| Part I Effects of Zichong granules(ZCKL)on in vitro fertilization and in vitro maturation outcome of repeated superovulation mice oocytes.OBJECTIVE: To investigate the effect of ZCKL on the oocytes quality of repeated superovulatory mice,and use it as a morphological indicator to evaluate oocytes quality.Method:90 female mice were adaptively fed for 5 days,and 75 mice with two consecutive and normal estrus cycles were included.Randomly they were divided into 5 groups: 5 times ovulation stimulation group(P/H×5 Group),single ovulation stimulation group(P/H×1 Group),natural cycle group(Control Group),5 times ovulation stimulation + ZCKL high dose Group(ZCKL High Group),5 times ovulation stimulation + ZCKL Low Group(ZCKL Low Group).15 mice were divided in each group.Modeling of repeated superovulation animal models:on the first day of each superovulation cycle,at 6 PM,mice were injected intraperitoneally with 5 IU of PMSG.After 48 hours,intraperitoneal injection of 5 IU of HCG was performed at 6 PM on the third day.This was a hyper-promoting ovulation cycle,which was repeated 3 days apart and repeated for 5 consecutive cycles.ZCKL High or Low Group was administered traditional Chinese medicine ZCKL,which was divided into the high and low doses,at the same time for ovulation induction.Stop Chinese medicine for one day on HCG day.14 hours after the last injection of HCG,the fallopian tubes were dissected,under the stereo microscope.The oviducts were torn off and the oviducts were taken from the ampulla to perform ovulation tests and cleavage experiments.The ovulation rate,IVF fertilization rate,2-cell,4-cell,8-cell,morula,and blastocyst formation rates were observed.The first polar body(PB1)excretion rate of in vitro maturation(IVM)of oocytes in GV-phase.To investigate the effect of ZCKL on the quality of mice oocytes,this was used as a morphological criterion for evaluating oocyte quality.Result: 1.Comparison of the number of oocytes retrieved by repeated superovulation.With the increase in the number of superovulation,the total number of excreted oocytes did not decrease,and the number of degenerated oocytes increased.The ratio increased,and 413 oocytes were obtained in the P/H×5 Group,of which 162 degenerated oocytes accounted for 39.2%.In the Control Group,98 oocytes were obtained,including 1 degenerated oocyte,accounting for 1%.With the increase of the number of superovulation,the number of oocytes in the normal morphology was decreased with the increase of the number of superovulation,and 303 MII-stage oocytes in the P/H×1 Group accounted for 78.7%;P/H × 5 Groups 251,accounting for 60.8%.With the increase in the number of ovulation promotion,the number of oocytes retrieved in MII-stage oocytes was 30.3±3.9 in the P/H×1 Group,and 25.1±2.2 in the P/H×5 Group,so the average number of oocytes retrieved decreased.Compared with the average of 25.1±2.2 of MII-stage oocytes in P/H×5 Group,the average number of MII-stage oocytes in the ZCKL High Group was 27.5±2.2,which was statistically significant(P<0.05).2.Comparison of the rate of fertilized oocytes which were produced by repeated superovulatory in each group.The fertilized oocytes rate was 100% in the Control Group,97% in the P/H×1 Group,94.0% in the P/H×5 Group,96.1% in the ZCKL Low Group,and 95.3% in the ZCKL High Group.There was no statistical difference among these groups(P>0.05).Comparison of in vitro development potential of ovulation with repeated superovulation on mice 1-cell fertilized oocytes in each group.The rate of 2-cell in each group: Control group: 100%;P/H×1 Group: 100%;P/H×5 Group: 94.6%;ZCKL Low Group: 96.2%;ZCKL High Group: 100%.There was no significant difference in the rate of 2-cell in each group(P>0.05).In each group embryos in the 4-cell,8-cell,morula,blastocyst stage: compared with the Control Group,the rates of 4-cell,8-cell,morulae,and blastocysts in the P/H×5 Group,the ZCKL Low,and the High Groups decreased significantly(P<0.01).In the 4-cell and 8-cell phase,the P/H×1 Group had a decreasing trend compared with the Control Group,but there was no statistical significance.In the stage of morula and blastocyst,the ratio of P/H×1 Group to Control Group decreased significantly(P<0.01,P<0.05).Compared with the P/H×5 Group,the 8-cell rate and blastocyst rate of the ZCKL High Group increased significantly(P=0.021,0.031).The effect of repeated superovulation on the in vitro maturation of mice oocytes: with the increase of the number of superovulation,thedischarge rate of PB1 had a decreasing trend.The rate of PB1 excretion in the ZCKL Low and High Groups increased,but there was no statistical difference(P>0.05).Summary: 1.As the frequency of repeated superovulation increased,degenerative oocyte rate rised,the number of MII-stage oocytes decreased and 4-cell,8-cell,morula,blastocyst formation rate decreased.2.Repeated superovulatory and oral administration of ZCKL,could increase the number of effective ovulation,indicating that nourishing kidney essence was the fundation for follicular development.3.Repeated superovulation and ZCKL intervention at the same time could reduce the incidence of degenerative oocytes,including 8-cell rate and blastocyst rate.ZCKL High Group was more obvious improvement.Kidney nourishing traditional Chinese medicine ZCKL could improve the quality of oocytes and promote embryonic development,with a dose-related relationship.Part Ⅱ Research on the molecular mechanism of ZCKL in improving the oocyte quality of repeatedly superovulated mice.Section I Effects of ZCKL on ovarian tissue morphology and sex hormone level in repeated superovulation mice.OBJECTIVE: Kidney nourishing Chinese medicine ZCKL previous experiments proved that it had estrogen-like effects,so whether it could regulate hormonal changes in repeated superovulation mice.Through the detection of hormone levels,pathological analysis of ovarian tissue structure changes,we tried to figure out the further elucidation of the mechanism.Methods: KM female mice,6 weeks old,a total of 40,grouping and modeling methods were as same as partⅠ.The mice were selected 12 hours after the last intraperitoneal injection of HCG.Control Group vaginal smear method was used for the monitoring of the situation,which was taken 12 hours after the mice proestrus.The mice blood was taken from the eyelids,and serum was prepared.The purpose was to detect the levels of E2 and P.After mechanical isolation of the ovaries from the mice,the weight of the ovaries was measured.The isolated ovaries were immediately fixed in 4% paraformaldehyde for HE staining.Result: 1.Effect of ZCKL on the number of follicles and corpus luteum in repeated superovulation mice.(1)There was no significant difference in the average number of primordial follicles(P>0.05).(2)Number of primary follicles and secondary follicles in each group: Compared with the Control group,the number of primary and secondary follicles in the P/H×5 Group,the ZCKL Low Group and the High Group was significantly reduced(P<0.01).Compared with P/H×1 Group,the results of P/H×5 Group,ZCKL Low Group and High Group were same as before(P<0.01);compared with P/H×5 Group,the number of primary follicles and secondary follicles in ZCKL High and Low Groups was significantly increased,and the difference was statistically significant.The ZCKL High Group was more effective in improving the number of secondary follicles(P<0.01).(3)Number of antral follicles:compared with the Control Group,the antral follicles in the P/H×1 and P/H×5 Groups were significantly reduced(P<0.01),and the antral follicles decreased in the ZCKL Low Group(P<0.05).The differences in the ZCKL High Group were not significant(P>0.05).Compared with the P/H×5 Group,there was no significant difference in the number of antral follicles between the High and Low ZCKL Groups(P>0.05).(4)The number of corpus luteum:compared with Control Group,the number of corpus luteum increased significantly in P/H×1 Group,P/H×5 Group,ZCKL Low Group and High Group(P<0.01).There was no significant difference in the number of corpus luteum among the P/H×5 Group,the ZCKL Low and High Groups(P>0.05).2.Effect of kidney nourishing Chinese medicine ZCKL on the weight of mice ovaries.Ovary weight:compared with the Control Group,P/H×1 Group,P/H×5 Group,ZCKL Low and High Group ovaries weight increased significantly(P<0.01).There was no significant difference in ovaries weight among P/H×5 Group,ZCKL High and Low Groups(P>0.05).3.Effects of kidney nourishing Chinese medicine ZCKL on the level of hormones in repeated superovulatory mice.Estrogens,progestins:compared with Control Group,P/H×5 Group,ZCKL Low and High Group E2,P levels were significantly lower(P<0.01),E2,P levels of P/H×1 Group increased(P<0.05,P<0.01);compared with P/H×1 Group,E2 and P levels in P/H×5 Group,ZCKL Low Group and High Group were significantly lower(P<0.01).Compared with P/H×5 Group,E2 and P levels increased in ZCKL High and Low Groups(P<0.05,P<0.01).Summary: 1.Repeated ovulation induction could result in a significant decrease in preantral follicles and antral follicles,and the number of corpus luteum increased significantly.2.With the increase in the number of ovulation induction,the ovary weight also increased,and E2 and P levels showed a downward trend.3.ZCKL,a kind of kidney nourishing Chinese medicine,had similar effects as estrogen and progesterone.It could affect the reproductive and endocrine environment of repeated superovulatory mice and increase the number of primary follicles and secondary follicles.The dose-effect relationship was obvious.The ZCKL High Group had better effects,and the number of secondary follicles increased more obviously.Section II Effect of ZCKL on the expression of OCT4 in repeated superovulation mice oocytes.OBJECTIVE:The purpose of this section of the study was to investigate whether pluripotency gene octamer-associated transcription factor 4(OCT4)was associated with the developmental potential of mice embryos after repeated superovulation.Effects of adding ZCKL,a traditional kidney nourishing Chinese medicine,on the expression of OCT4 in oocytes.Method:KM female mice,6 weeks old,a total of 250,grouping and modeling methods were as same as part Ⅰ.The experimental group selected mice after the last intraperitoneal injection of HCG for 4 hours.In the Control Group,the vaginal smear method was used to monitor the emotions.The mice were taken 4 hours after the early stage of estrus.The bilateral ovaries were removed and placed in M2 droplets in the dishes.Under a stereomicroscope,the ovarian tissues were chopped and retrieved,and approximately 500 pieces of COCs were collected.Treatment with 0.3 mg/ml hyaluronidase to dissociate cumulus cells,MII-stage oocytes were isolated in PBS droplets using an oviduct and washed repeatedly.About 200 oocytes from each group were selected for QPCR detection of related genes.More than 300 were used for the WB detection of related genes.The remaining M2 droplets that had been removed from the oocytes were transferred to a PCR tube and centrifuged at 1000 rpm for 5 min.The white substance on the bottom wall of the tube was cumulus granulosa cells.The cumulus granulosa cells from 200 COCs were used for QPCR detection of related genes.More than 300 COCs cumulus granulosa cells were used for WB detection of related proteins.Result: OCT4 protein expression in oocytes of each group:compared with the Control Group,P/H×5 Group,ZCKL Low and High Group,OCT4 protein and m RNA expression was significantly reduced(P<0.01).Compared with P/H×1 Group,OCT4 protein and m RNA expression in P/H×5 Group and ZCKL Low Group decreased significantly(P<0.01).Compared with P/H×5 Group,OCT4 protein and m RNA expression in ZCKL Low and High Groups increased significantly(P<0.01).Compared with the Low Group,the OCT4 protein and m RNA expression was significantly increased in the High ZCKL Group(P<0.05).Summary: 1.Down-regulation of OCT 4 m RNA and protein,which was the regulator of mice oocytes development,triggered the cellular apoptotic pathway.2.With the increase of superovulation cycles,OCT4 m RNA and protein expression in oocytes was reduced,which showed deterioration of oocytes quality after repeated superovulation.It was one of the possible mechanismsa for the decline of oocyte quality in the embryonic 4-cell stage of repeated superovulation.3.ZCKL could upregulate the expression of OCT4 protein and m RNA in oocytes,which provided a powerful experimental basis for the kidney to improve the quality of embryos.Section III Effect of ZCKL on the signaling pathway of oocyte maturation in repeated superovulation mice.OBJECTIVE:This study was based on the gonadotropin-directed EGFR/MAPK3/1 signaling pathway and the interaction between oocytes and surrounding cumulus cells GDF9/SMAD2 signaling pathway.Purpose of the study was to explore the molecular mechanism of ZCKL to improve oocyte quality.In this section,mice cumulus granulosa cells and oocytes were used as research objects to investigate the effect of ZCKL on the quality of oocytes in repeated superovulatory mice.Method: Same as the third part.Result: 1.Comparison of m RNA and protein expression of GDF9,SMAD2,CDC2 in mice oocytes of each group.Compared with Control group,m RNA and protein expression of GDF9,SMAD2,CDC2 in P/H×1 Group,P/H×5 Group,ZCKL Low and High Group were significantly decreased(P<0.01).Compared with P/H×1 Group,the above expression was significantly down-regulated in P/H×5 Group and ZCKL Low Group(P<0.05).Compared with the P/H×5 Group,the m RNA and protein expression of GDF9,SMAD2 and CDC2 in the Low,High Groups of ZCKL increased.The increase in ZCKL High Group was significant(P<0.01).2.Comparison of m RNA,protein expression and c AMP concentration of LHR,EGFR,PKA and MAPK3/1 in cumulus granulosa cells of mice in each group.Compared with the Control Group,m RNA and protein expression of LHR,EGFR,PKA,and MAPK3/1 in P/H×5 Group,ZCKL Low Group and High Group was significantly decreased(P<0.01).The c AMP concentration was also decreased.The expression of the above-mentioned gene protein in the P/H×1 Group had a decreasing trend,and the m RNA and protein expression of LHR,EGFR decreased significantly(P<0.01).Compared with P/H×5 Group,ZCKL High Group and Low Group increased the expression of these genes and proteins in different degrees,and the difference was statistically significant.The ZCKL High Group had better resulted in improving m RNA and protein levels of EGFR than the ZCKL Low Group(P<0.05).Summary: 1.Desensitization of LHR for multiple stimulation might be associated with loss of coupling of receptors,resulting in decreased c AMP concentration in granular cells,down-regulation of PKA,and decreased phosphorylation of key downstream effector protein MAPK3/1.After activation of MPF,the translocation to the nucleus presented a down-regulation trend,which caused the GV-stage oocytes to decompose to produce the MII-stage oocyte,so the number decreased and the fragmented oocytes increased,resulting in a decrease in the rate of subsequent cleavage embryos.2.Combined use of ZCKL and gonadotropin could increase the level of LHR,increase the concentration of c AMP in granule cells,activate PKA,promote the phosphorylation of key downstream effector protein MAPK3/1,and translocate to the nucleus after activation of MPF,so that GV-stage oocytes decomposed to produce MII-stage oocytes,ie nuclear maturation,to cytoplasm maturation,fertilization and embryonic development.The number of oocytes in the MII-stage increased,and the proportion of embryos in the cleavage-stage increased.3.The mechanism of ZCKL to improve the quality of oocytes might be achieved by up-regulating GDF9/SMAD2,which induced cumulus expansion and activated EGFR to promote gonadotropin-directed EGFR/MAPK3/1 signaling pathway.It enriched the scientific connotation of "kidney diominates reproduction".Conclusion: Repeated stimulation of ovulation increased the rate of degenerating oocytes and reduced the number of MII oocytes,which reduced the number and quality of effective oocytes and affected the further development of the embryo.Repeated stimulation of ovulation resulted in a significant decrease in the number of antral follicles and antral follicles in the ovary,and a significant increase in the number of corpus luteum.Ovarian weight also increased with the number of ovulation induction.Repeated stimulation of desensitization of LHR might be associated with loss of coupling of receptors,resulting in a decrease in c AMP concentration in granular cells,and a decrease in PKA,leading to a decrease in the phosphorylation of key downstream effector protein MAPK3/1.After MPF activation,the translocation to the nucleus presented a down-regulation tendency,which resulted in the decomposition of GV-stage oocytes into MII-stage oocytes,ie nuclear maturation,reduced numbers,and increased number of fragmented oocytes,resulting in a decrease in the ratio of embryos during the cleavage period.ZCKL could increase the 8-cell rate and blastocyst rate of IVF in MII oocytes.The ZCKL High Group improved more significantly.ZCKL could increase the number of primary follicles and secondary follicles,especially with secondary follicles.The ZCKL High Group worked better.Nourishing Kidney Chinese medicine ZCKL could improve the oocytes quality of repeated superovulation mice by increasing the level of LHR,increasing the concentration of c AMP in granulosa cells,activating PKA,or possibly up-regulating GDF9/SMAD2-induced cumulus expansion to activate EGFR and promoting key downstream effector proteins,phosphorylation of MAPK3/1,translocation of MPF to the nucleus,decomposing GV-stage oocytes to generate MII-stage oocytes.It enriched the scientific connotation of "kidney dominates reproduction". |
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