Para-phenylenediamine deteriorates oocyte quality by impairing mitochondrial function.

Several studies demonstrate that para-phenylenediamine (PPD) is often added to permanent oxidative hair dyes. Sub-chronic topical exposure to PPD in male rats damages their testicular function; however, little is known about the effects of PPD exposure on the female reproductive system, especially on oocyte quality. In this study, we found that PPD can affect the meiotic capacity of oocytes and their fertilization potential. In particular, PPD can damage the spindle/chromosome structure and prevent oocytes from developing and maturing normally. Furthermore, PPD exposure compromised the dynamics of cortical granules and their component, ovastacin. In addition to the protein level of Juno, the sperm receptors on the egg membrane, were substantially impaired in PPD-administered oocytes, thus leading to fertilization failure. Finally, we found that PPD exposure resulted in abnormal mitochondrial function, which led to oocyte degeneration, apoptosis, and increased ROS levels. Altogether, our study illustrates that mitochondrial dysfunction and redox perturbation are the major causes of the poor quality of oocytes exposed to PPD.

Sperm MMP-2 is indispensable for fast electrical block to polyspermy at fertilization in Xenopus tropicalis.

Sperm matrix metalloproteinase-2 (MMP-2) is necessary for frog fertilization. Monospermy is ensured by a fast, electrical block to polyspermy mediated by a positive fertilization potential. To determine the role of the MMP-2 hemopexin domain (HPX) in a fast block to polyspermy during fertilization of the frog, Xenopus tropicalis, we prepared mutant frogs deficient in mmp2 gene using the transcription activator-like effector nuclease method. mmp2 ΔHPX (-/-) sperm without MMP-2 protein were able to fertilize wild-type (WT; +/+) eggs. However, polyspermy occurred in some eggs. The mutant sperm generated a normal fertilization potential amounting to 10 mV, and were able to fertilize eggs at 10 mV, at which WT sperm never fertilized. Sensitivity during voltage-dependent fertilization decreased in mutant sperm. This study demonstrates for the first time that the genetic alteration of the MMP-2 molecule in sperm causes polyspermy during fertilization of a monospermic species. Our findings provide reliable evidence that sperm MMP-2 is indispensable for the fast, electrical block to polyspermy during Xenopus fertilization.

WGBS combined with RNA-seq analysis revealed that Dnmt1 affects the methylation modification and gene expression changes during mouse oocyte vitrification.

Understanding the molecular level changes of oocyte cryopreservation and the subsequent warming process is essential for improving the oocyte cryopreservation technologies. Here, we collected the mature metaphase II (MII) oocytes from mice and vitrified. After thawing, single-cell whole-genome bisulphite sequencing (scWGBS) and single-cell RNA sequencing (scRNA-seq) were used to investigate the molecular attributes of this process. Compared to the fresh oocytes, the vitrified oocytes had lower global methylation and gene expression levels, and 1426 genes up-regulated and 3321 genes down-regulated. The 1426 up-regulated differentially expressed genes (DEGs) in the vitrified oocytes were mainly associated with the histone ubiquitination, while the 3321 down-regulated genes were mainly enriched in the mitochondrion organisation and ATP metabolism processes. The differentially methylated regions (DMRs) were mainly located in promoter, intron and exon region of genes, and the length of DMRs in the vitrified oocytes were also significantly lower than that of the fresh oocytes. Notably, there were no significant difference in the expression levels of DNA demethylases (Tet1, Tet2 and Tet3) and methyltransferases (Dnmt3a and Dnmt3b) between two treatments of oocytes. However, Dnmt1 and kcnq1ot1, which are responsible for maintaining DNA methylation, were significantly down regulated in the vitrified oocytes. Gene regulatory network (GRN) analysis showed the Dnmt1 and kcnq1ot1 play a core role in regulating methylation and expression levels of downstream genes. Moreover, some genes associated with oocyte quality were significantly down-regulated in the vitrified oocytes. The present data provides a new perspective for understanding the impact of vitrification on oocytes.

Follicular fat-soluble vitamins as markers of oocyte competency.

The main aim of this prospective study was to investigate the effect of the concentration of fat-soluble vitamins A, D, E and K in individual follicles on oocyte quality and developmental competence. The analysis was performed on 313 follicular fluid (FF) samples from 50 patients undergoing ovarian stimulation with intracytoplasmic sperm injection. We demonstrated that the mean concentration of individual vitamins in FF correlated with their level in serum (p < 0.0001). The levels of vitamin D in FF were higher than in serum, while the opposite was observed for other analyzed vitamins. We did not observe a correlation between FF vitamin D concentration with fertilization success. However, we observed its association with embryo development status on day 3. Moreover, we showed a statistically significant negative correlation between the mean day 5 embryo score and the concentration of vitamin D in serum (rS = -0.68 p = 0.01) and follicular fluid (rS = -0.71 p = 0.01). Our study showed that FF concentration of vitamin A and E was helpful in the prediction of fertilization success of each individual oocyte. Moreover, vitamin A and E concentrations in FF were associated with status of embryo development on the third day of culture. Vitamin A was also associated with the embryo quality on day 2 and the embryo development status on day 5 after fertilization. In conclusion, a combination of FF vitamin analysis and routine morphological assessment could allow for a more accurate and sensitive method of determining embryonic developmental competence and enable the selection of a better embryo to transfer and perhaps translating into an increased chance of pregnancy.Abbreviations: in vitro fertilization: IVF; anti-Mullerian hormone: AMH; follicular fluid: FF; intracytoplasmic sperm injection: ICSI; top quality: TQ; vitamin D binding globulin level: VDBP; assisted reproductive technology: ART.

The Effect of Preincubation Time and Myo-inositol Supplementation on the Quality of Mouse MII Oocytes.

BACKGROUND: It is demonstrated that optimal preincubation time improves oocyte quality, fertilization potential and developmental rate. This study aimed to evaluate the effect of preincubation time in the simple and myo-inositol supplemented medium on the oocyte quality regarding oxidative stress and mitochondrial alteration. METHODS: Cumulus oocyte complexes (COCs) retrieved from superovulated NMRI mice were divided in groups of 0, 4 and 8 hr preincubation time in the simple and 20 mmol/L myo-inositol supplemented media. Intracellular reactive oxygen species (H2O2), glutathione (GSH), mitochondrial membrane potential (MMP), ATP content, and mitochondrial amount were measured and analyzed in experimental groups. One-way ANOVA and Kruskal-Wallis were respectively used for parametric and nonparametric variables. Statistical significance was defined as p<0.05. RESULTS: In comparison to control group, variables including ROS, GSH, mitochondrial amount, fertilization and developmental rates were significantly changed after 4 hr of preincubation in the simple medium, while MMP decreased following 8 hr of preincubation in the simple medium (p˂0.001). Preincubation of oocytes up to 8 hr in the simple medium could not decrease ATP content. For both 4 and 8 hr preincubation times, myo-inositole could decrease H2O2 and increase GSH and MMP levels and consequently could improve fertilization rate compared to oocytes preincubated in the simple culture. CONCLUSION: It seems that 4 hr or more preincubation time can decrease the oocyte quality and lead to reduced oocyte fertilization and developmental potential. Howevere, myo-inositol may prevent oocyte quality reduction and improve fertilization potential in comparision to the equivalent simple groups.

Early and later studies on action potential and fertilization potential of echinoderm oocytes and Ca2+ response of mammalian oocytes.

This is a personal essay starting from the early study on fertilization signals in echinoderm and mammalian oocytes. It presents actual examples showing that a unexpected discovery leads to unimaginable development of the research in diverse directions in later years and yields a common concept after long years' effort and accumulation. Those outcomes are the happiest gift for researchers. We also learn many precepts in our own research life.