Quantification of Histone Acetyltransferase and Histone Deacetylase Transcripts

Mammalian oocytes are very unique cells with an unlimited developmental potential. These totipotent cells are able to remove existing gene-expression patterns and to impose new ones.

However, genome reprogramming is still a mystery. Posttranslational modifications by acetylation of the N-termini portion of histones composing the nucleosome are involved in genome reprogramming. These modifications alter the higher-order chromatin structure to render the DNA accessible to the regulatory and transcriptional machinery.

In the present study, we have investigated, to our knowledge for the first time, precise expression patterns of seven genes involved in chromatin structure throughout bovine embryo development. Oocytes harvested from bovine ovaries were used for in vitro production of germinal vesicle oocytes, metaphase II oocytes, 2- and 8-cell embryos, and blastocysts. Total RNA was extracted from pools (triplicates) of 20 oocytes or from embryos of each developmental stage. By means of quantitative reverse transcription-polymerase chain reaction using SYBR Green to detect double-stranded DNA, mRNA expression profiles for histone deacetylases (HDAC1, HDAC2, HDAC3, and HDAC7), histone acetyltransferases (GCN5 and HAT1), and histone H2A were established.

Transcripts for all genes were detected at all stages from the oocyte to the blastocyst. The HDAC1, HDAC2 (class I HDAC), and HAT1 (type B HAT) revealed similar expression profiles. The HDAC3 (class I HDAC) tends to have an expression profile similar to those of HDAC1, HDAC2, and HAT1, whereas the HDAC7 (class II HDAC) and GCN5 (type A HAT) profiles were different from those three. These results indicate variable levels of histone deacetylases and histone acetyltransferases throughout embryonic development and may indicate the ones that are involved in somatic remodeling.

On the Nature and Origin of DNA Strand Breaks in Elongating Spermatids

Transient DNA strand breaks are generated in the whole population of elongating spermatids and are perfectly coincident with histone H4 hyperacetylation at chromatin-remodeling steps. Given the limited DNA repair capacity of elongating spermatids, chromatin remodeling may present a threat to genetic integrity of the male gamete. The nature of the DNA strand breakage, the enzymes involved, and the role of H4 hyperacetylation in the process must be determined to further investigate the potential mutagenic consequences of this important transition. We used the metachromatic dye acridine orange in combination with fluorescence-activated cell sorting to achieve separation of spermatids according to their condensation state. Using single-cell electrophoresis (comet assay), in both alkaline and neutral conditions, we demonstrated that double-stranded breaks account for most of the DNA fragmentation observed in purified elongating spermatids. DNA strand breaks were generated in round spermatids as a result of de novo histone hyperacetylation induced by trichostatin A, whereas an increase in endogenous DNA strand breaks was observed in elongating spermatids. Using a short-term culture of testicular cells, we demonstrated that DNA strand breaks in spermatids were abolished on incubation with two functionally different topoisomerase II inhibitors. Hence, topoisomerase II appears as the unique enzyme responsible for the transient double-stranded breaks in elongating spermatids but depends on histone hyperacetylation for its activity.

Regulation of supply and demand for maternal nutrients in mammals by imprinted genes

The placenta has evolved in eutherian mammals primarily to provide nutrients for the developing fetus. The genetic control of the regulation of supply and demand for maternal nutrients is not understood. In this review we argue that imprinted genes have central roles in controlling both the fetal demand for, and the placental supply of, maternal nutrients. Recent studies on Igf2 (insulin like growth factor 2) knockout mouse models provide experimental support for this hypothesis.

These show effects on placental transport capacity consistent with a role of IGF-II in modulating both the placental supply and fetal demand for nutrients. Imprinting of genes with such functions may have coevolved with the placenta and new evidence suggests that transporter proteins, as well as the regulators themselves, may also be imprinted. These data and hypotheses are important, as deregulation of supply and demand affects fetal growth and has long term consequences for health in mammals both in the neonatal period and, as a result of fetal programming, in adulthood.