Anacardic acid reduces acetylation of H4K12 in mouse Oocytes during vitrification

Objective: Over the last years, vitrification has been widely used for oocyte cryopreservation, in animals and humans; however, it frequently causes minor and major epigenetic modifications. The effect of oocyte vitrification on levels of acetylation of histone H4 at lysine 12 [AcH4K12], and histone acetyltransferase [Hat] expression, was previously assessed; however, little is known about the inhibition of Hat expression during oocyte vitrification. This study evaluated the effect of anacardic acid [AA] as a Hat inhibitor on vitrified mouse oocytes

Materials and Methods: In this experimental study, 248 mouse oocytes at metaphase II [MII] stage were divided in three experimental groups namely, fresh control oocytes [which were not affected by vitrification], frozen/thawed oocytes [vitrified] and frozen/thawed oocytes pre-treated with AA [treatment]. Out of 248 oocytes, 173 oocytes were selected and from them, 84 oocytes were vitrified without AA [vitrified group] and 89 oocytes were pretreated with AA, and then vitrified [treatment group]. Fresh MII mouse oocytes were used as control group. Hat expression and AcH4K12 levels were assessed by using real-time quantitative polymerase chain reaction [PCR] and immunofluoresce staining, respectively. In addition, survival rate was determined in vitrified and treatment oocytes

Results: Hat expression and AcH4K12 modification significantly increased [4.17 +/- 1.27 [P.0.001] and 97.57 +/- 6.30 [P<0.001], respectively] in oocytes that were vitrified, compared to the fresh oocytes. After treatment with AA, the Hat mRNA expression and subsequently H4K12 acetylation levels were significantly reduced [0.12 +/- 0.03 [P.0.001] and 89.79 +/- 3.20 [P.0.05], respectively] in comparison to the vitrified group. However, the survival rate was not significantly different between the vitrified [90.47%] and treatment [91.01%] groups [P>0.05]

Conclusion: The present study suggests that AA reduces vitrification risks caused by epigenetic modifications, but does not affect the quality of vitrification. In fact, AA as a Hat inhibitor was effective in reducing the acetylation levels of H4K12

Melatonin modifies histone acetylation during in vitro maturation of mouse oocytes

Objective: We evaluated the effect of melatonin, as a potent antioxidant agent, on glutathione [GSH] and reactive oxygen species [ROS] levels, as well as histone H3 lysine 9 [H3K9], and H4 lysine 12 [H4K12] acetylation when added to oocytes culture medium

Materials and Methods: In this experimental study, two in vitro and in vivo groups were used. In the in vitro group, cumulus oocyte complexes [COCs] from the ovaries of B6D2F1 mice were cultured in maturation medium containing two doses of melatonin [10-9 and 10-6 M] and without melatonin [control group treated with dimethyl sulfoxide [DMSO]] for 22-24 hour. The cumulus expansion and nuclear status were monitored by an inverted microscope. Next, COCs were isolated from the oviducts of superovulated mice and studied as the in vivo group. In in vitro and in vivo matured oocytes, GSH and ROS levels were assessed by monochlorobimane [MCB] and 2-7-dichlorodihydrofluorescein diacetate [H2DCFDA] staining, respectively. Changes in histone acetylation were examined by immunofluorescent staining with specific antibodies against acetylated H3K9 and H4K12

Results: The H4K12 acetylation and ROS levels were significantly higher in the oocytes matured in the in vitro group compared to the in vivo group [P<0.05]. Furthermore, glutathione levels in the in vitro group were considerably lower than that of the in vivo group [P<0.05]. Melatonin at the concentration of 10-6 M had the most substantial effect on nuclear maturation and histone acetylation as well as glutathione and ROS levels in the in vitro group [P<0.05]

Conclusion: Exogenous melatonin improves the competence of mouse oocytes during in vitro maturation [IVM]

Histone modification of embryonic stem cells produced by somatic cell nuclear transfer and fertilized blastocysts

Nuclear transfer-embryonic stem cells [NT-ESCs] are genetically identical to the donor's cells; provide a renewable source of tissue for replacement, and therefore, decrease the risk of immune rejection. Trichostatin A [TSA] as a histone deacetylase inhibitor [HDACi] plays an important role in the reorganization of the genome and epigenetic changes. In this study, we examined whether TSA treatment after somatic cell nuclear transfer [SCNT] can improve the developmental rate of embryos and establishment rate of NT-ESCs line, as well as whether TSA treatment can improve histone modification in NT-ESCs lines. In this experimental study, mature oocytes were recovered from BDF1 [C57BL/6xDBA/2] mice and enucleated by micromanipulator. Cumulus cells were injected into enucleated oocytes as donor. Reconstructed embryos were activated in the presence or absence of TSA and cultured for 5 days. Blastocysts were transferred on inactive mouse embryonic fibroblasts [MEF], so ESCs lines were established. ESCs markers were evaluated by reverse transcription-polymerase chain reaction [RT-PCR]. Histone modifications were analyzed by enzyme linked immunosorbent assay [ELISA]. Result of this study showed that TSA treatment after SCNT can improve developmental rate of embryos [21.12 +/- 3.56 vs.8.08 +/- 7.92], as well as establishment rate of NT-ESCs line [25 vs.12.5]. We established 6 NT-ESCs in two experimental groups, and three embryonic stem cells [ESCs] lines as control group. TSA treatment has no effect in H3K4 acetylation and H3K9 tri-methylation in ESCs. TSA plays a key role in the developmental rate of embryos, establishment rate of ESC lines after SCNT, and regulation of histone modification in NT-ESCs, in a manner similar to that of ESCs established from normal blastocysts