Contribution of the α1-Acid Glycoprotein in Drug Pharmacokinetics: The Usefulness of α1-Acid Glycoprotein-Knockout Mice.

α1-Acid glycoprotein (AGP) is a primary binding protein for many basic drugs in plasma. The number of drugs that bind to AGP, such as molecular target anticancer drugs, has been continuously increasing. Since the plasma level of AGP fluctuates under various pathological conditions such as inflammation, it is important to evaluate the contribution of AGP to drug pharmacokinetics. Here, we generated conventional AGP-knockout (AGP-KO) mice and used them to evaluate the contribution of AGP. The pharmacokinetics of drugs that bind to two AGP variants (F1*S or A variants) or albumin were evaluated. Imatinib (a F1*S-binding drug) and disopyramide (an A-binding drug) or ibuprofen (an albumin-binding drug) were administered to wild-type (WT) and AGP-KO. The plasma level of imatinib and disopyramide decreased rapidly in AGP-KO as compared to WT. In AGP-KO, AUC and t1/2 were decreased, then CLtot was increased. Compared with disopyramide, imatinib pharmacokinetics showed more marked changes in AGP-KO as compared to WT. The results seemed to be due to the difference in plasma level of each AGP variant (F1*S:A = 2-3:1). No differences were observed in ibuprofen pharmacokinetics between the WT and AGP-KO mice. In vitro experiments using plasma from WT and AGP-KO showed that unbound fractions of imatinib and disopyramide were higher in AGP-KO. These results suggest that the rapid elimination of imatinib and disopyramide in AGP-KO could be due to decreased protein binding to AGP. Taken together, the AGP-KO mouse could be a potential animal model for evaluating the contribution of AGP to the pharmacokinetics of various drugs.

Time elapsed between ovulation and insemination determines the quality of fertilized rat oocytes.

Genetically modified rats are valuable models in human disease research. We recently developed an improved system for rat sperm cryopreservation and in vitro fertilization (IVF) that facilitates the efficient production and preservation of genetically modified rats. In the IVF procedure performed using frozen-thawed rat sperm, the IVF schedule is fixed to ensure timely hormone administration and oocyte collection. To enhance the flexibility of the IVF schedule, possible periods of postovulated rat oocytes with normal fertility and developmental abilities should be determined. Therefore, in this study, we examined the fertilization and developmental ability of incubated oocytes 1-13 h after oocyte collection at 9:00 AM. The fertilization rate decreased 7 h after oocyte collection, and abnormally fertilized oocytes appeared 10 h after oocyte collection. The developmental rate also decreased 7 h after oocyte collection; however, live pups were obtained from oocytes 12 h after oocyte collection. In summary, ovulated rat oocytes exhibited a high developmental ability after IVF for up to 4 h after oocyte collection.

High-concentration bovine serum albumin enhances fertilization ability of cold-stored rat sperm.

Cold transport of the cauda epididymides is a useful technique for shipping laboratory rat sperm. Cold transport of rat sperm avoids potential risks of microbiological infection, animal escape or death, and animal welfare issues. Previously, we reported that a cold-storage solution containing dimethyl sulfoxide and quercetin maintained the fertility of cold-stored rat sperm. However, cold-stored rat sperm exhibited a decreased fertilization rate after 24-h storage. To recover the fertility of cold-stored sperm, we focused on the effects of bovine serum albumin (BSA), a cholesterol acceptor that induces sperm capacitation. We sought to determine the optimal concentration of BSA in fertilization medium based on the fertility of cold-stored rat sperm. High concentrations of BSA (40 mg/ml) enhanced the fertilization rate of cold-stored rat sperm and maintained sperm fertility for 144 h. Embryos derived from cold-stored and BSA-treated sperm normally developed into pups after embryo transfer. In summary, high BSA concentrations enhanced the fertility of cold-stored rat sperm and prolonged the storage period to 144 h, thereby expanding the transportable region for genetically engineered rats.

Sexual fate of murine external genitalia development: Conserved transcriptional competency for male-biased genes in both sexes.

Testicular androgen is a master endocrine factor in the establishment of external genital sex differences. The degree of androgenic exposure during development is well known to determine the fate of external genitalia on a spectrum of female- to male-specific phenotypes. However, the mechanisms of androgenic regulation underlying sex differentiation are poorly defined. Here, we show that the genomic environment for the expression of male-biased genes is conserved to acquire androgen responsiveness in both sexes. Histone H3 at lysine 27 acetylation (H3K27ac) and H3K4 monomethylation (H3K4me1) are enriched at the enhancer of male-biased genes in an androgen-independent manner. Specificity protein 1 (Sp1), acting as a collaborative transcription factor of androgen receptor, regulates H3K27ac enrichment to establish conserved transcriptional competency for male-biased genes in both sexes. Genetic manipulation of MafB, a key regulator of male-specific differentiation, and Sp1 regulatory MafB enhancer elements disrupts male-type urethral differentiation. Altogether, these findings demonstrate conservation of androgen responsiveness in both sexes, providing insights into the regulatory mechanisms underlying sexual fate during external genitalia development.

Prostaglandin F2α Affects the Cycle of Clock Gene Expression and Mouse Behavior.

Prostaglandins are bioactive compounds, and the activation of their receptors affects the expression of clock genes. However, the prostaglandin F receptor (Ptgfr) has no known relationship with biological rhythms. Here, we first measured the locomotor period lengths of Ptgfr-KO (B6.129-Ptgfrtm1Sna) mice and found that they were longer under constant dark conditions (DD) than those of wild-type (C57BL/6J) mice. We then investigated the clock gene patterns within the suprachiasmatic nucleus in Ptgfr-KO mice under DD and observed a decrease in the expression of the clock gene cryptochrome 1 (Cry1), which is related to the circadian cycle. Moreover, the expression of Cry1, Cry2, and Period2 (Per2) mRNA were significantly altered in the mouse liver in Ptgfr-KO mice under DD. In the wild-type mouse, the plasma prostaglandin F2α (PGF2α) levels showed a circadian rhythm under a 12 h cycle of light-dark conditions. In addition, in vitro experiments showed that the addition of PTGFR agonists altered the amplitude of Per2::luc activity, and this alteration differed with the timing of the agonist addition. These results lead us to hypothesize that the plasma rhythm of PGF2α is important for driving clock genes, thus suggesting the involvement of PGF2α- and Ptgfr-targeting drugs in the biological clock cycle.

Dimethyl-α-cyclodextrin induces capacitation by removing phospholipids from the plasma membrane of mouse sperm†.

Capacitation is an important event in the completion of fertilization by mammalian sperm. Cholesterol efflux is a trigger of capacitation. In general, cholesterol acceptors of albumin and ß-cyclodextrins are used to induce capacitation during in vitro fertilization. Previously, we reported that methyl-ß-cyclodextrin (MBCD), which is composed of seven glucoses, had a higher ability to induce capacitation than bovine serum albumin (BSA) in frozen-thawed mouse sperm. Comparison of albumin and cyclodextrins is helpful for understanding the mechanism of capacitation. In this study, we examined the effects of albumin, MBCD, and a different type of cyclodextrin, dimethyl-α-cyclodextrin (DMACD), which is composed of six glucoses, on several events of sperm capacitation. We showed that DMACD induced sperm capacitation and promoted fertilization ability. The time required to increase the fertilization rate differed among BSA, MBCD, and DMACD. BSA and MBCD enhanced cholesterol and phospholipid efflux, whereas DMACD enhanced only phospholipid efflux. BSA, MBCD, and DMACD increased sperm membrane fluidity, rearrangement of the lipid raft, and the acrosome reaction. These findings suggest that phospholipid efflux is a novel trigger of capacitation. Increasing the choice of sperm capacitation inducers may be useful for improving in vitro fertilization (IVF) techniques not only in mice, but also in various species in which it has been difficult to produce embryos by IVF.

Asian Mouse Mutagenesis Resource Association (AMMRA): mouse genetics and laboratory animal resources in the Asia Pacific.

The Asian Mouse Mutagenesis Resource Association (AMMRA) is a non-profit organization consisting of major resource and research institutions with rodent expertise from within the Asia Pacific region. For more than a decade, aiming to support biomedical research and stimulate international collaboration, AMMRA has always been a friendly and passionate ally of Asian and Australian member institutions devoted to sharing knowledge, exchanging resources, and promoting biomedical research. AMMRA is also missioned to global connection by working closely with the consortiums such as the International Mouse Phenotyping Consortium and the International Mouse Strain Resource. This review discusses the emergence of AMMRA and outlines its many roles and responsibilities in promoting, assisting, enriching research, and ultimately enhancing global life science research quality.

Species Difference in Hydrolysis of an Ester-type Prodrug of Levodopa in Human and Animal Plasma: Different Contributions of Alpha-1 Acid Glycoprotein.

Previously, we found that ONO-2160, an ester-type prodrug of levodopa (3-hydroxy-l-tyrosine), was mainly hydrolyzed in human plasma by α1-acid glycoprotein (AGP) with a partial contribution of albumin. In this study, we investigated whether ONO-2160 was hydrolyzed in the plasma of preclinical species (dog, rabbit, rat, and mouse) and humans and whether AGP and albumin are involved in its hydrolysis. ONO-2160 was hydrolyzed to some extent in the plasma of all tested species with the order of magnitude mouse > human > rabbit > rat > dog. Except for dogs, ONO-2160 was partially hydrolyzed by animal AGP and albumin. This indicated that, similar to albumin, AGP possesses esterase-like activity in mice, rats, and rabbits, as well as humans. A comparison of the values of intrinsic clearance per milliliter of plasma demonstrated that AGP was the major contributor to the hydrolysis of ONO-2160 in rabbit plasma, whereas albumin was primarily responsible for the hydrolysis of ONO-2160 in mouse plasma. This was confirmed by experiments using AGP-knockout mouse plasma. This study reports the first evidence for the existence of species differences in the hydrolysis of ONO-2160 in plasma related to the different contributions of AGP and albumin.

Intracerebroventricular Treatment with 2-Hydroxypropyl-ß-Cyclodextrin Decreased Cerebellar and Hepatic Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB) Expression in Niemann-Pick Disease Type C Model Mice.

Niemann-Pick disease type C (NPC) is a recessive hereditary disease caused by mutation of the NPC1 or NPC2 gene. It is characterized by abnormality of cellular cholesterol trafficking with severe neuronal and hepatic injury. In this study, we investigated the potential of glycoprotein nonmetastatic melanoma protein B (GPNMB) to act as a biomarker reflecting the therapeutic effect of 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) in an NPC mouse model. We measured serum, brain, and liver expression levels of GPNMB, and evaluated their therapeutic effects on NPC manifestations in the brain and liver after the intracerebroventricular administration of HP-ß-CD in Npc1 gene-deficient (Npc1-/-) mice. Intracerebroventricular HP-ß-CD inhibited cerebellar Purkinje cell damage in Npc1-/- mice and significantly reduced serum and cerebellar GPNMB levels. Interestingly, we also observed that the intracerebral administration significantly reduced hepatic GPNMB expression and elevated serum ALT in Npc1-/- mice. Repeated doses of intracerebroventricular HP-ß-CD (30 mg/kg, started at 4 weeks of age and repeated every 2 weeks) drastically extended the lifespan of Npc1-/- mice compared with saline treatment. In summary, our results suggest that GPNMB level in serum is a potential biomarker for evaluating the attenuation of NPC pathophysiology by intracerebroventricular HP-ß-CD treatment.

Azithromycin Inhibits Constitutive Airway Epithelial Sodium Channel Activation in Vitro and Modulates Downstream Pathogenesis in Vivo.

Epithelial sodium channel (ENaC) is an amiloride-sensitive sodium ion channel that is expressed in epithelial tissues. ENaC overexpression and/or hyperactivation in airway epithelial cells cause sodium over-absorption and dysregulated ciliary movement for mucus clearance; however, the agents that suppress constitutive airway ENaC activation are yet to be clinically available. Here, we focused on macrolides, which are widely used antibiotics that have many potential immunomodulatory effects. We examined whether macrolides could modulate constitutive ENaC activity and downstream events that typify cystic fibrosis (CF) and chronic obstructive pulmonary diseases (COPD) in in vitro and in vivo models of ENaC overexpression. Treatment of ENaC-overexpressing human bronchial epithelial cells (ß/γENaC-16HBE14o- cells) with three macrolides (erythromycin, clarithromycin, azithromycin) confirmed dose-dependent suppression of ENaC function. For in vivo studies, mice harboring airway specific ßENaC overexpression (C57BL/6J-ßENaC-transgenic mice) were treated orally with azithromycin, a well-established antimicrobial agent that has been widely prescribed. Azithromycin treatment modulated pulmonary mechanics, emphysematous phenotype and pulmonary dysfunction. Notably, a lower dose (3 mg kg-1) of azithromycin significantly increased forced expiratory volume in 0.1 s (FEV0.1), an inverse indicator of bronchoconstriction. Although not statistically significant, improvement of pulmonary obstructive parameters such as emphysema and lung dysfunction (FEV0.1%) was observed. Our results demonstrate that macrolides directly attenuate constitutive ENaC function in vitro and may be promising for the treatment of obstructive lung diseases with defective mucociliary clearance, possibly by targeting ENaC hyperactivation.

Auto-measure emphysematous parameters and pathophysiological gene expression profiles in experimental mouse models of acute and chronic obstructive pulmonary diseases.

Pulmonary emphysema, inflammation and senescence-like phenotype are pathophysiological characteristics of chronic obstructive pulmonary disease (COPD). Recently, a murine model of COPD has been established by inducing airway-specific overexpression of epithelial Na+ channel ß subunit (ßENaC-Tg mice). However, little is known about the histological and biochemical differences between ßENaC-Tg mice and an existing acute emphysematous mouse model (elastase-induced model). Here, we first utilized whole lung image-based quantification method for histological analysis to determine auto-measure parameters, including alveolar area, alveolar perimeter, (major axis + minor axis)/2 and Feret diameter. Even though the extent of emphysema was similar in both models, the coefficient of variation (CV) of all histological parameters was smaller in ßENaC-Tg mice, indicating that ßENaC-Tg mice show homogeneous emphysema as compared with elastase-induced acute model. Expression analysis of lung tissue RNAs further revealed that elastase-induced model exhibits transient changes of inflammation markers (Kc, Il-6, Lcn2) and senescence-related markers (Sirt1, p21) at emphysema-initiation stage (1 day), which does not last until emphysema-manifestation stage (3 weeks); while the up-regulation is stable at emphysema-manifestation stage in ßENaC-Tg mice (14-week old). Thus, these studies demonstrate that ßENaC-Tg mice exhibit diffuse-type emphysema with stable expression of inflammatory and senescence-like markers.

In Vitro and In Vivo Evaluation of 6-O-α-Maltosyl-ß-Cyclodextrin as a Potential Therapeutic Agent Against Niemann-Pick Disease Type C.

Niemann-Pick disease Type C (NPC) is a rare lysosomal storage disease characterized by the dysfunction of intracellular cholesterol trafficking with progressive neurodegeneration and hepatomegaly. We evaluated the potential of 6-O-α-maltosyl-ß-cyclodextrin (G2-ß-CD) as a drug candidate against NPC. The physicochemical properties of G2-ß-CD as an injectable agent were assessed, and molecular interactions between G2-ß-CD and free cholesterol were studied by solubility analysis and two-dimensional proton nuclear magnetic resonance spectroscopy. The efficacy of G2-ß-CD against NPC was evaluated using Npc1 deficient Chinese hamster ovary (CHO) cells and Npc1 deficient mice. G2-ß-CD in aqueous solution showed relatively low viscosity and surface activity; characteristics suitable for developing injectable formulations. G2-ß-CD formed higher-order inclusion complexes with free cholesterol. G2-ß-CD attenuated dysfunction of intercellular cholesterol trafficking and lysosome volume in Npc1 deficient CHO cells in a concentration dependent manner. Weekly subcutaneous injections of G2-ß-CD (2.9 mmol/kg) ameliorated abnormal cholesterol metabolism, hepatocytomegaly, and elevated serum transaminases in Npc1 deficient mice. In addition, a single cerebroventricular injection of G2-ß-CD (21.4 µmol/kg) prevented Purkinje cell loss in the cerebellum, body weight loss, and motor dysfunction in Npc1 deficient mice. In summary, G2-ß-CD possesses characteristics favorable for injectable formulations and has therapeutic potential against in vitro and in vivo NPC models.

STAT3 inhibition attenuates the progressive phenotypes of Alport syndrome mouse model.

Background: Alport syndrome (AS) is a hereditary, progressive nephritis caused by mutation of type IV collagen. Previous studies have shown that activation of signal transducer and activator of transcription 3 (STAT3) exacerbates other renal diseases, but whether STAT3 activation exacerbates AS pathology is still unknown. Here we aim to investigate the involvement of STAT3 in the progression of AS. Method: Phosphorylated STAT3 expression was assessed by immunoblotting analysis of kidneys and glomeruli of an AS mouse model (Col4a5 G5X mutant). To determine the effect of blocking STAT3 signaling, we treated AS mice with the STAT3 inhibitor stattic (10 mg/kg i.p., three times per week for 10 weeks; n = 10). We assessed the renal function [proteinuria, blood urea nitrogen (BUN), serum creatinine] and analyzed the glomerular injury score, fibrosis and inflammatory cell invasion by histological staining. Moreover, we analyzed the gene expression of nephritis-associated molecules. Results: Phosphorylated STAT3 was upregulated in AS kidneys and glomeruli. Treatment with stattic ameliorated the progressive renal dysfunction, such as increased levels of proteinuria, BUN and serum creatinine. Stattic also significantly suppressed the gene expression levels of renal injury markers (Lcn2, Kim-1), pro-inflammatory cytokines (Il-6, KC), pro-fibrotic genes (Tgf-ß, Col1a1, α-Sma) and Mmp9. Stattic treatment decreased the renal fibrosis congruently with the decrease of transforming growth factor beta (TGF-ß) protein and increase of antifibrosis-associated markers p-Smad1, 5 and 8, which are negative regulators of TGF-ß signaling. Conclusion: STAT3 inhibition significantly ameliorated the renal dysfunction in AS mice. Our finding identifies STAT3 as an important regulator in AS progression and provides a promising therapeutic target for AS.

History of cryobiology, with special emphasis in evolution of mouse sperm cryopreservation.

Confucius said study the past if you would define the future and a popular statement says that history depends on who writes it. To talk about history it is necessary to find and define a milestone where to start the narration. The intention of this quick review is to take the reader through moments and selected publications; part and pieces of memories showing how the concept of cryopreservation, specifically for mouse sperm, was conceived and sustained as we know it today. Beginning with the development of the microscope (1677) and continuing through the 17th century with the first documented observation by L. Spallanzani describing that sperm could maintain the motility under cold conditions. As J. Sherman suggested, we divide the cryopreservation evolution into two sequences, previous to and after 1949 when Polge, Smith and Parkes discovered the property of glycerol as cryoprotectant. Later, in 1972, D. Whittingham, S. Leibo, and P. Mazur applying a slow freezing process achieved the first embryo freezing (mouse). During that time many theories were scientifically confirmed. Among those, Peter Mazur demonstrated the relation between the speed of freezing and intracellular ice formation, and Stanley Leibo that each cell type has their unique freezing curve. In 1950, after the discovery of the protective aspect of glycerol, sperm from many mammals were frozen, except from the mouse. It was in the early 90's when the mouse sperm freezing becomes important and it was a real challenge for many groups, nevertheless, the technique using skim milk and raffinose modified by Dr Nakagata was the beginning of a different story ….

Lipocalin 2 binds to membrane phosphatidylethanolamine to induce lipid raft movement in a PKA-dependent manner and modulates sperm maturation.

Mammalian sperm undergo multiple maturation steps after leaving the testis in order to become competent for fertilization, but the molecular mechanisms underlying this process remain unclear. In terms of identifying factors crucial for these processes in vivo, we found that lipocalin 2 (Lcn2), which is known as an innate immune factor inhibiting bacterial and malarial growth, can modulate sperm maturation. Most sperm that migrated to the oviduct of wild-type females underwent lipid raft reorganization and glycosylphosphatidylinositol-anchored protein shedding, which are signatures of sperm maturation, but few did so in Lcn2 null mice. Furthermore, we found that LCN2 binds to membrane phosphatidylethanolamine to reinforce lipid raft reorganization via a PKA-dependent mechanism and promotes sperm to acquire fertility by facilitating cholesterol efflux. These observations imply that mammals possess a mode for sperm maturation in addition to the albumin-mediated pathway.

Dimethyl sulfoxide and quercetin prolong the survival, motility, and fertility of cold-stored mouse sperm for 10 days.

Technology for preserving sperm is useful for disseminating valuable male genetic traits. Cold storage is suitable for easily transporting sperm as an alternative to the shipment of live animals. However, there is a technical limitation in that the fertility of cold-stored sperm declines within 3 days. To overcome this problem, we examined the protective effects of quercetin and dimethyl sulfoxide (DMSO). DMSO and quercetin maintained the fertility and motility of cold-stored sperm for 10 days. In addition, quercetin attenuated the reduction of mitochondrial membrane potential of cold-stored sperm during sperm preincubation, allowing the induction of capacitation, and it localized to the midpiece of sperm. Furthermore, DMSO and quercetin enhanced the level of tyrosine phosphorylation of cold-stored sperm. DMSO and quercetin have life-prolonging effects on sperm during cold storage. Cold storage using DMSO and quercetin will provide a robust system for internationally transporting valuable sperm samples.

Fertility of cold-stored mouse sperm is recovered by promoting acrosome reaction and hyperactivation after cholesterol efflux by methyl-beta-cyclodextrin.

Cold storage of the cauda epididymis, a male reproductive organ, is an efficient means of transporting genetically modified mice, an alternative to live animal shipment. The fertility of cold-stored sperm decreases in a time-dependent manner. However, the cause of the reduction in the fertility of sperm after cold storage remains unclear. It is known that cholesterol efflux from the sperm membrane is a trigger of capacitation. The dysfunction of cholesterol efflux due to changes in the membrane state in low temperatures may explain the reduced fertility. In this study, we examined the fertility (fertilization rate, acrosome reaction, and motility) and the amount of cholesterol and membrane fluidity in cold-stored sperm after treatment with two cholesterol acceptors, bovine serum albumin (BSA), and methyl-beta-cyclodextrin (MBCD). MBCD-treated sperm exhibited the highest rate of fertilization. Two-cell embryos derived from in vitro fertilization using 0.75-mM-MBCD-treated sperm after cold storage for 72 h developed to offspring. MBCD also displayed greater ability to remove cholesterol from the sperm membrane than BSA. The percentage of viable sperm with membrane destabilization was increased by MBCD in cold-stored sperm. The acrosome reaction strongly occurred in MBCD-treated sperm. In motility analysis, MBCD improved the lateral amplitude of head movement and beat frequency. These results suggest that MBCD-induced cholesterol efflux enhances membrane fluidity and promotes acrosome reaction and hyperactivation, resulting in improved fertility of cold-stored sperm.

GANP protein encoded on human chromosome 21/mouse chromosome 10 is associated with resistance to mammary tumor development.

Human chromosome 21 is known to be associated with the high risk of hematological malignancy but with resistance to breast cancer in the study of Down syndrome. In human cancers, we previously observed the significant alterations of the protein expression encoded by the ganp/MCM3AP gene on human chromosome 21q22.3. Here, we investigated GANP protein alterations in human breast cancer samples (416 cases) at various stages by immunohistochemical analysis. This cohort study clearly showed that expression of GANP is significantly decreased in human breast cancer cases with poor prognosis as an independent risk factor (relapse-free survival, hazard ratio = 2.37, 95% confidence interval, 1.27-4.42, P = 0.007 [univariate analysis]; hazard ratio = 2.70, 95% confidence interval, 1.42-5.13, P = 0.002 [multivariate analysis]). To investigate whether the altered GANP expression is associated with mammary tumorigenesis, we created mutant mice that were conditionally deficient in the ganp/MCM3AP gene using wap-cre recombinase transgenic mice. Mammary gland tumors occurred at a very high incidence in female mammary gland-specific GANP-deficient mice after severe impairment of mammary gland development during pregnancy. Moreover, tumor development also occurred in female post parous GANP-heterodeficient mice. GANP has a significant role in the suppression of DNA damage caused by estrogen in human breast cancer cell lines. These results indicated that the GANP protein is associated with breast cancer resistance.

N-acetyl cysteine prolonged the developmental ability of mouse two-cell embryos against oxidative stress at refrigerated temperatures.

Cold storage of two-cell embryos at refrigerated temperatures is a useful means to ship genetically engineered mice. We previously reported that M2 medium maintained the developmental ability of two-cell embryos for 48 h at 4 °C, and offspring were obtained from embryos transported by a courier service under refrigerated temperatures. The limitation of 48 h practically restricts the shipping destination of the embryos. To enhance the applicability of the cold-storage technique, prolonging the time to maintain developmental ability of the embryos is required. Oxidative stress may be a cause of the declining developmental ability of cold-stored embryos. However, the effect of oxidative stress on developmental ability of embryos has not been investigated. We examined intracellular glutathione (GSH) levels of cold-stored two-cell embryos to evaluate the effect of oxidative and investigated the efficacy of adding N-acetyl cysteine (NAC) to the preservation medium on the developmental ability of cold-stored embryos and transported two-cell embryos at refrigerated temperatures. Intracellular GSH levels of two-cell embryos decreased by cold storage for longer than 72 h, whereas NAC recovered this reduction and improved the developmental ability of embryos cold-stored for 96 h. In the transport experiment, the developmental rate of transported two-cell embryos to offspring was increased by adding NAC to the preservation medium. We found that NAC prolonged the storage period of two-cell embryos and maintained the developmental ability by alleviating the reduction of intracellular GSH. These findings will improve the technique of cold-storage of two-cell embryos to facilitate efficient transport of genetically engineered mice worldwide.