DOI: 10.5061/dryad.ksn02v705
title: Sex affects immunolabeling for histone 3 K27me3 in the trophectoderm of the bovine blastocyst but not labeling for histone 3 K18ac

description: 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.
description: 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 10<sup>6</sup> sperm/mL. After
 12-18 h at 38.5°C in a humidified atmosphere of 5% (v/v)
 CO<sub>2</sub> 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%
 CO<sub>2</sub>, 5% O<sub>2</sub>, 90%
 N<sub>2</sub> 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).