10.5061/DRYAD.KSN02V705
Hansen, Peter
0000-0003-3061-9333
University of Florida
Carvalheira, Luciano
Universidade Federal de Minas Gerais
Tribulo, Paula
University of Florida
Borges, Alan
Universidade Federal de Minas Gerais
Sex affects immunolabeling for histone 3 K27me3 in the trophectoderm of
the bovine blastocyst but not labeling for histone 3 K18ac
Dryad
dataset
2019
Embryos
Blastocysts
Histones
Epigenetics
DNA methylation
Oocytes
Sperm
Histone modification
National Institutes of Health
https://ror.org/01cwqze88
HD088352
LE "Red" Larson endowment
Coordenação de Aperfeicoamento de Pessoal de Nível Superior
https://ror.org/00x0ma614
2020-01-03T00:00:00Z
2020-01-03T00:00:00Z
en
https://doi.org/10.1371/journal.pone.0223570
45327 bytes
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CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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).