Toru Takeo

Methyl-Beta-Cyclodextrin Improves Fertilizing Ability of C57BL/6 Mouse Sperm after Freezing and Thawing by Facilitating Cholesterol Efflux from the Cells

Abstract Sperm cryopreservation provides an economical means of storing genetically engineered mouse strains in resource facilities. In general, relatively high fertilization rates are obtained for frozen/thawed sperm of the CBA/JN, DBA/2N, and C3H inbred strains and some F1 hybrid strains. However, the fertilization rate for frozen/thawed sperm of C57BL/6, which is the main strain of genetically engineered mice, remains very low. Therefore, it is necessary to establish an in vitro fertilization (IVF) method for cryopreserved C57BL/6 sperm that can obtain a high rate of fertilization after thawing. In the present study, we focused on the effects of methyl-beta-cyclodextrin (MBCD) on the fertilizing ability of frozen/thawed C57BL/6 sperm. Our results have shown that the highest fertilization rate for frozen/thawed sperm was obtained with MBCD at 1.0 mM for 30 min (63.7% ± 11.0%), but the effects were attenuated by long-term incubation for 120 min at 1.0 or 2.0 mM. The embryos with frozen/thawed sperm showed good developmental potential, and the offspring had normal fertility. The efflux of cholesterol from frozen/thawed sperm was increased by MBCD in a dose-dependent manner and occurred much earlier and to a greater extent than bovine serum albumin. The localization of cholesterol labeled by filipin in the sperm plasma membrane was drastically decreased by MBCD. In summary, we suggest that MBCD is useful for developing an IVF method for frozen/thawed C57BL/6 mouse sperm achieving a high fertilization rate, being involved in the capacity to sequester cholesterol from sperm membrane.

Decrease of Fertilizing Ability of Mouse Spermatozoa after Freezing and Thawing Is Related to Cellular Injury

Abstract In general, the fertilizing ability of cryopreserved mouse spermatozoa is less than that of fresh spermatozoa. This ability is especially low in C57BL/6, the main strain used for the production of transgenic mice. To solve this problem, the relationship between cell damage and fertilizing ability in cryopreserved mouse spermatozoa was examined in this study. Sperm motility analysis revealed no significant difference among the motilities of cryopreserved C57BL/6J, BALB/cA, and DBA/2N sperm (67.6%, 43.4%, and 60.0%, respectively) after thawing. However, the results of in vitro fertilization (IVF), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) showed a strong correlation between the frequency of aberrant spermatozoa (FAS) and fertilization rates (FR; C57BL/6J: FAS, 83.7%; FR, 17.0%; BALB/cA: FAS, 67.2%; FR, 24.2%; and DBA/2N: FAS, 10.2%; FR, 93.6%), and damage to spermatozoa was localized particularly in the acrosome of the head and mitochondria.

LGR4 Regulates the Postnatal Development and Integrity of Male Reproductive Tracts in Mice

Abstract The roles of the leucine-rich repeat domain containing G protein-coupled receptor (GPCR) 4 (Lgr4), which is one of the orphan GPCRs, were analyzed with the Lgr4 hypomorphic mutant mouse line (Lgr4Gt). This homozygous mutant had only one-tenth the normal transcription level; furthermore, 60% of them survived to adulthood. The homozygous male was infertile, showing morphologic abnormalities in both the testes and the epididymides. In the testes, luminal swelling, loss of germinal epithelium in the seminiferous tubules, and rete testis dilation were observed. Cauda epididymidis sperm were immotile. Rete testis dilation was due to a water reabsorption failure caused by a decreased expression of an estrogen receptor (ESR1) and SLC9A3 in the efferent ducts. Although we found differential regulation of ESR1 expression in the efferent ducts and the epididymis, the role of ESR1 in the epididymis remains unclear. The epididymis contained short and dilated tubules and completely lacked its initial segment. In the caput region, we observed multilamination and distortion of the basement membranes (BMs) with an accumulation of laminin. Rupture of swollen epididymal ducts was observed, leading to an invasion of macrophages into the lumen. Male infertility was probably due to the combination of a developmental defect of the epididymis and the rupture of the epithelium resulting in the immotile spermatozoa. These results indicate that Lgr4 has pivotal roles to play in the regulation of ESR1 expression, the control of duct elongation through BM remodeling, and the regional differentiation of the caput epididymidis.

Reduced Glutathione Enhances Fertility of Frozen/Thawed C57BL/6 Mouse Sperm after Exposure to Methyl-Beta-Cyclodextrin

Sperm cryopreservation is useful for the effective storage of genomic resources derived from genetically engineered mice. However, freezing the sperm of C57BL/6 mice, the most commonly used genetic background for genetically engineered mice, considerably reduces its fertility. We previously reported that methyl-beta-cyclodextrin dramatically improved the fertility of frozen/thawed C57BL/6 mouse sperm. Recently, it was reported that exposing sperm to reduced glutathione may alleviate oxidative stress in frozen/thawed mouse sperm, thereby enhancing in vitro fertilization (IVF); however, the mechanism underlying this effect is poorly understood. In the present study, we examined the combined effects of methyl-beta-cyclodextrin and reduced glutathione on the fertilization rate of IVF with frozen/thawed C57BL/6 mouse sperm and the characteristic changes in the zona pellucida induced by reduced glutathione. Adding reduced glutathione to the fertilization medium increased the fertilization rate. Methyl-beta-cyclodextrin and reduced glutathione independently increased fertilization rates, and their combination produced the strongest effect. We found that reduced glutathione increased the amount of free thiols in the zona pellucida and promoted zona pellucida enlargement. Finally, 2-cell embryos produced by IVF with the addition of reduced glutathione developed normally and produced live offspring. In summary, we have established a novel IVF method using methyl-beta-cyclodextrin during sperm preincubation and reduced glutathione during the IVF procedure to enhance fertility of frozen/thawed C57BL/6 mouse sperm.

Combination medium of cryoprotective agents containing l-glutamine and methyl-β-cyclodextrin in a preincubation medium yields a high fertilization rate for cryopreserved C57BL/6J mouse sperm

Recently, a vast number of genetically-engineered mice have been created in various laboratories worldwide, all of which need to be effectively archived. The cryopreservation of mouse sperm provides a simple and economical means of storing the mice in mouse resource facilities. The current protocol for sperm cryopreservation using 18% raffinose pentahydrate and 3% skim milk (R18S3) has been adopted in most laboratories. In general, we can attain relatively high fertilization rates for frozen/thawed sperm in many inbred and F1 hybrid strains. However, the sperm of C57BL/6J mice shows an extremely low fertility rate after freezing and thawing (0–20%). In this study, we attempted to improve the low fertility of frozen/thawed C57BL/6J mouse sperm. Our results showed that a combination of R18S3 containing l-glutamine and methyl-β-cyclodextrin (MBCD) in a preincubation medium dramatically increased the rate of fertilization (69.2 ± 12.2%). Furthermore, the developmental potencies of two-cell embryos produced by frozen/thawed sperm to live young were normal (fresh: 46.0 ± 8.2%, frozen/thawed: 51.5 ± 11.1%). In summary, we conclude that a new method of sperm cryopreservation and in vitro fertilization using modified R18S3 with l-glutamine and MBCD in a preincubation medium yields a high fertilization rate for frozen/thawed C57BL/6J strain sperm. Furthermore, the new method provides a reliable archiving and reproducing system for genetically-engineered mice using sperm cryopreservation.

Efficacy of 2-hydroxypropyl-β-cyclodextrin in Niemann-Pick disease type C model mice and its pharmacokinetic analysis in a patient with the disease

Niemann-Pick type C disease (NPC), an autosomal recessive lysosomal storage disorder, is an inherited disease characterized by the accumulation of intracellular unesterified cholesterol. A solubilizing agent of lipophilic compounds, 2-hydroxypropyl-β-cyclodextrin (HPBCD), is an attractive drug candidate against NPC disease. However, establishment of the optimum dosage of HPBCD remains to be determined. In this study, we evaluated the effective dosage of HPBCD in NPC model (Npc1(-/-)) mice, and determined serum HPBCD concentrations. Subcutaneous injection of 1000-4000 mg/kg HPBCD improved the lifespan of Npc1(-/-) mice. In addition, liver injury and cholesterol sequestration were significantly prevented by 4000 mg/kg HPBCD in Npc1(-/-) mice. Serum HPBCD concentrations, when treated at the effective dosages (1000-4000 mg/kg), were approximately 1200-2500 µg/mL at 0.5 h after subcutaneous injection, and blood HPBCD concentrations were immediately eliminated in Npc1(-/-) mice. Furthermore, we examined serum HPBCD concentrations when treated at 40000 mg (approximately 2500 mg/kg) in a patient with NPC. We observed that the effective concentration in the in vivo study using Npc1(-/-) mice was similar to that in the patient. In the patient, systemic clearance and the volume of distribution of HPBCD were in accordance with the glomerular filtration rate and extracellular fluid volume, respectively. These results could provide useful information for developing the optimal dosage regimen for HPBCD therapy when administered intravenously to NPC patients.

Akt1-Mediated Skeletal Muscle Growth Attenuates Cardiac Dysfunction and Remodeling after Experimental Myocardial Infarction

Background— It is appreciated that aerobic endurance exercise can attenuate unfavorable myocardial remodeling following myocardial infarction. In contrast, little is known about the effects of increasing skeletal muscle mass, typically achieved through resistance training, on this process. Here, we utilized transgenic (TG) mice that can induce the growth of functional skeletal muscle by switching Akt1 signaling in muscle fibers to assess the impact of glycolytic muscle growth on post-myocardial infarction cardiac remodeling. Methods and Results— Male-noninduced TG mice and their nontransgenic littermates (control) were subjected to left anterior coronary artery ligation. Two days after surgery, mice were provided doxycycline in their drinking water to activate Akt1 transgene expression in a skeletal muscle-specific manner. Myogenic Akt1 activation led to diminished left ventricular dilation and reduced contractile dysfunction compared with control mice. Improved cardiac function in Akt1 TG mice was coupled to diminished myocyte hypertrophy, decreased interstitial fibrosis, and increased capillary density. ELISA and protein array analyses demonstrated that serum levels of proangiogenic growth factors were upregulated in Akt1 TG mice compared with control mice. Cardiac eNOS was activated in Akt1 TG mice after myocardial infarction. The protective effect of skeletal muscle Akt activation on cardiac remodeling and systolic function was abolished by treatment with the eNOS inhibitor L-NAME. Conclusions— Akt1–mediated skeletal muscle growth attenuates cardiac remodeling after myocardial infarction and is associated with an increased capillary density in the heart. This improvement appears to be mediated by skeletal muscle to cardiac communication, leading to activation of eNOS-signaling in the heart.

Superovulation using the combined administration of inhibin antiserum and equine chorionic gonadotropin increases the number of ovulated oocytes in C57BL/6 female mice.

Superovulation is a reproductive technique generally used to produce genetically engineered mice. Superovulation in mice involves the administration of equine chorionic gonadotropin (eCG) to promote follicle growth and then that of human chorionic gonadotropin (hCG) to induce ovulation. Previously, some published studies reported that inhibin antiserum (IAS) increased the number of ovulated oocytes in ddY and wild-derived strains of mice. However, the effect of IAS on the C57BL/6 strain, which is the most widely used inbred strain for the production of genetically engineered mice, has not been investigated. In addition, the combined effect of IAS and eCG (IASe) on the number of ovulated oocytes in superovulation treatment has not been examined. In this study, we examined the effect of IAS and eCG on the number of ovulated oocytes in immature female mice of the C57BL/6 strain in superovulation treatment. Furthermore, we evaluated the quality of obtained oocytes produced by superovulation using IASe by in vitro fertilization (IVF) with sperm from C57BL/6 or genetically engineered mice. The developmental ability of fresh or cryopreserved embryos was examined by embryo transfer. The administration of IAS or eCG had a similar effect on the number of ovulated oocytes in C57BL/6 female mice. The number of ovulated oocytes increased to about 3-fold by the administration of IASe than by the administration of IAS or eCG alone. Oocytes derived from superovulation using IASe normally developed into 2-cell embryos by IVF using sperm from C57BL/6 mice. Fresh or cryopreserved 2-cell embryos produced by IVF between oocytes of C57BL/6 mice and sperm from genetically engineered mice normally developed into live pups following embryo transfer. In summary, a novel technique of superovulation using IASe is extremely useful for producing a great number of oocytes and offspring from genetically engineered mice.

Applications of cryopreserved unfertilized mouse oocytes for in vitro fertilization

Since the first successful reports into oocyte freezing, many papers concerning the cryopreservation of mouse oocytes have been published. However, a simple and practical cryopreservation method for unfertilized C57BL/6 mouse oocytes, and an IVF system using these cryopreserved oocytes have yet to be established, in spite of the fact that C57BL/6 is the prevalent inbred strain and is used for large-scale knockout programs. In this study, unfertilized C57BL/6 mouse oocytes were cryopreserved via a simple vitrification method. After warming, IVF was performed using cryopreserved unfertilized oocytes and fresh sperm, cryopreserved unfertilized oocytes and cold-stored sperm, cryopreserved unfertilized oocytes and frozen sperm (C57BL/6 strain sperm), and cryopreserved unfertilized oocytes and frozen sperm derived from GEM strains (C57BL/6 background GEM strains). Nearly all of the cryopreserved oocytes were recovered, of which over 90% were morphologically normal. Those oocytes were then used for in vitro fertilization, resulting in 72-97% of oocytes developing into 2-cell embryos. A portion of the 2-cell embryos were transferred to recipients, resulting in live young being produced from 32-49% of the embryos. In summary, we established the simple and practical method of mouse oocyte vitrification with high survivability and developmental ability and the IVF using the vitrified-warmed oocytes with fresh, cold-stored or cryopreserved sperm with high fertility.

Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging

BackgroundNeural crest cells (NCCs) are embryonic, multipotent stem cells. Their long-range and precision-guided migration is one of their most striking characteristics. We previously reported that P0-Cre/CAG-CAT-lacZ double-transgenic mice showed significant lacZ expression in tissues derived from NCCs.ResultsIn this study, by embedding a P0-Cre/CAG-CAT-EGFP embryo at E9.5 in collagen gel inside a culture glass slide, we were able to keep the embryo developing ex vivo for more than 24 hours; this development was with enough NCC fluorescent signal intensity to enable single-cell resolution analysis, with the accompanying NCC migration potential intact and with the appropriate NCC response to the extracellular signal maintained. By implantation of beads with absorbed platelet-derived growth factor-AA (PDGF-AA), we demonstrated that PDGF-AA acts as an NCC-attractant in embryos.We also performed assays with NCCs isolated from P0-Cre/CAG-CAT-EGFP embryos on culture plates. The neuromediator 5-hydroxytryptamine (5-HT) has been known to regulate NCC migration. We newly demonstrated that dopamine, in addition to 5-HT, stimulated NCC migration in vitro. Two NCC populations, with different axial levels of origins, showed unique distribution patterns regarding migration velocity and different dose-response patterns to both 5-HT and dopamine.ConclusionsAlthough avian species predominated over the other species in the NCC study, our novel system should enable us to use mice to assay many different aspects of NCCs in embryos or on culture plates, such as migration, division, differentiation, and apoptosis.