Effect of microgravity on mammalian embryo development evaluated at the International Space Station.

Mammalian embryos differentiate into the inner cell mass (ICM) and trophectoderm at the 8-16 cell stage. The ICM forms a single cluster that develops into a single fetus. However, the factors that determine differentiation and single cluster formation are unknown. Here we investigated whether embryos could develop normally without gravity. As the embryos cannot be handled by an untrained astronaut, a new device was developed for this purpose. Using this device, two-cell frozen mouse embryos launched to the International Space Station were thawed and cultured by the astronauts under microgravity for 4 days. The embryos cultured under microgravity conditions developed into blastocysts with normal cell numbers, ICM, trophectoderm, and gene expression profiles similar to those cultured under artificial-1 g control on the International Space Station and ground-1 g control, which clearly demonstrated that gravity had no significant effect on the blastocyst formation and initial differentiation of mammalian embryos.

Development of a new device for manipulating frozen mouse 2-cell embryos on the International Space Station.

Whether mammalian embryos develop normally under microgravity remains to be determined. However, embryos are too small to be handled by inexperienced astronauts who orbit Earth on the International Space Station (ISS). Here we describe the development of a new device that allows astronauts to thaw and culture frozen mouse 2-cell embryos on the ISS without directly contacting the embryos. First, we developed several new devices using a hollow fiber tube that allows thawing embryo without practice and observations of embryonic development. The recovery rate of embryos was over 90%, and its developmental rate to the blastocyst were over 80%. However, the general vitrification method requires liquid nitrogen, which is not available on the ISS. Therefore, we developed another new device, Embryo Thawing and Culturing unit (ETC) employing a high osmolarity vitrification method, which preserves frozen embryos at -80°C for several months. Embryos flushed out of the ETC during thawing and washing were protected using a mesh sheet. Although the recovery rate of embryos after thawing were not high (24%-78%) and embryonic development in ETC could not be observed, thawed embryos formed blastocysts after 4 days of culture (29%-100%) without direct contact. Thus, this ETC could be used for untrained astronauts to thaw and culture frozen embryos on the ISS. In addition, this ETC will be an important advance in fields such as clinical infertility and animal biotechnology when recovery rate of embryos were improved nearly 100%.

Paternally inherited H3K27me3 affects chromatin accessibility in mouse embryos produced by round spermatid injection.

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 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.

Healthy cloned offspring derived from freeze-dried somatic cells.

Maintaining biodiversity is an essential task, but storing germ cells as genetic resources using liquid nitrogen is difficult, expensive, and easily disrupted during disasters. Our aim is to generate cloned mice from freeze-dried somatic cell nuclei, preserved at -30 °C for up to 9 months after freeze drying treatment. All somatic cells died after freeze drying, and nucleic DNA damage significantly increased. However, after nuclear transfer, we produced cloned blastocysts from freeze-dried somatic cells, and established nuclear transfer embryonic stem cell lines. Using these cells as nuclear donors for re-cloning, we obtained healthy cloned female and male mice with a success rate of 0.2-5.4%. Here, we show that freeze-dried somatic cells can produce healthy, fertile clones, suggesting that this technique may be important for the establishment of alternative, cheaper, and safer liquid nitrogen-free bio-banking solutions.

Mouse in vivo-derived late 2-cell embryos have higher developmental competence after high osmolality vitrification and -80°C preservation than IVF or ICSI embryos.

Mammalian embryos are most commonly cryopreserved in liquid nitrogen; however, liquid nitrogen is not available in special environments, such as the International Space Station (ISS), and vitrified embryos must be stored at -80°C. Recently, the high osmolarity vitrification (HOV) method was developed to cryopreserve mouse 2-cell stage embryos at -80°C; however, the appropriate embryo is currently unknown. In this study, we compared the vitrification resistance of in vivo-derived, in vitro fertilization (IVF)-derived, and intracytoplasmic sperm injection (ICSI)-derived mouse 2-cell embryos against cryopreservation at -80°C. The ICSI embryos had lower survival rates after warming and significantly lower developmental rates than the in vivo and IVF embryos. Further, IVF embryos had a lower survival rate after warming, but a similar rate to the in vivo embryos to full-term development. This result was confirmed by simultaneous vitrification of in vivo and IVF embryos in the same cryotube using identifiable green fluorescent protein-expressing embryos. We also evaluated the collection timing of the in vivo embryos from the oviduct and found that late 2-cell embryos had higher survival and developmental rates to full-term than early 2-cell embryos. Some early 2-cell embryos remained in the S-phase, whereas most late 2-cell embryos were in the G2-phase, which may have affected the tolerance to embryo vitrification. In conclusion, when embryos must be cryopreserved under restricted conditions, such as the ISS, in vivo fertilized embryos collected at the late 2-cell stage without long culture should be employed.

Evaluating the long-term effect of space radiation on the reproductive normality of mammalian sperm preserved on the International Space Station.

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.

Characterizing the role of, and physical and emotional burden on caregivers of patients with heart failure: Results from a cross-sectional survey in Japan.

The aim of this cross-sectional survey was to characterize the role of and burden on caregivers of heart failure (HF) patients in Japan, since such data are limited at present. Data from 126 caregivers whose average age was 63.5 years were analyzed. Helping to prepare meals/cooking was the most frequently reported activity (47% of caregivers); 24% found this the most burdensome. The most frequently reported physical consequence of caregiving was feeling physically tired (44%); emotionally worrying about the patient (62%) was the most frequent psychological consequence. Approximately half of the caregivers reported that caring for patients impacted their lifestyle. Although 40% of caregivers asked questions to physicians regarding diet or lifestyle modifications, 19% did not ask any. Caregivers play a crucial role in the management of HF patients in Japan but experience physical and emotional burden. Solutions are required to reduce the caregiver burden associated with HF.

Novel gene Merlot inhibits differentiation and promotes apoptosis of osteoclasts.

Extended osteoclast longevity is deeply involved in the pathogenesis of bone diseases such as osteoporosis and rheumatoid arthritis, though the mechanisms that determine osteoclast lifespan are not fully understood. Here we present findings indicating that the newly characterized gene Merlot, which encodes a highly conserved yet uncharacterized protein in vertebrates, is an important regulator for termination of osteoclastogenesis via induction of apoptosis. Mice lacking Merlot exhibited low bone mass due to increased osteoclast and bone resorption. Furthermore, osteoclast precursors overexpressing Merlot failed to differentiate into mature osteoclasts, while Merlot deficiency led to hyper-nucleation and prolonged survival of osteoclasts, accompanied by sustained nuclear localization of nuclear factor of activated T cell c1 (NFATc1) and derepression of glycogen synthase kinase-3ß (GSK3ß) activity, known to regulate NFATc1 activity and induce apoptosis. Merlot-deficient osteoclasts were found to represent suppression of caspase-3-mediated apoptosis and Merlot deficiency caused transcriptional downregulation of a proapoptotic cascade, including Bax, Bak, Noxa, and Bim, as well as the executor caspase members Casp-3, -6, and -7, and upregulation of anti-apoptotic Bcl2, resulting in a low apoptotic threshold. Thus, Merlot regulates osteoclast lifespan by inhibition of differentiation and simultaneous induction of apoptosis via regulation of the NFATc1-GSK3ß axis.

Exposure of Free-Ranging Wild Carnivores and Domestic Dogs to Canine Distemper Virus and Parvovirus in the Cerrado of Central Brazil.

Human population growth around protected areas increases the contact between wild and domestic animals, promoting disease transmission between them. This study investigates the exposure of free-ranging wild carnivores and domestic dogs to canine distemper virus (CDV) and parvovirus in Emas National Park (ENP) in the Cerrado savanna of central Brazil. Serum samples were collected from 169 wild carnivores, including the maned wolf (Chrysocyon brachyurus), crab-eating fox (Cerdocyon thous), hoary fox (Pseudalopex vetulus), puma (Puma concolor), ocelot (Leopardus pardalis), pampas cat (Leopardus colocolo), jaguarundi (Herpailurus yagouaroundi), striped hog-nosed skunk (Conepatus semistriatus) and coati (Nasua nasua), and from 35 domestic dogs living on rural properties bordering ENP. Serological tests showed that 10.6% of wild carnivores (maned wolves, crab-eating foxes and ocelots) and 71.4% of domestic dogs were exposed to CDV, and 56.8% of wild carnivores, including all species sampled except coatis, and 57.1% of domestic dogs were exposed to parvovirus. This report is the first to indicate that the free-ranging pampas cat, jaguarundi and striped hog-nosed skunk are exposed to parvovirus. CDV and parvovirus deserve attention in ENP, and it is extremely important to monitor the health of carnivore populations and perform molecular diagnosis of the viruses to determine the possible involvement of the domestic dog in their transmission.

Encapsulation of beraprost sodium in nanoparticles: analysis of sustained release properties, targeting abilities and pharmacological activities in animal models of pulmonary arterial hypertension.

Prostaglandin I2 (PGI2) and its analogues (such as beraprost sodium, BPS) are beneficial for the treatment of pulmonary arterial hypertension (PAH). The encapsulation of BPS in nanoparticles to provide sustained release and targeting abilities would improve both the therapeutic effect of BPS on PAH and the quality of life of patients treated with this drug. BPS was encapsulated into nanoparticles prepared from a poly(lactic acid) homopolymer and monomethoxy poly(ethyleneglycol)-poly(lactide) block copolymer. The accumulation of nanoparticles in damaged pulmonary arteries was examined using fluorescence-emitting rhodamine S-encapsulated nanoparticles. The monocrotaline-induced PAH rat model and the hypoxia-induced mouse model were used to examine the pharmacological activity of BPS-encapsulated nanoparticles. A nanoparticle, named BPS-NP, was selected among various types of BPS-encapsulated nanoparticles tested; this was based on the sustained release profile in vitro and blood clearance profile in vivo. Fluorescence-emitting rhodamine S-encapsulated nanoparticles were prepared in a similar manner to that of BPS-NP, and showed accumulation and prolonged residence in monocrotaline-damaged pulmonary peripheral arteries. Intravenous administration of BPS-NP (once per week, 20µg/kg) protected against monocrotaline-induced pulmonary arterial remodeling and right ventricular hypertrophy. The extent of this protection was similar to that observed with oral administration (once per day, 100µg/kg) of BPS alone. The once per week intravenous administration of BPS-NP (20µg/kg) also exhibited an ameliorative effect on hypoxia-induced pulmonary arterial remodeling and right ventricular hypertrophy. The beneficial effects of BPS-NP on PAH animal models seem to be mediated by its sustained release and tissue targeting profiles. BPS-NP may be useful for the treatment of PAH patients due to reduced dosages and frequency of BPS administration.

Diclofenac protects cultured human corneal epithelial cells against hyperosmolarity and ameliorates corneal surface damage in a rat model of dry eye.

PURPOSE: Dry eye syndrome (DES) is characterized by an increase in tear osmolarity and induction of the expression and nuclear localization of an osmoprotective transcription factor (nuclear factor of activated T-cells 5 [NFAT5]) that plays an important role in providing protection against hyperosmotic tears. In this study, we screened medicines already in clinical use with a view of finding compounds that protect cultured human corneal epithelial cells against hyperosmolarity-induced cell damage. METHODS: Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and cellular NFAT5 level was measured by immunoblotting. The rat model for DES was developed by removal of the lacrimal glands, with an assessment of corneal surface damage based on levels of fluorescein staining and epithelial apoptosis. RESULTS: Some nonsteroidal anti-inflammatory drugs (NSAIDs), including diclofenac sodium (diclofenac), were identified during the screening procedure. These NSAIDs were able to suppress hyperosmolarity-induced apoptosis and cell growth arrest. In contrast, other NSAIDs, including bromfenac sodium (bromfenac), did not exert such a protective action. Treatment of cells with diclofenac, but not bromfenac, stimulated both the nuclear localization and expression of NFAT5 under hyperosmotic conditions. In the rat model for DES, topical administration of diclofenac (but not bromfenac) to eyes reduced corneal surface damage without affecting the volume of tear fluid. CONCLUSIONS: Diclofenac appears to protect cells against hyperosmolarity-induced cell damage and NFAT5 would play an important role in this protective action. The findings reported here may also indicate that the topical administration of diclofenac to eyes may be therapeutically beneficial for DES patients.

Prostaglandin E1 -containing nanoparticles improve walking activity in an experimental rat model of intermittent claudication.

OBJECTIVES: Due to the low stability of lipid emulsions, a lipid emulsion of prostaglandin E1 (Lipo-PGE1 ) necessitates daily intravenous drip infusions. To overcome this issue, we developed nanoparticles containing PGE1 (Nano-PGE1 ). Nano-PGE1 showed a good sustained-release profile of PGE1 from the nanoparticles in vitro, which may permit a longer-lasting therapeutic effect to be achieved. We here examined the pharmacological activity of Nano-PGE1 in a rat experimental model of intermittent claudication induced by femoral artery ligation. METHODS: The walking activity of the rat was tested on a rodent treadmill. Tissue levels of PGE1 were determined by enzyme immunoassay, and skeletal muscle angiogenesis (capillary growth) was monitored by immunohistochemical analysis. KEY FINDINGS: PGE1 could be detected in the lesion site one day after the intravenous administration of Nano-PGE1 but not of Lipo-PGE1 . An increased accumulation of Nano-PGE1 in the lesion site compared with control (unlesioned) site was also observed. The ligation procedure reduced the walking activity, which in turn was improved by a single administration of Nano-PGE1 but not of Lipo-PGE1 . The single administration of Nano-PGE1 also stimulated angiogenesis in the skeletal muscle around the ligated artery. CONCLUSIONS: The findings of this study suggest that Nano-PGE1 improves the walking activity of femoral artery-ligated rats through the accumulation and sustained release of PGE1 .