and CSF2. It was found that amounts of immunolabeled H3K27me3 were greater (P=0.030) for male embryos than female embryos.  Additionally, labeling for H3K27me3 and H3K18ac depended upon the bull from which embryos were derived. Although CSF2 reduced the proportion of embryos developing to the blastocyst, there was no effect of CSF2 on labeling for H3K27me3 or H3K18ac.  Results indicate that the blastocyst trophoctoderm  can be modified epigenetically by embryo sex and paternal inheritance through alterations in histone epigenetic marks.

Matured COC were washed in HEPES-TALP and transferred in groups of 30 into microdrops of medium consisting of 50 µL IVF-HEPES and 3.5 µL of a solution of 0.05 mM penicillamine, 0.25 mM hypotaurine, and 25 μM epinephrine covered by mineral oil. Each drop was fertilized with 20 µL X or Y-sorted semen that had been purified using Puresperm 40/80 as described by the manufacturer (Nidacon International, Mölndal, Sweden), at a final concentration of ~ 2 x 10⁶ sperm/mL. After 12-18 h at 38.5°C in a humidified atmosphere of 5% (v/v) CO₂ in air, presumptive zygotes were removed from fertilization drops and denuded by vortexing for 5 min in a tube consisting of 100 µL 10,000 U/mL hyaluronidase in 0.9% (w/v) NaCl and 600 µL of HEPES-TALP. The denuded putative zygotes were washed two times in HEPES-TALP, once in SOF-BE2, placed in groups of 25-30 in 45 µL drops of SOF-BE2 medium covered with mineral oil, and incubated at 38.5°C in 5% CO₂, 5% O₂, 90% N₂ and humidified air in a benchtop incubator (EVE, WTA, College Station, TX, USA). At day 5 post-insemination [120 h after insemination (hpi)], 5 µL of SOF-BE2 containing either 100 ng/mL CSF2 (a gift from CIBA-GEIGY, Basle, Switzerland) or vehicle [Dulbecco’s phosphate-buffered saline (DPBS) containing 1 mg/ml fatty-acid free bovine serum albumin (BSA) (Sigma-Aldrich)] was added to each culture drop result in a final concentration of 10 ng/mL CSF2. Cleavage and blastocyst rate were evaluated at day 3.5 post insemination (84 hpi) and at day 7.5 (180 hpi), respectively. The blastocysts at 7.5 were harvested, fixed at in 4% (w/v) paraformaldehyde for 15 min and stored in 50 µL DPBS containing 1% (w/v) polyvinylpyrrolidone at 4°C until processing for histone labeling.

Embryos were produced in a total of 20 replicates involving 5907 oocytes. For each replicate, X and Y-sorted sperm from one or more bulls (ST Genetics, Navasota, TX, USA) was used to fertilize oocytes, and embryos were treated with CSF2 or vehicle. A total of three different Holstein bulls (ST Genetics, Navasota, TX, USA) was used in the experiment. Bull A was used in 8 replicates, bull B in 8 replicates and bull C in 7 replicates For histone labeling,a total of 17 replicates (4789 oocytes) was performed and each replicate utilized sperm from a single bull for fertilization. Bull A  was used in 6 replicates, bull B in 6 replicates and bull C in 5 replicates.

Image J software (version 1.51j8, National Institutes of Health, Bethesda, MD, USA) was utilized to quantify immunofluorescence. Blastocyst nuclei were identified and counted based on Hoechst 33342 labeling. The region of the inner cell mass (ICM) was identified as an aggregation of cells in the blastocyst and the remaining cells were considered as TE cells. Because of difficulties in distinguishing between ICM cells and overlying TE cells in the area of the ICM, analysis was performed only for nuclei that were clearly in the TE region. With the freehand selection tool, a perimeter was manually drawn around each individual nucleus to measure immunofluorescent intensity for H3, H3K27me3, or H3K18ac (green) as well as DNA (blue). Background intensity in the green channel was measured in a random dark area close to the embryo and this value was subtracted from the value for histone intensity. Mean intensity of nuclear labeling for H3, H3K27me3 and H3H18ac was determined for each blastocyst.  The total number of blastocysts analyzed was 123 for H3 (17 replicates), 125 for H3K27me3 (17 replicates) and 128 for H3H18ac (16 replicates).