Methyl-beta-cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after freezing and thawing by facilitating cholesterol efflux from the cells

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.     

Life expectancy, its relation to sexual activity and body weight in male inbred mice.

Body weight of male mice of five inbred strains caged in groups of five was determined three times between the ages of 80 and 100 days, representing the individual fully grown body weight. Sexual activity of each mouse (number of ejaculations and intromissions) was estimated under competitive conditions between the ages of 120 and 150 days in eleven repetitions. Within all inbred strains only about half of the males displayed sexual activity when confronted with an estric female. The other do not. The animals remained in their cage groups until natural death after 727 +/- 215 days (C57BL/6), 638 +/- 260 days (BALB/c), 630 +/- 187 days (CBA), 560 +/- 230 days (DBA/2) and 317 +/- 62 (AKR) days. Only amongst the sexually active animals did individual life span correlate with the number of ejaculations. This was seen in C57BL/6, CBA and DBA/2. Particularly sexually successful animals, carrying out the most ejaculations, live 10-20% longer than their (subdominant) competitors displaying less sexual success. Sexually inactive males (characterised by no ejaculations and less other sexual activities) show that this characteristic of their personalities imposes no limitation upon their life expectancy. Fully grown body weight and the individual life span correlates within the strains DBA/2, C57BL/6 and BALB/c. Medium-sized animals have a greater life expectancy than small or large ones.     

Immune responses of different mouse strains after challenge with equivalent lethal doses of Toxoplasma gondii

Most immunological studies that utilize different strains of inbred mice following T. gondii infection fail to compensate for differences in host susceptibility to the size of the parasite innoculum. To address this concern, susceptible C57BL/6 and resistant CBA/J mice were orally infected with either an equivalent 50% lethal dose (LD50) of brain cysts of the 76K strain of T. gondii (15 cysts in C57BL/6, 400 cysts in CBA/J) or the same dose of parasites in each mouse strain. C57BL/6 mice receiving 400 cysts (LD50 of CBA/J mice) died post infection, whereas CBA/J mice that received 15 cysts (LD50 of C57BL/6 mice) survived. Parasite loads in the brains and serum Toxoplasma-specific IgG1 titers of LD50-infected C57BL/6 mice were significantly higher than those in LD50- or 15 cysts-infected CBA/J mice, whereas splenocyte proliferation to Toxoplasma antigen and the percentage of CD8 alpha+ T cells were reduced in LD50-infected C57BL/6 mice. In contrast, serum IgG2a and IgM titers, the percentage of gamma delta T cells and IFN-gamma expression of spleen of LD50-infected CBA/J mice were higher than those of either 15 cysts-infected CBA/J mice or LD50-infected C57BL/6 mice. These observations demonstrate that the immune response between LD50-infected C57BL/6 and CBA/J mice was more prominent when compared to C57BL/6 or CBA/J mice receiving the same parasite inoculum. These observations would suggest that caution must be excersized in the planning and interpretation of data when the size of the parasite inoculum has not been adjusted for mouse strain.     

Apoptosis in the preimplantation mouse embryo: effect of strain difference and in vitro culture.

Cell death by apoptosis occurs predominantly in the inner cell mass (ICM) of the blastocyst, the cell population which carries the germ line and gives rise to the foetus. The frequency of apoptosis in blastocysts varies widely within outbred species such as human and cow. We have addressed the basis of this variation by examining the relative influence of strain difference and in vitro culture conditions on apoptosis, using embryos from two different strains of mice (MF1 and C57BL6/CBA) in two different culture media (M16 and kSOM). In both strains and all crosses apoptosis was first detected by nuclear fragmentation or TUNEL [Terminal deoxynucleotidyl transferase mediated d-UTP nick end-labelling] labelling at the early blastocyst stage. This was true for embryos which had developed in vivo, and in vitro in both M16 and kSOM. The apoptotic index in blastocysts was found to be significantly different between both media and strain (P < 0.0001). Blastocysts from MF1 x MF1 at equivalent stages had an apoptotic index of 32.4% in M16 and 20.3% in kSOM. Blastocysts from C57BL6/CBA x C57BL6/CBA had an apoptotic index of 19.3% in M16 and 14.4% in kSOM. When embryos of similar cell number were compared, a significantly greater apoptotic index was found for cultured MF1 x MF1 embryos with a cell number between 40 and 59 compared to similar directly flushed C57BL6/CBA embryos (P = 0.001), and MF1 embryos (P < 0.0005). MF1 x MF1 embryos and C57BL6/CBA x MF1 embryos of 60-79 cells had a greater apoptotic index in M16 than kSOM (P < 0.0005) but the difference between media was not significant for C57BL6/CBA x C57BL6/CBA. When strain was compared MF1 x MF1 embryos of 60-79 cells had a significantly greater apoptotic index than C57BL6/CBA x MF1 in both media (P < 0.0005 M16; P = 0.002 kSOM) and than C57BL6/CBA x C57BL6/CBA in M16 (P = 0.019). Our data suggest that genetic make-up and the chemical composition of simple medium are equally important in determining the level of apoptosis.     

Genetic Determination of the Developmental Program for Mouse Liver β-GALACTOSIDASE: Involvement of Sites Proximate to and Distant from the Structural Gene

The identification and mode of action of genetic loci that program gene expression during development are important for understanding differentiation in higher organisms. Previous work from this laboratory has identified two patterns for the postnatal development of liver beta-galactosidase among inbred mouse strains: type I, where activity levels remain constant after about 30 days of age, is found in strains DBA/2J, CBA/J, and BALB/cJ, among others; type II, where activity levels increase between 25 and 50 days of age to reach a new adult level, is found in strain C57BL/6J and related strains. It has been shown that the type I vs. type II developmental difference between strains C57BL/6J and DBA/2J is due to variation at a locus, Bgl-t, that maps with the beta-galactosidase complex, [Bgl], on chromosome 9. In the present study, we have confirmed the existence of Bgl-t as a temporal locus within [Bgl] by analysis of both a congenic strain carrying the beta-galactosidase complex of strain CBA/J in the C57BL/6J genetic background and a cross of strains CBA/J and C57BL/6J. The existence of additional temporal loci for beta-galactosidase that segregate independently of the structural gene and participate in determination of the type I vs. type II difference was revealed by analysis of: (1) a congenic strain containing the beta-galactosidase complex of strain BALB/cJ in the C57BL/10Sn background; (2) recombinant inbred lines derived from progenitor strains C57BL/6ByJ and BALB/cByJ; and (3) a genetic cross between strains C57BL/6ByJ and BALB/cByJ. Thus, for these pairs of strains, the type I vs. type II developmental difference is due to variation at a temporal locus (or loci) unlinked to the enzyme structural gene, and not at Bgl-t. These facts, together with information gathered from an examination of the distribution of beta-galactosidase phenotypes among over 100 inbred strains (Breen, Lusis and Paigen 1977), have led us to conclude that the postnatal developmental pattern for liver beta-galactosidase is determined by a set of interacting temporal genes. One of these, Bgl-t, is located within [Bgl], and one or more are separable from [Bgl] by recombination. A possible mode of interaction among the temporal and instructural loci is suggested.     

Intracytoplasmic sperm injection is more efficient than in vitro fertilization for generating mouse embryos from cryopreserved spermatozoa

Efficient and dependable mouse cryopreservation methods are urgently needed because the production of mice with transgenes and disrupted and mutant genes is now commonplace. Preservation of these unique genomes provides an essential safeguard for future research. Unfortunately, mouse spermatozoa appear more vulnerable to freezing than other species, e.g., bovine and human. In this study, we examined the efficiency of intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) in generating embryos from mouse spermatozoa frozen with 18% raffinose and 3% skim milk for cryoprotection. A comparison was made between the inbred strain C57BL/6J, commonly used in mutagenic and transgenic studies, and a hybrid strain B6D2F1 (C57BL/6J x DBA/2J). C57BL/6J spermatozoa are known to be more sensitive to freezing than B6D2F1. Fertilization of oocytes after IVF was significantly lower with C57BL/6J spermatozoa when compared with B6D2F1 spermatozoa for both fresh and frozen spermatozoa (fresh, 89 vs. 55%; frozen, 56 vs. 9%). Freezing also reduced the fertility of B6D2F1 spermatozoa (89 vs. 56%). Fertilization improved dramatically after ICSI with fresh and frozen C57BL/6J spermatozoa (90 and 85%) and also with frozen B6D2F1 spermatozoa (87%). The development of two-cell embryos to the blastocyst stage was lower for C57BL/6J than B6D2F1 (42-61% and 84-98%) in all treatments but similar for embryos within each strain. The normality of chromosomes from fresh and frozen spermatozoa was assessed in oocytes prior to first cleavage. The majority of oocytes had normal chromosomes after IVF (98-100%) and ICSI (87-95%), indicating that chromosomal abnormalities were not responsible for the poorer development in vitro of C57BL/6J embryos. In conclusion, our data show that ICSI is a more efficient and effective technique than IVF for generating embryos from frozen spermatozoa. More important, ICSI is especially valuable for strains where IVF with fresh spermatozoa produces few or no embryos.     

The susceptibility of inbred mice to infection with Brachylaima cribbi (Digenea: Brachylaimidae)

Susceptibility to infection with Brachylaima cribbi was studied in eight strains of inbred mice (AKR, C3H/HeJ, CBA/CaH, BALB/c, DBA/2J, SJL/J, A/J, C57BL/6J) and Swiss albino outbred mice by quantifying faecal egg excretion over the period of the infection. Preliminary experiments indicated that a combination of filtration/sedimentation/diethyl ether sedimentation was the most sensitive and reliable technique for quantification of eggs in faeces. Mice were infected with 13-15 wild-type B. cribbi metacercariae from naturally infected Cernuella virgata and in a second experiment with human-derived B. cribbi from laboratory-reared Helix aspersa. In both experiments C57BL/6J mice were the most susceptible having the highest egg excretion levels and the longest duration of infection. Worm burdens were assessed at 12 wpi for the wild-type and at 9 wpi for the human-derived infections, when the majority of mice were no longer excreting eggs. The numbers of worms recovered from the small intestine were few and there were no significant differences among the inbred or outbred groups of mice. We have found that C57BL/6J mice were the most susceptible to Brachylaima cribbi infection as assessed by excretion of eggs and provide a suitable model for a laboratory life-cycle.     

不同品系小鼠胚胎玻璃化冷冻保存的比较研究

目的　研究甘油作为冷冻保护剂、不同基因型小鼠对胚胎玻璃化冷冻的影响。方法　采用 6 5mol L的甘油作为冷冻保护剂 ,采用二步法对CBA、NOD、C57BL 6J、ICR及CD1小鼠 3 5d的胚胎进行玻璃化冷冻 ,并比较了不同品系小鼠胚胎的复苏率及移植受孕率。结果和结论　CBA、NOD、C57BL 6J,ICR及CD1的复苏率分别为 5 7 6 %、4 8%、31 3%、86 5 %及 88% ,移植受孕率为 2 1%、2 3 5 %、11%、38%和 35 5 % ,封闭群小鼠的胚胎复苏率、移植受孕率均显著高于近交系小鼠。这提示胚胎的复苏率及移植受孕率可能与小鼠的不同基因型有关。五个品系中 ,桑椹胚及早期囊胚的体外复苏率均显著高于扩张囊胚。这说明不同基因型及胚胎的不同发育阶段对胚胎玻璃化冷冻效果有影响     

Influence of the maternal effect on allogenic inhibition of hematopoietic stem cells]

Bone marrow cells (0,5-10(6)) of female mice of CBA or C57BL strains were injected intravenously to lethally irradiated CBA, C57BL/6, (femaleCBA X maleC57BL/6)F1 and (femaleC57BL/6 X maleCBA)F1 mice. Spleen of recipients as assayed for colony count on the 9th day after bone marrow transplantation by the method of Till and McCullouch. Stem cells of CBA mice demonstrated failure of allogenic inhibition in (CBA X C57BL/6)F1 hybrid mice and formed the same number of colonies as in the spleen of syngenic recipients. The level of allogenic inhibition of CBA stem cells transplanted to (C57BL/6 X X CBA)F1 hybrid mice was 50%. Bone marrow cells of C57BL/6 mice formed colonies in spleen of (CBA X C57BL/6)F1 mice at least in 20 times less than in syngenic combination. In the transplantation of bone marrow from C57BL/6 mice to (C57BL/6 X CBA)F1 hybrid mice the allogenic inhibition was less pronounced (77-85%) as compared with the transfer of cells to (CBA X C57BL/6)F1 hybrid mice (95%). The sex of a recipient did not influence the number of formed colonies. The different level of allogenic inhibition of parental stem cells can not be explained by the effect of linkage with sex as the female of reciprocal hybrid mice have identical structure of sex chromosomes (X(CBA)XC57BL/6). The data obtained indicate that the maternal effect affects allogenic inhibition of stem cells in parent--F1 system. It is possible that the maternal influence may be determined by cytoplasmic factors of inheritance which affect the expressivity of recessive genes Hh, controlling the inheritance of specific haematopoietic cell antigens.     

The development of diploid parthenogenetic mouse embryos of the inbred C57BL and CBA strains]

We studied preimplantation development in vitro and postimplantation development in vivo of diploid parthenogenetic mouse embryos of C57BL/6 and CBA strains, as well as of (CBA x C57BL/6)F1 hybrids. Development to blastocyst stage of diploid eggs obtained from C57BL/6, CBA, and hybrid mice was observed in 90, 15, and 73% cases, respectively. After implantation, C57BL/6 embryos did not develop to somite stages, while CBA and hybrid embryos reached various stages of somite formation in 45 and 30% cases, respectively. Cultivation of embryos beginning from one-cell stage in the medium containing 2% newborn calf serum increased the yield of blastocysts from 15 to 59% in CBA embryos and from 73 to 90% in hybrids; However, such effect was not observed with C57BL/6 embryos. The latest stages of development observed in CBA and hybrid diploid parthenogenetic embryos were 33-35 somites and 25-30 somites, respectively. Imprinting patterns in chromosomes of CBA and C57BL/6 gametes are discussed.     

Comparison of intracytoplasmic sperm injection for inbred and hybrid mice

We compared the results of intracytoplasmic sperm injection (ICSI) that leads to full term development of hybrid (B6C3F1 and B6D2F1) and inbred (C57BL/6) mouse embryos. Although fertilization and pre-implantation development of C57BL/6 eggs were similar to those of F1 hybrid eggs, post-implantation development of the embryos from C57BL/6 females was significantly poorer than those of the eggs from hybrid females. Reciprocal crosses of C57BL/6 and B6C3F1 gametes revealed that the low rate of post-implantation development of C57BL/6 embryos was due to oocyte factor(s), rather than the sperm factor.     

Effects of 5-azacytidine on the development of parthenogenetic mouse embryos

This study describes the effects of 5-azacytidine (5-azaC) on the development of diploid parthenogenetic embryos (PE) of CBA, C57BL/6 and (CBA × C57BL/6)F₁ mice in vitro at the 1-cell or the blastocyst stage or in vivo after implantation. Our findings indicate that genomic imprinting is modulated by genetic background. Non-fertilized C57BL/6 eggs form diploid parthenogenetic blastocysts at a much higher frequency than CBA eggs. Eggs from F₁ hybrid females form parthenogenetic blastocysts at an approximately intermediate level between these inbred strains of mice. C57BL/6 PE do not develop to the somite stages. In contrast, CBA PE and F₁ PE develop to various somite stages. Following administration of 5–azaC at 1.0 μmol/L in vitro at the 1- -cell stage, the number of implantations of C57BL/6 PE transferred to pseudopregnant females increased. In contrast, the number of implantations and somite F₁ PE did not significantly change following exposure to 5–azaC. However, administration of 5-azaC at the 1-cell stage stimulates development of somite F₁ PE. Administration of 5-azaC at 0.2 and 1.0 μmol/L in vitro at the blastocyst stage did not change the number of implantations of C57BL/6 PE. However, the number of implantations and somite CBA PE decreased. After injection of 5azaC at 0.24mg/kg in vivo at day 8 of gestation, some F₁ PE developed to 26–35 somites compared with a maximum of 25 somites in controls. The different effects of 5-azaC on the development of PE depend upon the mouse strain used and the stage of development.     

Exploratory activity of mice of different genetic strains after olfaction disruption by 3-methylindole (skatole)

The behavior of mice of two inbred strains (C57BL/6J and CBA) and their F1 hybrids was evaluated in the open field test after intraperitoneal administration of 3-methylindole (skatole) disrupting epithelium of the main olfactory system. High motor and exploratory activities and emotional sensitivity was observed in intact C57BL/6J mice compared to CBA mice and their hybrids. Anosmia induced by intraperitoneal administration of skatole changed the behavior of C57BL/6J and CBA mice. The direction of the observed changes in the orientation and exploratory behavior of anosmic animals was different. Anosmia decreased motor and exploratory activities in C57BL/6J mice and increase them in CBA mice. Intact hybrid mice demonstrated the predominance of the CBA genotype in the orientation and exploratory activity in the test used. Anosmia in hybrid animals had no significant effect on the orientation and exploratory behavior.     

Schistosoma japonicum: Infection characteristics in mice of various strains and a difference in the response to eggs

Female mice of 12 inbred strains were exposed to 20–25 cercariae of Schistosoma japonicum and infection status determined at day 40 by counting numbers of adult worms, eggs in faeces and eggs in a segment of liver. Most mouse strains appeared to be ‘permissive’ hosts although at least one strain (129/J) was shown to be relatively resistant in terms of day 40 adult worm numbers. In a radioisotopic lung assay for sensitivity to eggs, and developed as a rapid means of assessing granuloma formation, CBA/H mice were shown to differ from C57BL/6 mice in being non-responders. Histological examination of lungs of sensitized CBA/H and C57BL/6 mice injected intravenously with eggs established that granuloma formation was much more intense in C57BL/6 than CBA/H mice. Preliminary indications are that infected CBA/H mice are also low anti egg circumoval precipitin (COP) responders. Analysis of immune responses to isolated egg antigens in these two strains, and identification of the antigens of eggs to which such responses are directed in C57BL/6 mice, should provide insights into immunological disease processes (such as granulomatous inflammation) in this model system of japonicum schistosomiasis.     

Spermatogenic effects of male-fertile translocations in the mouse

Four male-fertile translocations, T(2;4)13H, T(2.8)26H, T(7;18)50H and T(1;13)70H were crossed to the inbred strains CBA/H and C57BL/6J. F1 heterozygotes were compared with wild-type litter-mates for signs of spermatogenic impairment, in view of previous reports that the C57BL strain had this effect in the T(14;15)6Ca translocation. There was a general tendency for body-weights to be slightly reduced in translocation carriers vs. wild-type. Mean testis weights were significantly reduced on the C57BL background with all four translocations as compared to wild-type, but also significantly increased in T26H on CBA. Sperm counts were also reduced on the C57BL background in T13H, T50H and T70H (significantly so in the last two) but were significantly increased in T13H on a CBA background. Only in T50H did the frequency of sperm-head abnormalities show any marked change in the translocation heterozygotes, being approximately doubled with both CBA and C57BL backgrounds although still remaining at a low level. It was concluded that the deleterious effects of C57BL strains on spermatogenesis in translocation heterozygotes were not confined to T6Ca but were probably widespread. Some inconclusive evidence suggested that this might be because some genetic factors associated with C57BL tended to reduce chiasma frequencies in translocation heterozygotes.     

Simple and efficient in vitro fertilization with cryopreserved C57BL/6J mouse sperm

Archiving of mouse stocks by cryopreservation of sperm has great potential, because it is simple, rapid, and cheap. However, for some of the most commonly used inbred strains, including C57BL/6J, the postthaw fertility of the sperm (0%-12%) is too low to be useful without recourse to zona nicking or intracytoplasmic sperm injection to aid penetration of the zona pellucida. In the present study, nonmotile sperm and cell debris were removed from thawed suspensions of C57BL/6J mouse sperm, and the remaining, largely progressively motile sperm were used for in vitro fertilization. These sperm fertilized 38%-88% of denuded, zona-intact eggs, and when 2-cell embryos were transferred to pseudopregnant recipient mice, 40%-63% produced live-born young. The production of 2-cell embryos and the birth of live pups at these rates indicate that cryopreservation of sperm is a practical way to archive the haploid genome of genetically altered C57BL/6J mice.     

Separation of motile populations of spermatozoa prior to freezing is beneficial for subsequent fertilization in vitro: a study with various mouse strains

Success with in vitro fertilization (IVF) using inbred strains of mice varies considerably and appears to be related to the proportion of motile spermatozoa present in epididymal sperm samples of different strains. In this study, motile spermatozoa were separated from the original samples using a column of Sephadex G25. IVF rates were compared between separated and nonseparated samples of epididymal spermatozoa before and after cryopreservation. Oocytes and spermatozoa were obtained from FVB, DBA/2, C57BL/6J, and BALB/c inbred mice; and from F1 (C57BL/6J;ts DBA/2) hybrid mice, and isogenic gametes were used for IVF. These strains of mice were chosen because of their common use in transgenesis and mutagenesis studies. Dulbecco PBS was used for sperm separation on Sephadex, 18% raffinose, and 3% skim milk for cryopreservation; T6 medium for IVF; and mKSOM(AA) for embryo culture. There was a marked improvement in the rate of fertilization using fresh spermatozoa after motile spermatozoa were separated in C57BL/6J and BALB/c strains (92% vs. 58%, 79% vs. 44%) but no differences were found in fertilization rates between separated and nonseparated spermatozoa in F1, FVB, and DBA/2 strains (99% vs. 83%, 95% vs. 93%, 86% vs. 87%, respectively). After cryopreservation, higher rates of fertilization were obtained with separated motile samples in all strains; the greatest improvements were obtained with spermatozoa from C57BL/6J and BALB/c strains (40% vs. 16% and 51% vs. 14% for separated and nonseparated spermatozoa, respectively). No differences were found between the proportions of 14.5-day fetuses developing from embryos derived from separated and nonseparated spermatozoa with or without cryopreservation (33% to 46%). In conclusion, the fertility of frozen-thawed mouse epididymal spermatozoa improves significantly when highly motile populations of spermatozoa are separated for freezing.     

Muscarinic receptors in brain from four strains of inbred mice

The density of brain muscarinic receptors from four strains of inbred mice was determined. C57BL/6J mice had a significantly higher density of muscarinic receptors in the forebrain than Balb/cJ or C57BL/10J mice. In the midbrain, C57BL/6J mice also had the highest density of receptors and in the hindbrain, C57BL/6J and AKR/J mice had a two fold higher receptor density compared to the other two strains. These findings demonstrate that inbred strains of mice which exhibit a range of genetically-determined behaviors, have varying densities of muscarinic receptors.     

Strain-dependent beta-adrenergic receptor function influences myocardial responses to isoproterenol stimulation in mice

Recently, we showed that compared with the A/J inbred mouse strain, C57BL/6J (B6) mice have an athlete's cardiac phenotype. We postulated that strain differences would result in greater left ventricular (LV) hypertrophy in response to isoproterenol in B6 than A/J mice and tested the hypothesis that a differential response could be explained partly by differences in beta-adrenergic receptor (beta-AR) density and/or coupling. A/J and B6 mice were randomized to receive daily isoproterenol (100 mg/kg sc) or isovolumic vehicle for 5 days. Animals were studied using echocardiography, tail-cuff blood pressure, histopathology, beta-AR density and percent high-affinity binding, and basal and stimulated adenylyl cyclase activities. One hundred twenty-eight mice (66 A/J and 62 B6) were studied. Isoproterenol-treated A/J mice demonstrated greater percent increases in echocardiographic LV mass/body weight (97 +/- 11 vs. 20 +/- 10%, P = 0.001) and in gravimetric heart mass/body weight versus same-strain controls than B6 mice. Histopathology scores (a composite of myocyte hypertrophy, nuclear changes, fibrosis, and calcification) were greater in isoproterenol-treated A/J vs. B6 mice (2.8 +/- 0.2 vs.1.9 +/- 0.3, P < 0.05), as was quantitation of myocyte damage (22.3 +/- 11.5 vs. 4.3 +/- 3.5%). Interstrain differences in basal beta-AR density, high-affinity binding, and adenylyl cyclase activity were not significant. However, whereas isoproterenol-treated A/J mice showed nonsignificant increases in all beta-AR activity measures, isoproterenol-treated B6 mice had lower beta-AR density (57 +/- 6 vs. 83 +/- 8 fmol/mg, P < 0.05), percent high-affinity binding (15 +/- 2 vs. 26 +/- 3%, P < 0.005), and GTP + isoproterenol-stimulated adenylyl cyclase activity (10 +/- 1.1 vs. 5.8 +/- 1.5 pmol cAMP.mg(-1).min(-1)) compared with controls. High-dose, short-term isoproterenol produces greater macro- and microscopic cardiac hypertrophy and injury in A/J than B6 mice. A/J mice, unlike B6 mice, do not experience beta-AR downregulation or uncoupling in response to isoproterenol. Abnormalities in beta-adrenergic regulation may contribute to strain-related differences in the vulnerability to isoproterenol-induced cardiac changes.     

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.