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Implications of histone modification and DNA
methylation during mouse spermatogenesis
Ning Song, Takehiko Koji
Department of Histology and Cell Biology, Nagasaki University
Graduate School of Biomedical Sciences
Epigenetic factors such as histone modification and DNA methylation
have been implicated in the regulation of gene expression. However,
the association of those factors with a specific stage of germ cells
during spermatogenesis has not been fully understood. In the present
study, we analysed DNA methylation states quantitatively in each
germ cell by a novel method, HELMET. Histone H3 modifications
were examined by immunohistochemistry. The methylation ratio
of CCGG sequence gradually increased to a maximum in the
stage from pachytene spermatocytes to round spermatids, and then
declined. The increase in DNA methylation seemed to be correlated
with deacetylation of histone H3, indicating genes are generally
inactivated transcriptionally. We also found the promotion of histone
H3 acetylation in parallel with DNA demethylation in elongating
spermatids, may facilitate histone-protamine exchange. Interestingly,
in vivo treatment of mice with 5-azadC reduced DNA methylation,
but also induced H3K4me3 in spermatogonia accompanying with
frequent apoptosis. These results indicate coordinate changes in the
modifications of DNA and histone, and their close association with
spermatogenesis.
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Unique integration of fertilizationrelated protein
Equatorin EQT into the acrosomal membrane
during spermatogenesis
Chizuru Ito, Kenji Yamatoya, Kiyotaka Toshimori
Dept. Anatomy and Developmental Biology, Grad. Sch. Med., Univ.
Chiba
EQT is a widely distributed fertilization-related acrosomal protein in
mammalian sperm. During the acrosome reaction some quantity of
EQT is translocated on the plasma membrane covering the equatorial
segment (ES) where sperm fuses with an oolemma, while the
majority remains on the inner acrosomal membrane (IAM) until male
pronucleus is formed. To analyze the nature of the EQT it is important
to investigate its origin and expression during spermatogenesis. In
this study we show the integration process of EQT in wild and EQT-
EGFP transgenic mice using immunoelectron microscopy and highresolution
light microscopy including STED nanoscopy. EQT mRNA
was first detected in stage I-VII pachytene spermatocytes and then
in step1-7 round spermatids. EQT protein was initially detected on
the nascent outer acrosomal membrane of step 2 round spermatids
and then gradually accumulated mainly on the IAM of the ES in
elongating spermatids. EQT was not found acrosomal granules. We
also found EQT forms complex with acrosomal matrices and the
perinuclear theca (PT) by MS/MS analysis after immunoprecipitation.
This evidence suggests EQT co-translocates associating with
acrosomal matrices and PT.
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3 4
5 3
Hoxa11/d11/d12
Hox
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1996