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.

Toll-like receptor4 as a modulator of fertilization and subsequent pre-implantation development following in vitro maturation in mice.

PROBLEM: The use of in vitro maturation (IVM) as an alternative approach to the conventional assisted reproductive technique in clinical practice is limited due to low fertilization rate. The expression of Toll-like receptors (TLRs) as members of an innate immune system in cumulus cells are thought to affect fertilization. This study was aimed to evaluate the effect of IVM on TLR4 gene expression and fertilization rate in oocytes derived from IVM in comparison with in vivo matured one. METHOD OF STUDY: Cumulus cells are collected from oocytes derived IVM and in vivo. The expression level of Tlr4 in cumulus cells of both groups was assessed by quantitative real-time PCR. To examine the protein expression level of TLR4, immunocytochemistry and Western blotting techniques were carried out. TLR4 receptor functions were also confirmed by blockade assay and TLR4 receptor activation with lipopolysaccharide. RESULT: There was a substantial decrease in fertilization and blastulation rate in the IVM group in comparison with the in vivo one. The mRNA expression and protein levels of TLR4 declined in cumulus cells following IVM. Anti-TLR4 blocking antibody dramatically decreased the rate of fertilization and blastocyst formation compared to the in vivo group. In contrast, the fertilization rate was enhanced significantly in the presence of LPS as a TLR4 ligand compared to the control group. CONCLUSION: Low expression level of Tlr4 following IVM and higher fertilization rate through TLR4 receptor activation with LPS proposed that alteration in TLR4 expression and subsequent cytokine section could be a possible cause of low fertilization rate in IVM-derived oocytes.

Does sperm DNA fragmentation affect the developmental potential and the incidence of apoptosis following blastomere biopsy?

Common methods employed in assisted reproduction technology (ART) include intracytoplasmic sperm injection (ICSI) with an unspecified level of sperm DNA fragmentation (SDF) and preimplantation genetic diagnosis (PGD). The aim of this study was to investigate the impact of SDF on human preimplantation embryo development and the incidence of apoptosis following a single blastomere biopsy. Using sperm chromatin dispersion (SCD) to assess SDF, a total of 20 processed semen samples were categorized into two groups; group I: SDF ≤30% and group II: SDF >30%. After ICSI, fertilization, cleavage, and embryo quality score were assessed. A single blastomere was biopsied from day 3 embryos and development was monitored on day 4. The frequency of apoptosis in biopsied embryos was assayed by TUNEL and the level of BCL-2, BAX, hsa-mir-15a, and hsa-mir-16-1 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). SCD was found to be negatively correlated with sperm motility and normal form spermatozoa (p < 0.05). The rate of fertilization, cleavage, and embryo quality score were not significantly different between the two groups (all p > 0.05). SDF >30% had no negative effect on potential development and did not increase the proportion of apoptotic cells and the level of apoptosis-related genes and microRNAs (miRNAs) in group II vs. group I (p > 0.05). It appears that at the levels assessed paternal genome damage had little if any negative effect on preimplantaton embryo development and apoptosis following single blastomere biopsy. This may reflect the selection of morphologically normal sperm for ICSI and the repair capacity of the oocyte.

Regulation of embryonic development and apoptotic-related gene expression by brain-derived neurotrophic factor in two different culture conditions in ovine.

In the present study, we aimed to evaluate effects of brain-derived neurotrophic factor (BDNF) which is a member of neurotrophic factor family on developmental competence of oocytes in sheep. In vitro maturation was performed in presence of various concentrations (0, 10, and 100 ng/mL) of BDNF. Meiotic maturation, levels of intracellular glutathione, embryonic developmental potential after parthenogenetic activation, number of total and apoptotic cells in blastocysts, and expression of Bax and Bcl-2 genes in blastocyst cells were determined. Under unstressed condition, while at 100 ng/mL concentration, BDNF increased the IVM rate; an increase of glutathione level was observed at 10 ng/mL concentration. Moreover, when BDNF-treated oocytes were used for parthenogenetic activation, more blastocyst at both 10 and 100 ng/mL levels was obtained in comparison with the untreated group. Under heat stress (HS), the blastocyst rate was dramatically reduced in untreated oocytes compared to that obtained from 10 ng/mL BDNF groups. Total cell number in blastocysts was not affected by the treatment groups. The mean of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei in blastocysts was not influenced by addition of BDNF in medium and that presence or absence of thermal stress during IVM than the control group. Moreover, our data revealed that the expression of Bax and Bcl-2 genes in blastocysts was affected by both BDNF concentration and HS. Conclusively, supplementation of IVM medium with 10 ng/mL BDNF had a beneficial effect on sheep oocyte competence by increasing the rate of blastocyst especially when HS exists.

Effect of L-carnitine supplementation on maturation and early embryo development of immature mouse oocytes selected by brilliant cresyle blue staining.

PURPOSE: The present study was designed to investigate the effect of L-carnitine treatment during IVM on nuclear and cytoplasmic maturation of immature oocytes selected by Brilliant Cresyle Blue (BCB) staining, and their subsequent developmental competence. MATERIALS & METHODS: Compact cumulus-oocyte complexes (COCs) were collected from NMRI mice ovaries and stained with BCB staining. BCB+ (colored cytoplasm) oocytes were then cultured in tissue culture medium (TCM) 199 with 0.0, 0.3 and 0.6 mg/ml L-carnitine. RESULTS: The both L-carnitine concentrations significantly increased the intracellular glutathione (P<0.001), nuclear maturation (P<0.01) and expression levels of cyclin-dependent kinase1 (CDK1) (P<0.05). Moreover, treated oocytes with 0.6 mg/ml L-carnitine showed increased (P < 0.05) expression of mitogen-activated protein kinase1 (MAPK1) mRNA. Also, adding L-carnitine (0.6 mg/ml) to IVM medium significantly increased the cleavage rate (P<0.05). The blastocyst development rate (BDR) in the both L-carnitine treated groups was significantly higher (P<0.001) than the control group. L-carnitine had no significant effect on total blastocyst cell numbers. CONCLUSIONS: These data indicated that L-carnitine supplementation during IVM of immature BCB+ oocytes improved preimplantation developmental competence of oocytes after IVF, probably by accelerating cytoplasmic and nuclear maturation of oocytes. It may provide a novel approach to improving ART outcomes in infertile couples.

Melatonin modulates the expression of BCL-xl and improve the development of vitrified embryos obtained by IVF in mice.

PURPOSE: Antioxidant and anti-apoptotic effects of melatonin on development of in vitro fertilization (IVF)/vitrified two-cell mouse embryos were evaluated in this study. METHODS: The IVF two-cell embryos were vitrified by cryotop, and were cultured in KSOM medium in different concentrations of melatonin (10(-6), 10(-9), 10(-12) M) and without melatonin. The blastocyst cell number, apoptotic cells and glutathione (GSH) level were evaluated by differential, TUNEL and cell tracker blue staining, respectively. The expression of Bax and Bcl-xl genes was evaluated by qPCR. The expression of melatonin receptors (Mtnr1a and Mtnr1b) in mouse 2-cell embryos and blastocysts was evaluated by RT-PCR. RESULTS: Melatonin increased the rate of cleavage and blastulation at 10(-12) M concentration (p < 0.05). The number of trophectoderm and inner cell mass showed a significant increase (p < 0.05) in 10(-9) M melatonin. The 10(-9) M and 10(-12) M melatonin treatments significantly reduced (p < 0.05) the apoptotic index. The significant increase in the expression of Bcl-xl observed at 10(-9) M concentration however, reduced expression of Bax was not statistically significant. The levels of GSH in 10(-9) and 10(-12) M groups were significantly improved relative to the control group (p < 0.05). The Mtnr1a was expressed in 2-cell embryos and blastocysts in all groups, but the expression of Mntr1b was not detected. CONCLUSION: Melatonin may have a special role against oxidative stress in protection of IVF/vitrified embryos.

Intracellular glutathione content, developmental competence and expression of apoptosis-related genes associated with G6PDH-activity in goat oocyte.

PURPOSE: To associate glucose-6-phosphate dehydrogenase (G6PDH) activity in goat oocytes with intracellular glutathione (GSH) content, meiotic competence, developmental potential, and relative abundance of Bax and Bcl-2 genes transcripts. METHODS: Goat oocytes were exposed to brilliant cresyl blue (BCB) staining test and categorized into BCB(+) (blue-cytoplasm), and BCB(-) (colorless-cytoplasm) groups. A group of oocytes were not exposed to BCB test and was considered as a control group. After maturation in vitro, a group of oocytes were used for determination of nuclear status and intracellular GSH content while another group was subjected to parthenogenetic activation followed by in vitro embryo culture. RESULTS: We found that BCB(+) oocytes not only yielded higher rate of maturation, but also showed an increased level of intracellular GSH content than BCB(-) and control oocytes. Furthermore, BCB(+) oocytes produced more blastocysts than BCB(-) and control oocytes. Our data revealed that the expression of anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) genes were interacted with G6PDH-activity in mature oocyte, their surrounding cumulus cells, and blastocyst-stage embryos. CONCLUSIONS: The results of this study demonstrate that selection of goat oocytes based on G6PDH-activity through the BCB test improves their developmental competence, increases intracellular GSH content, and affects the expression of the apoptosis-related genes.