Tolerance of the freeze-dried mouse sperm nucleus to temperatures ranging from −196 °C to 150 °C

It has long been believed that tolerance against extreme environments is possible only for ‘lower’ groups, such as archaea, bacteria or tardigrades, and not for more ‘advanced’ species. Here, we demonstrated that the mammalian sperm nucleus also exhibited strong tolerance to cold and hot temperatures. When mouse spermatozoa were freeze-dried (FD), similar to the anhydrobiosis of Tardigrades, all spermatozoa were ostensibly dead after rehydration. However, offspring were obtained from recovered FD sperm nuclei, even after repeated treatment with conditions from liquid nitrogen to room temperature. Conversely, when FD spermatozoa were heated at 95 °C, although the birth rate was decreased with increasing duration of the treatment, offspring were obtained even for FD spermatozoa that had been heat-treated for 2 h. This period was improved up to 6 h when glucose was replaced with trehalose in the freeze-drying medium, and the resistance temperature was extended up to 150 °C for short periods of treatment. Randomly selected offspring grew into healthy adults. Our results suggest that, when considering the sperm nucleus/DNA as the material that is used as a blueprint of life, rather than cell viability, a significant tolerance to extreme temperatures is present even in ‘higher’ species, such as mammals

Involvement of Multidrug Resistance-Associated Protein 1 in Intestinal Toxicity of Methotrexate

金沢大学医薬保健研究域薬学系Purpose: Methotrexate (MTX) causes dose-limiting gastrointestinal toxicity due to exposure of intestinal tissues, and is a substrate of the multidrug resistance-associated protein (MRP) 1. Here we examine the involvement of MRP1, which is reported to be highly expressed in the proliferative crypt compartment of the small intestine, in the gastrointestinal toxicity of MTX. Methods: MTX was intraperitonealy administered to mrp1 gene knockout (mrp1(-/-)) and wild-type (mrp1(+/+)) mice. Body weight, food and water intake were monitored, intestinal histological studies and pharmacokinetics of MTX were examined. Results: mrp1(-/-) mice more severely decreased body weight, food and water intake than mrp1(+/+) mice. Almost complete loss of villi throughout the small intestine in mrp1(-/-) mice was observed, whereas the damage was only partial in mrp1(+/+) mice. Plasma concentration and biliary excretion profiles of MTX were similar in mrp1(-/-) and mrp1(+/+) mice, though accumulation of MTX in immature proliferative cells isolated from mrp1(-/-) mice was much higher compared to mrp1(+/+) mice. Immunostaining revealed localization of Mrp1 in plasma membrane of the intestinal crypt compartment in mrp1(+/+) mice, but not in mrp1(-/-) mice. Conclusion: Mrp1 determines the exposure of proliferative cells in the small intestine to MTX, followed by gastrointestinal toxicity. © 2009 Springer Science+Business Media, LLC

Gene Knockout and Metabolome Analysis of Carnitine/Organic Cation Transporter OCTN1

金沢大学医薬保健研究域薬学系Purpose: Solute carrier OCTN1 (SLC22A4) is an orphan transporter, the physiologically important substrate of which is still unidentified. The aim of the present study was to examine physiological roles of OCTN1. Methods: We first constructed octn1 gene knockout (octn1-/-) mice. Metabolome analysis was then performed to identify substrates in vivo. The possible association of the substrate identified with diseased conditions was further examined. Results: The metabolome analysis of blood and several organs indicated complete deficiency of a naturally occurring potent antioxidant ergothioneine in octn1-/- mice among 112 metabolites examined. Pharmacokinetic analyses after oral administration revealed the highest distribution to small intestines and extensive renal reabsorption of [3H]ergothioneine, both of which were much reduced in octn1-/- mice. The octn1-/- mice exhibited greater susceptibility to intestinal inflammation under the ischemia and reperfusion model. The blood ergothioneine concentration was also much reduced in Japanese patients with Crohn\u27s disease, compared with healthy volunteers and patients with another inflammatory bowel disease, ulcerative colitis. Conclusions: These results indicate that OCTN1 plays a pivotal role for maintenance of systemic and intestinal exposure of ergothioneine, which could be important for protective effects against intestinal tissue injuries, providing a possible diagnostic tool to distinguish the inflammatory bowel diseases. © 2010 Springer Science+Business Media, LLC

Mitochondrial Physiology and Gene Expression Analyses Reveal Metabolic and Translational Dysregulation in Oocyte-Induced Somatic Nuclear Reprogramming

While reprogramming a foreign nucleus after somatic cell nuclear transfer (SCNT), the enucleated oocyte (ooplasm) must signal that biomass and cellular requirements changed compared to the nucleus donor cell. Using cells expressing nuclear-encoded but mitochondria-targeted EGFP, a strategy was developed to directly distinguish maternal and embryonic products, testing ooplasm demands on transcriptional and post-transcriptional activity during reprogramming. Specifically, we compared transcript and protein levels for EGFP and other products in pre-implantation SCNT embryos, side-by-side to fertilized controls (embryos produced from the same oocyte pool, by intracytoplasmic injection of sperm containing the EGFP transgene). We observed that while EGFP transcript abundance is not different, protein levels are significantly lower in SCNT compared to fertilized blastocysts. This was not observed for Gapdh and Actb, whose protein reflected mRNA. This transcript-protein relationship indicates that the somatic nucleus can keep up with ooplasm transcript demands, whilst transcription and translation mismatch occurs after SCNT for certain mRNAs. We further detected metabolic disturbances after SCNT, suggesting a place among forces regulating post-transcriptional changes during reprogramming. Our observations ascribe oocyte-induced reprogramming with previously unsuspected regulatory dimensions, in that presence of functional proteins may no longer be inferred from mRNA, but rather depend on post-transcriptional regulation possibly modulated through metabolism

Detrimental effects of microgravity on mouse preimplantation development in vitro.

Sustaining life beyond Earth either on space stations or on other planets will require a clear understanding of how the space environment affects key phases of mammalian reproduction. However, because of the difficulty of doing such experiments in mammals, most studies of reproduction in space have been carried out with other taxa, such as sea urchins, fish, amphibians or birds. Here, we studied the possibility of mammalian fertilization and preimplantation development under microgravity (microG) conditions using a three-dimensional (3D) clinostat, which faithfully simulates 10(-3) G using 3D rotation. Fertilization occurred normally in vitro under microG. However, although we obtained 75 healthy offspring from microG-fertilized and -cultured embryos after transfer to recipient females, the birth rate was lower than among the 1G controls. Immunostaining demonstrated that in vitro culture under microG caused slower development and fewer trophectoderm cells than in 1G controls but did not affect polarization of the blastocyst. These results suggest for the first time that fertilization can occur normally under microG environment in a mammal, but normal preimplantation embryo development might require 1G

Parental competition for the regulators of chromatin dynamics in mouse zygotes

Quantification of chromatin dynamics in paternal and maternal pronuclei reveals opposite effects on chromatin dynamics by oocyte- and sperm derived factors