If humans ever start to live permanently in space, assisted reproductive technology using preserved spermatozoa will be important for producing offspring; however, radiation on the International Space Station (ISS) is more than 100 times stronger than that on Earth, and irradiation causes DNA damage in cells and gametes.Here we examined the effect of space radiation on freeze-dried mouse spermatozoa held on the ISS for 9 mo at -95°C, with launch and recovery at room temperature. DNA damage to the spermatozoa and male pronuclei was slightly increased, but the fertilization and birth rates were similar to those of controls. Nextgeneration sequencing showed only minor genomic differences between offspring derived from space-preserved spermatozoa and controls, and all offspring grew to adulthood and had normal fertility. Thus, we demonstrate that although space radiation can damage sperm DNA, it does not affect the production of viable offspring after at least 9 mo of storage on the ISS.International Space Station | preservation | freeze-dry | spermatozoa | fertilization S ince the "space dog" Laika (Лайка) was first placed into orbit in 1957 (1), many humans and animals have been to space or stayed on the International Space Station (ISS) for more than 6 mo. In the future, humans likely will live on largescale space stations or in other space habitats for several years or even over many generations. At that time, assisted reproductive technology (ART) likely will be used to produce humans in space habitats, given that the use of ART by infertile couples has increased year by year and that ART can be performed with cryopreserved spermatozoa or embryos (2, 3). In a similar way, domestic animals likely will be generated by artificial insemination (AI) in space, because many domestic animals are already produced by AI using long-term cryopreserved spermatozoa (4). In addition, genetic diversity is very important for maintaining a species, especially in small colonies, and this could be achieved by cryopreserving a diverse range of gamete cells. The environment in space is very different from that on Earth, however, including high levels of space radiation and microgravity, and the effects of these factors on mammalian reproduction are largely unknown. Although with current technology, producing offspring in such an environment can be difficult or dangerous (5), the study of reproduction in space is a very important subject for our future.So far, the effects of microgravity on early development have been studied using sea urchins, fish, amphibians, and birds (6-12). These studies have concluded that microgravity does not prevent animal reproduction. However, because of the difficulty in maintaining mammals and performing experiments in space, studies of mammal reproduction in space have not progressed as well as in other animals, and only a few papers have been published (13-18). Those studies and our previous study (19) have suggested that mammalian reproduction in space under conditions of microgravity cannot be easil...

Mice and cattle use distinct pathways for the first cell segregation into inner cell mass (ICM) and trophectoderm (TE) lineages at the blastocyst stage. However, limited knowledge is available regarding the reliable transcriptional networks that orchestrate the complex developmental processes at this stage in nonrodent species. In order to elucidate the site-dominant transcriptomic properties of bovine blastocysts, we separated cell samples into the ICM and TE using both mechanical and chemical methods and performed in silico prescreening for candidate genes that were site-dominantly expressed in bovine blastocysts. We further performed quantitative real-time PCR and in situ hybridization using the site-specific cell samples. As a result, we identified seven ICM-dominant genes and five TE-dominant genes not found in earlier studies. Our findings provide novel insights into the mechanism of cell-fate specification in the pre-implantation bovine embryo.

Platelets participate in not only thrombosis and hemostasis but also other pathophysiological processes, including tumor metastasis and inflammation. However, the putative role of platelets in the development of solid organs has not yet been described. Here, we report that platelets regulate lung development through the interaction between the platelet-activation receptor, C-type lectin-like receptor-2 (Clec-2; encoded by ), and its ligand, podoplanin, a membrane protein. Clec-2 deletion in mouse platelets led to lung malformation, which caused respiratory failure and neonatal lethality. In these embryos, α-smooth muscle actin-positive alveolar duct myofibroblasts (adMYFs) were almost absent in the primary alveolar septa, which resulted in loss of alveolar elastic fibers and lung malformation. Our data suggest that the lack of adMYFs is caused by abnormal differentiation of lung mesothelial cells (luMCs), the major progenitor of adMYFs. In the developing lung, podoplanin expression is detected in alveolar epithelial cells (AECs), luMCs, and lymphatic endothelial cells (LECs). LEC-specific podoplanin knockout mice showed neonatal lethality and-like lung developmental abnormalities. Notably, these -like lung abnormalities were also observed after thrombocytopenia or transforming growth factor-β depletion in fetuses. We propose that the interaction between Clec-2 on platelets and podoplanin on LECs stimulates adMYF differentiation of luMCs through transforming growth factor-β signaling, thus regulating normal lung development.

The first segregation at the blastocyst stage is the symmetry-breaking event to characterize two cell components; namely, inner cell mass (ICM) and trophectoderm (TE). TEA domain transcription factor 4 (TEAD4) is a well-known regulator to determine TE properties of blastomeres in rodent models. However, the roles of bovine TEAD4 in blastocyst development have been unclear. We here aimed to clarify the mechanisms underlining TE characterization by TEAD4 in bovine blastocysts. We first found that the mRNA expression level was greater in TE than in ICM, which was further supported by TEAD4 immunofluorescent staining. Subsequently, we examined the expression patterns of TE-expressed genes;, and, in the -knockdown (KD) blastocysts. These expression levels significantly decreased in the KD blastocysts compared with controls. Of these downregulated genes, the expression level decreased the most. We further analyzed the expression levels of TE-expressed genes;, and in the KD blastocysts. Strikingly, the KD blastocysts showed the downregulation of , and Furthermore, the ratio of TE-to-ICM cell numbers in the KD blastocysts significantly decreased compared to controls. To our knowledge, this is the first study showing the regulation of expression thorough in mammalian embryos. Not only that, this study also provides evidence that reciprocal regulation of and is required for TE development with appropriate gene expression in bovine blastocysts.

Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38–77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal.