Takuji Nishihara

GSE Is a Maternal Factor Involved in Active DNA Demethylation in Zygotes

After fertilization, the sperm and oocyte genomes undergo extensive epigenetic reprogramming to form a totipotent zygote. The dynamic epigenetic changes during early embryo development primarily involve DNA methylation and demethylation. We have previously identified Gse (gonad-specific expression gene) to be expressed specifically in germ cells and early embryos. Its encoded protein GSE is predominantly localized in the nuclei of cells from the zygote to blastocyst stages, suggesting possible roles in the epigenetic changes occurring during early embryo development. Here, we report the involvement of GSE in epigenetic reprogramming of the paternal genome during mouse zygote development. Preferential binding of GSE to the paternal chromatin was observed from pronuclear stage 2 (PN2) onward. A knockdown of GSE by antisense RNA in oocytes produced no apparent effect on the first and second cell cycles in preimplantation embryos, but caused a significant reduction in the loss of 5-methylcytosine (5mC) and the accumulation of 5-hydroxymethylcytosine (5hmC) in the paternal pronucleus. Furthermore, DNA methylation levels in CpG sites of LINE1 transposable elements, Lemd1, Nanog and the upstream regulatory region of the Oct4 (also known as Pou5f1) gene were clearly increased in GSE-knockdown zygotes at mid-pronuclear stages (PN3-4), but the imprinted H19-differential methylated region was not affected. Importantly, DNA immunoprecipitation of 5mC and 5hmC also indicates that knockdown of GSE in zygotes resulted in a significant reduction of the conversion of 5mC to 5hmC on LINE1. Therefore, our results suggest an important role of maternal GSE for mediating active DNA demethylation in the zygote.

Oral melatonin supplementation improves oocyte and embryo quality in women undergoing in vitro fertilization-embryo transfer.

Abstract The aim of this study was to evaluate the efficacy of oral melatonin supplementation on oocyte and embryo quality in patients in an assisted reproductive technologies program. All patients were treated for at least 2 weeks with melatonin (3 mg/day). To evaluate the cumulative effect of melatonin supplementation, we compared cycle outcomes between the first (no supplementation) and second cycles (melatonin supplementation) of patients who completed two treatment cycles. There were no significant differences in maturation rates (p = 0.50), blastocyst rates (p = 0.75), and the rate of good quality blastocysts (p = 0.59) between the first and second cycles. The fertilization rate of ICSI was higher in the second cycle than that in the first cycle (69.3 versus 77.5%). Being limited to patients with a low fertilization rate in the first cycle (<60%), the fertilization rate dramatically increased after melatonin treatment (35.1 versus 68.2%). The rate of good quality embryos also increased (48.0 versus 65.6%). An important finding in our study was that oral melatonin supplementation can have a beneficial effect on the improvement of fertilization and embryo quality and this may have occurred due to a reduction in oxidative damage.

Possible Role of ZPAC, Zygote-specific Proteasome Assembly Chaperone, During Spermatogenesis in the Mouse

In the mammalian testis, the ubiquitin-proteasome system plays important roles in the process that promotes the formation of mature sperm. We recently identified zygote-specific proteasome assembly phaperone (ZPAC), which is specifically expressed in the mouse gonads and zygote. ZPAC mediates a unique proteasome assembly pathway in the zygote, but the expression profile and function of ZPAC in the testis is not fully understood. In this study, we investigated the possible role of ZPAC during mouse spermatogenesis. First, we analyzed the expression of ZPAC and 20S proteasome subunit α4/PSMA7 in the adult mouse testis. ZPAC and α4 were expressed in spermatogonia, spermatocytes, and round spermatids. In elongating spermatids, ZPAC was expressed until step 10, whereas expression of α4 persisted until step 12. We then examined the expression profile of ZPAC and α4 in a mouse model of experimental unilateral cryptorchidism. Consistent with appearance of morphologically impaired germ cells following cryptorchidism, the ZPAC protein level was significantly decreased at 4 days post induction of experimental cryptorchidism (D4) compared with the intact testis, although the amount of α4 protein persisted at least until D10. Moreover, intense ZPAC staining was co-localized with staining of annexin V, an early indicator of apoptosis in mammalian cells, in germ cells of cryptorchid testis, but ZPAC was also expressed in germ cells showing no detectable expression of annexin V. These results suggest that ZPAC plays a role during spermatogenesis and raises the possibility that 20S proteasome mediated by ZPAC may be involved in the regulation of germ cell survival during spermatogenesis.

Functional Analysis of Nocturnin, a Circadian Deadenylase, at Maternal-to-zygotic Transition in Mice

Abstract Degradation of maternally stored mRNAs after fertilization is an essential process for mammalian embryogenesis. Maternal mRNA degradation depending on deadenylases in mammalian early embryos has been mostly speculated, rather than directly demonstrated. Previously, we found that gene expression of nocturnin, which functions as a circadian clock-controlled deadenylase in mammalian cells, was clearly changed during the maternal-to-zygotic transition (MZT). Here, we investigated the possible role of nocturnin during mouse MZT. First, we examined the expression profile and localization of nocturnin in mouse oocytes and early embryos. The abundance of Nocturnin mRNA level was significantly decreased from the MII to 4-cell stages and slightly increased from the 8-cell to blastocyst stages, whereas the Nocturnin protein level was almost stable in all examined cells including GV and MII oocytes and early embryos. Nocturnin was localized in both the cytoplasm and the nucleus of all examined cells. We then examined the effect of loss or gain of Nocturnin function on early embryonic development. Knockdown of Nocturnin by injection of Nocturnin antisense expression vector into 1-cell embryos resulted in the delay of early embryonic development to the early blastocyst stage. Moreover, Nocturnin-overexpressed embryos by injection of Nocturnin expression vector impaired their development from the 1-cell to 2-cell or 4-cell stages. These results suggest that precise expression of nocturnin is critical to proper development of early mouse embryos. Functional analysis of nocturnin may contribute to the understanding of the possible role of the deadenylase at mouse MZT.

Evaluation of transfer media containing different concentrations of hyaluronan for human in vitro fertilization

The aim of the present prospective study was to investigate the efficacy of hyaluronan (HA) using two different concentrations as embryo transfer media for in vitro fertilization embryo transfer cycles.

Evaluation of antioxidant status and oxidative stress markers in follicular fluid for human in vitro fertilization outcome

Antioxidant status and oxidative stress markers in human follicular fluid (FF) surrounding oocytes may be related to the outcomes of in vitro fertilization and embryo transfer (IVF‐ET). Therefore, we herein examined the relationship between antioxidant status and oxidative stress markers in FF and the outcomes of IVF‐ET.