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

effects of in vitro maturation technique on the expression of genes involved in embryonic genome activation of human embryos

Objective: In vitro maturation technique [IVM] is shown to have an effect on full maturation of immature oocytes and the subsequent embryo development. Embryonic genome activation [EGA] is considered as a crucial and the first process after fertilization. EGA failure leads to embryo arrest and possible implantation failure. This study aimed to determine the role of IVM in EGA-related genes expression in human embryo originated from immature oocytes and recovered from women receiving gonadotrophin treatment for assisted reproduction

Materials and Methods: In this experimental study, germinal vesicle [GV] oocytes were cultured in vitro. After intracytoplasmic sperm injection of the oocytes, fertilization, cleavage and embryo quality score were assessed in vitro and in vivo. After 3-4 days, a single blastomere was biopsied from the embryos and then frozen. Afterwards, the expression of EGA-related genes in embryos was assayed using quantitative reverse transcriptase-polymerase chain reaction [PCR]

Results: The in vitro study showed reduced quality of embryos. No significant difference was found between embryo quality scores for the two groups [P=0.754]. The in vitro group exhibited a relatively reduced expression of the EGA- related genes, when compared to the in vivo group [all of them showed P=0.0001]

Conclusion: Although displaying the normal morphology, the IVM process appeared to have a negative influence on developmental gene expression levels of human preimplanted embryos. Based on our results, the embryo normal morphology cannot be considered as an ideal scale for the successful growth of embryo at implantation and downstream processes

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]

Expression of folliculogenesis-related genes in vitrified human ovarian tissue after two weeks in vitro culture

Objective: This study was designed to evaluate the effects of vitrification and in vitro culture of human ovarian tissue on the expression of oocytic and follicular cell-related genes

Materials and Methods: In this experimental study, ovarian tissue samples were obtained from eight transsexual women. Samples were cut into small fragments and were then assigned to vitrified and non-vitrified groups. In each group, some tissue fragments were divided into un-cultured and cultured [in alpha-MEM medium for 2 weeks] subgroups. The normality of follicles was assessed by morphological observation under a light microscope using hematoxylin and eosin [H and E] staining. Expression levels of factor in the germ line alpha [FIGLA], KIT ligand [KL], growth differentiation factor 9 [GDF-9] and follicle stimulating hormone receptor [FSHR] genes were quantified in both groups by real-time reverse transcriptase polymerase chain reaction [RT-PCR] at the beginning and the end of culture

Results: The percentage of normal follicles was similar between non-cultured vitrified and non-vitrified groups [P>0.05], however, cultured tissues had significantly fewer normal follicles than non-cultured tissues in both vitrified and non-vitrified groups [P<0.05]. In both cultured groups the rate of primary and secondary follicles was significantly higher than non-cultured tissues [P<0.05]. The expression of all examined genes was not significantly altered in both non-cultured groups. Whiles, in comparison with cultured tissues non-cultured tissues, the expression of FIGLA gene was significantly decreased, KL gene was not changed, GDF-9 and FSHR genes was significantly increased [P<0.05]

Conclusion: Human ovarian vitrification following in vitro culture has no impairing effects on follicle normality and development and expression of related-genes. However, in vitro culture condition has deleterious effects on normality of follicles

Exposure of neonatal mice to Sevoflurane may induce male germ cell apoptosis in testicular tissue after puberty

Background: common use of sevoflurane in congenital defects during repeated surgeries may have detrimental effects on spermatogenesis after puberty

Objective: this study investigated sevoflurane effects on spermatogenesis process in male mature mice after exposure in prepubertal time

Materials and Methods: 24 neonatal NMRI male mice were randomly classified in three groups. Experimental 1 and 2 groups [exposure to 1 minimum alveolar concentration [MAC] and 2 MAC sevoflurane, respectively in 2 lit/min oxygen [O[2]] for 7 days [30 min, daily] and control. All groups were sacrificed after 2 months. Histological assessment, immunohistochemistry and apoptosis process was done. Bax and Bcl2 expression was evaluated in the testicular tissue by real time Poly Chain Reaction

Results: our results showed that the integrity of testicular tissue was preserved in both experimental groups. Count of spermatogonial cells had significant decrease in group 2 compared to others. The rate of apoptosis in spermatogonial cells was 15+/-3% and 9+/-2% in the group 2 and 1, respectively. Also, Bax/Bcl[2] ratio was 0.2615, 1.0070 and 9.3657 in control, experimental group 1 and 2, respectively. This result was significant [p

Conclusion: continuous exposure of 2 MAC sevoflurane in 2 lit/min O[2] simultaneous during prepubertal may create more testicular tissue damage in terms of cellular and molecular function compared to continuous exposure to lower level of sevoflurane by increase in ratio of Bax/Bcl[2] and apoptosis in germ cells after puberty

Oligoclonal expression of T-cell receptor beta variable genes in normal human endometrium

In spite of their key role in various immunological processes occurring in the endometrium, T cells- especially alpha-beta+ subtype- residing in this mucosal tissue, have not been extensively explored. We present here the profile of expressed genes for variable region of ??chain of T cell receptor [TCR] in normal endometrium as compared to peripheral blood. Samples from endometrium were taken from normal fertile women during routine check-up by Pipelle pipette or after hysterectomy operation. Total RNA from both blood and endometrial samples was extracted and RT-PCR using BV gene specific primers was performed. After southern blotting, hybridization with radiolabelled specific probe and autoradiography, relative expression of each BV family was determined. Clonal expansions of the over-expressed genes were studied by determining their CDR3 length polymorphism. A total of 12 blood and 14 endometrial samples were collected. Only one TCRBV gene [TCRBV7] was expressed significantly more and 3 genes less frequently in the endometrium compared to blood. Also, two other genes [TCRBV10 and 12] were found marginally more frequent in the endometrium. As for their clonality, all 3 TCRBV genes examined here showed a rather restricted [oligoclonal] and in some cases, very restricted [probably monoclonal] pattern in the endometrium in contrast to polyclonal patterns in the blood. Our results indicate the similarities between T cells residing in different mucosal tissues and support their common recruitment and functional potentials. Moreover, our findings provide a basis for future investigations about endometrial T cell involvement and their antigen specificities in different gynecological problems