| Objective To study the effect of traditional chinese medicine for reinforcing the kidney on mouse oocyte in vitro maturation.Method Inmature oocytes were retreived from 90 ICR murine ovaries stimulated by pregnant mare serum gonadotropin (PMSG).245 mouse cumulus oocyte complexes (COCs) were collected and randomly divided into five groups, rat serum containing traditional chinese medicine 1#,2#,3# for reinforcing the kidney, blank serum(rat serum without traditional chinese medicine) and blank group, and microdrop matured in M199 media for 24 h with 2% rat serum containing traditional chinese medicine 1#,2#,3# for reinforcing the kidney,2% blank serum and medium without rat serum, separately. Oocyte maturation rate were counted. About 238 mouse COCs were collected and randomly divided into four groups, medicated serum 1#, follicle-stimulating hormone (FSH), blank serum and blank group, and matured in media with 2% medicated serum 1#,100IU/L FSH,2% blank serum and medium without rat serum, separately. Oocyte maturation rate, fertilization rate and cleavage rate were counted. The experiment was repeated 3 times. The mouse COCs were collected and randomly divided into four groups, medicated serum 1#, follicle-stimulating hormone (FSH), blank serum and blank group. The expression of oocyte cytoskeleton (spindle and chromosome) and regulatory subunit Cyclin B of mature promoting factor (MPF) were detected by immunofluorescence and observed by reflection fluorescence microscope. And the expression intensity of Cyclin B1 was analyzed by fluorescence testing instrument. The mouse COCs were collected and randomly divided into two groups, medicated serum 1# and blank serum group. Semi-quantity RT-PCR method was used to observe the expression of Cyclin B1 mRNA in mouse oocytes. FSH and E2 in rat blood were determinated by enzyme immunoassay (EIA).Results1. The oocyte maturation rate in the medicated serum 1#,2# groups were higher than that in the blank group (42.31%,36.17% vs 16.67%, P<0.01 or P<0.05). The oocyte maturation rate in the medicated serum 1# group was higher than that in the blank serum group (42.31% vs 22.92%, P<0.05). germinal vesicle breakdown and the first polar body (GVBD+PB1) in the medicated serum 1# group was higher than that in the blank group (84.62% vs 66.67%, P<0.05). There was no significant difference between the medicated serum 1#,2#,3# groups.2. The oocyte maturation rate in the FSH group and the medicated serum 1# group were higher than that in the blank group (33.69%,40.98% vs 17.71%, P<0.01), The oocyte maturation rate in the FSH group and the medicated serum 1# group were higher than that in the blank serum group (33.69%,40.98% vs 22.81%, P <0.05 or P<0.01). GVBD+PB1 in the FSH group and the medicated serum 1# group were higher than that in the blank group (80.75%,83.61% vs 64.00%, P<0.01). GVBD+PB1 in the FSH group and the medicated serum 1# group were higher than that in the blank serum group (80.75%,83.61% vs 71.35%, P<0.05 or P<0.01) There was no significant difference between the FSH group and the medicated serum 1# group.The experiment was repeated threel times to get the basically consistent outcome. The oocyte maturation rate in the FSH group and the medicated serum 1# group were higher than that in the blank group, respectively, P<0.05, P<0.05 or P<0.01. The oocyte maturation rate in the FSH group and the medicated serum 1" group were higher than that in the blank serum group, P<0.05. GVBD+PB1 in the FSH group and the medicated serum 1# group were higher than that in the blank group, respectively, P<0.05,P<0.05 or P<0.01. There was no significant difference between the FSH group and the medicated serum 1# group.3. Fluorescence intensity of Cyclin B1 in the FSH group and the medicated serum 1# group were higher than that in the blank group and the blank serum group (15069.00±3908.88,17936.50±4717.42 vs 5029.50±1656.61,6625.33±1777.41, P<0.01).There was no significant difference between the FSH group and the medicated serum 1" group. the expression of Cyclin B1 mRNA in the medicated serum 1# group (Cyclin B1/GAPDH=2.78) were higher than that in the blank serum group (Cyclin B1/GAPDH=2) There was no significant, difference of oocyte cytoskeleton between medicated serum 1", follicle-stimulating hormone (FSH), blank serum and blank group. The levels of FSH and E2 in the medicated serum 1#,2#,3# groups were higher than that in the blank serum group, but there was no significant difference between the four groups.Conclusion Rat serum containing traditional chinese medicine for reinforcing the kidney 1# has the effect of improving the mouse oocyte development in vitro. The medicated serum 1# has no adverse effect on the cytoskeleton configuration of the mature oocytes. The effect of medicated serum 1" for improving the mouse oocyte in vitro maturation may be ralated to the effect on increasing the expression of maturation promoting factor regulatory subunit Cyclin B1.
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