Space radiation may cause DNA damage to cells and concern for the inheritance of mutations in offspring after deep space exploration. However, there is no way to study the long-term effects of space radiation using biological materials. Here, we developed a method to evaluate the biological effect of space radiation and examined the reproductive potential of mouse freeze-dried spermatozoa stored on the International Space Station (ISS) for the longest period in biological research. The space radiation did not affect sperm DNA or fertility after preservation on ISS, and many genetically normal offspring were obtained without reducing the success rate compared to the ground-preserved control. The results of ground x-ray experiments showed that sperm can be stored for more than 200 years in space. These results suggest that the effect of deep space radiation on mammalian reproduction can be evaluated using spermatozoa, even without being monitored by astronauts in Gateway.

Mouse oocytes are generally collected after euthanasia. However, if oocytes were collected without euthanasia, then mice could be used to collect oocytes again after recovery. This condition is especially useful for mice that are genotypically rare. In this study, we examined the reusability of mice after collecting oocytes via a surgical operation. When oocytes were collected using medetomidine/midazolam/butorphanol combination anesthesia and examined for the quality of oocytes after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), they could develop to full term at the same rate as controls. When oocytes were collected from those mice a second time, the average number of oocytes was reduced by nearly 1/3. However, the blastocyst and offspring rates of those oocytes after IVF or ICSI were the same as those of the control regardless of the recovery day period. Although germinal vesicle (GV) oocytes can be collected from all reused mice, the final number of offspring did not increase. Interestingly, when oocytes were collected from the front position of the ampulla, 76% of the oviducts possessed oocytes after reuse, and the average number of oocytes significantly increased to a level comparable to that of the control. Finally, we examined whether reused mice can be used as recipient females, and then healthy offspring were obtained similarly as the control recipients. In conclusion, we provide a new method to collect a sufficient number of oocytes from reused mice without concern.

Artificial oocyte activation is important for assisted reproductive technologies, such as fertilization with round spermatids (ROSI) or the production of cloned offspring by somatic cell nuclear transfer (SCNT). Recently, phospholipase Cζ (PLCζ)-cRNA was used to mimic the natural process of fertilization, but this method required the serial injection of PLCζ-cRNA and was found to cause damage to the manipulated oocytes. Here we tried to generate offspring derived from oocytes that were fertilized using round spermatid or somatic cell nuclear transfer with the co-injection of PLCζ-cRNA. After co-injecting round spermatids and 20 ng/µL of PLCζ-cRNA into the oocytes, most of them became activated, but the activation process was delayed by more than 1 h. With the co-injection method, the rate of blastocyst formation in ROSI embryos was higher (64%) compared with that of the serial injection method (55%). On another note, when SCNT was performed using the co-injection method, the cloned offspring were obtained with a higher success rate compared with the serial-injection method. However, in either ROSI or SCNT embryos, the birth rate of offspring via the co-injection method was similar to the Sr activation method. The epigenetic status of ROSI and SCNT zygotes that was examined showed no significant difference among all activation methods. The results indicated that although the PLCζ-cRNA co-injection method did not improve the production rate of offspring, this method simplified oocyte activation with less damage, and with accurate activation time in individual oocytes, it can be useful for the basic study of oocyte activation and development.

Round spermatid injection (ROSI) results in a lower birth rate than intracytoplasmic sperm injection, which has hampered its clinical application. Inefficient development of ROSI-embryos has been attributed to epigenetic abnormalities. However, the chromatin-based mechanism that underpins the low birth rate in ROSI remains to be determined. Here, we show that a repressive histone mark H3K27me3 persists from mouse round spermatids into zygotes in ROSI and that round spermatid-derived H3K27me3 is associated with less accessible chromatin and impaired gene expression in ROSI-embryos. These loci are initially marked by H3K27me3 but undergo histone modification remodelling in spermiogenesis, resulting in reduced H3K27me3 in normal spermatozoa. Therefore, the absence of the epigenetic remodelling, presumably mediated by histone turnover during spermiogenesis, leads to dysregulation of chromatin accessibility and transcription in ROSI-embryos. Thus, our results unveil a molecular logic, in which chromatin states in round spermatids impinge on chromatin accessibility and transcription in ROSI-embryos, highlighting the importance of epigenetic remodelling during spermiogenesis in successful reproduction.

The underlying mechanism for parental asymmetric chromatin dynamics is still unclear. To reveal this, we investigate chromatin dynamics in parthenogenetic, androgenic, and several types of male germ cells-fertilized zygotes. Here we illustrate that parental conflicting role mediates the regulation of chromatin dynamics. Sperm reduces chromatin dynamics in both parental pronuclei (PNs). During spermiogenesis, male germ cells acquire this reducing ability and its resistance. On the other hand, oocytes can increase chromatin dynamics. Notably, the oocytes-derived chromatin dynamics enhancing ability is dominant for the sperm-derived opposing one. This maternal enhancing ability is competed between parental pronuclei. Delayed fertilization timing is critical for this competition and compromises parental asymmetric chromatin dynamics and zygotic transcription. Together, parental competition for the maternal factor enhancing chromatin dynamics is a determinant to establish parental asymmetry, and paternal repressive effects have supporting roles to enhance asymmetry.