Improvement In The Method For Breeding Sm22? Knockout (Sm22?^-/-) Mice And Study On Sm22? In The Contraction Of Uterus

Objective: SM22??smooth muscle 22?,SM22??is a 22-kDa actin-associated protein serving to regulate differentiation and phenotypic modulation of vascular smooth muscle cells?VSMCs?.Sm22? knockout(Sm22?^-/-)mice has been extensively used to study the human diseases.However,the birthrate of homozygous Sm22?^-/-mice is lower,and the pregnant Sm22?^-/-mice usually die of uterine inertia during parturition.To increase the birthrate of Sm22?^-/-mice,we sought to improve the traditional breeding method,and investigated the mechanism of uterine inertia in Sm22?^-/-mice.Method:1 Improvement in the method for breeding Sm22?^-/-miceMale Sm22?^-/-mice were mated with female Sm22?^-/-mice?homozygous breeding?.When the female showed some signs of delivery,caesarean section was performed and female ICR mice served as foster mother for the newborn litters.Meanwhile,male Sm22?^-/-mice were mated with female Sm22?+/-mice as control?homozygous breeding?.The female pregnancy rate and survival rate after weaning were compared between the two groups.2 Identification of the mice genotypesGenomic DNA was extracted from tails or ears tissues of the offspring mice,and analyzed by PCR.The PCR products were separated by 1% agarose gels,and then proportions of genotypes in offspring were analyzed.3 The effect of SM22? on the development of uterine smooth muscleTo evaluate the functional role of SM22? in the development of uterine smooth muscle,the uteri were harvested from wild type(Sm22?^+/+)and Sm22?^-/-female mice,and examined morphometrically,respectively.The cross-sections of the uterine were stained with haematoxylin-eosin.The relative thickness of uterine medial muscle was calculated with the Image-Pro Morphometric System.4 The effect of SM22? on the formation of F-actin in uterine smooth muscleF-actin was evaluated by reacting uterine smooth muscle tissues with phalloidin conjugated with fluorescent labels.5 Primary culture and identification of uterine smooth muscle cells?SMCs?SMCs were prepared from mice uterine myometrium using tissue explant method with some modifications.The cells were observed by phase contrast microscope and verified by immunofluorescence staining for smooth muscle ?-actin?SM ?-actin?.6 The effect of SM22? on the formation of actin stress fiber in uterine SMCsDouble fluorescence staining for SM22? and F-actin in uterine SMCs was used to analyze the effect of Sm22? knockout on the formation of actin stress fiber.7 Statistical analysesThe data were analyzed using SPSS 13.0 statistical software.Quantitative data were expressed as the mean ± SD and t test was performed.Categorical data were shown as percentage and tested with the ?2 test.P < 0.05 was considered statistically significant.Results: 1 There were not significantly different in both female pregnancy rate?50% and 60%?and survival rate after weaning?87.70% and 88.60%?between homozygous and heterozygous breeding method indicated?P>0.05?;2 Homozygote rate of the offspring?100%?in homozygous breeding method was higher that of heterozygous breeding method?47%?;3 The relative thickness of medial smooth muscle of Sm22?^-/-mice was less than that of WT mice?P<0.05?;4 Analysis of relative intensity of fluorescence showed that the content of F-actin was significantly reduced in Sm22?^-/-uterine smooth muscle compared with WT mice?P<0.05?;5 The primary uterine SMCs grew with spindle and showed typically “hill-valley” pattern under phase contrast microscope.Immunofluorescence analysis for SM ?-actin showed that positive reaction in the monolayer cells was more than 95%;6 Fluorescence staining showed that abundant actin stress fibers co-localized with SM22? and arranged in parallel bundles in the uterine SMCs of WT mice,while the actin stress fibers were sparse and scattered in the Sm22?^-/-uterine SMCs.Conclusions:1 Homozygous breeding is an effective way for rapid increase in the number of homozygote.2 Disruption of SM22? inhibits the formation of F-actin and decreases the contractility of uterine smooth muscle.