The Effects Of Doxorubicin On Oocyte Maturation And Ovarian Function In Mouse

With the optimization and upgrading of chemotherapy drugs and treatment schemes,the prognosis of malignant tumor patients is constantly improving,and some patients can be cured or survive for a long time.However,the toxic effects of chemotherapy drugs on ovarian function and fertility may lead to premature ovarian failure for a long time,and even seriously affect the quality of life of patients.Fertility protection has become a scientific problem and livelihood issue that needs to be solved urgently at present.Doxorubicin(DOX)is widely used in the treatment of various malignant tumors,and its gonadal toxicity and its influence on female fertility have attracted much attention.It is difficult to evaluate the gonadal toxicity of DOX alone because of drug combination,and its effects on oocyte maturation and ovarian function and its related mechanism are still unclear.In this study,mouse oocytes and animal models were used to simulate chemotherapy for adolescent women to explore the unclear question,so as to provide new clues and laboratory theoretical basis for fertility protection of patients who are about to receive anti-tumor therapy and face the risk of iatrogenic premature ovarian failure and fertility loss.This study includes two parts: the toxic effects of DOX on the meiotic maturation and quality of mouse oocytes in vitro;The toxicity study of single dose DOX after 28 d on the reproductive activities of female mice,such as estrus cycle,serum reproductive hormones,reproductive performance(embryo implantation number and litter size),ovarian follicle development,and the sequencing and analysis of ovarian transcriptome.The main results are as follows:1.The effects of DOX on meiotic maturation of mouse oocytes in vitro.(1)Mouse oocytes at germinal vesicle(GV)stage were cultured and treated with 0,10 n M and 100 n M DOX in vitro for 14 hours.The results showed that the first polar body extrusion(PBE)was significantly inhibited with 100 n M DOX(32.9 ± 5.6% vs control78.5 ± 3.5%,P < 0.0001),but not affected significantly with 10 n M DOX(69.7 ± 9.6%,P >0.05).These results indicated that DOX with certain concentration can significantly inhibit PBE of mouse oocytes.(2)The spindle morphology showed that the percentage of the first metaphase of Meiosis I(MI)arrest of oocytes was significantly increased in oocytes treated with 100 n M DOX for 14 h(46.6 ± 5.5% vs control 10.0 ± 1.8%,P < 0.0001).After the oocytes without germinal vesicle breakdown were removed,the oocytes were cultured for 12 h after DOX treatment,the PBE of oocytes was significantly inhibited,and DOX induced the MI arrest of oocytes(53.3 ± 3.3% vs control 16.8 ± 2.5%,P < 0.0001).After mouse oocytes at GV stage were treated with 100 n M DOX for 8 h,the spindle structure of oocytes was normal in two group,but the arrangement of chromosomes was disordered,and the width of chromosome MI plate increased(14.54 ± 0.53 μm vs control 12.11 ± 0.33 μm,P < 0.0001).The ratio of chromosome width to spindle length increased significantly(0.639 ± 0.025%vs control 0.518 ± 0.019%,P < 0.0001).These results indicated that DOX did not significantly affect germinal vesicle breakdown and MI spindle structure,while caused the disorder of chromosome arrangement of MI oocytes.(3)The expression and localization of kinetochore protein CREST and α-tubulin,showed that the proportion of abnormal kinetochore-microtubule structure of oocytes in MI stage increased significantly after DOX treatment(57.9 ± 3.0% vs control 23.9 ± 1.1%,P < 0.0001).After the oocytes at GV stage were cultured for 4.5 h and 10 h,the Bub R1 signal was located near the kinetochore at pro-Metaphase I stage in two groups.This signal disappeared at Anaphase/Telophase I stage in the control group,but was still found in the DOX group;The rate of oocytes with positive Bub R1 signal significantly increased in the DOX group(73.0 ± 8.6% vs control 25.6 ± 4.2%,P < 0.01).These results indicated that DOX treatment destroyed the kinetochore-microtubule structure of MI oocytes,and hindered the transition from metaphase to anaphase of oocyte meiosis by mediating the continuous activation of spindle assembly checkpoint.(4)DOX accumulated in the nucleus of oocytes after treatment with DOX.After 100 n M DOX treatment for 1 ～ 3 h,the percentage of oocytes with more than 10 positive foci of γ-H2 A.X increased significantly with the treatment time(P < 0.0001),these results showed that DOX induced DNA double-strand break.The fluorescence intensity of Annexin-V of oocytes in the DOX group enhanced significantly(19.34 ± 1.06 vs control12.47 ± 1.87,P < 0.01).The proportion of abnormal spindle in oocytes of the DOX group increased significantly when the development continued to MII stage(75.4 ± 1.5% vs control 12.6 ± 2.0%,P < 0.0001),and the spindle assembly was seriously affected followed by the disorder of chromosome arrangement.These results indicate that DOX can directly act on oocytes,induce DNA damage and early cell apoptosis in oocytes,disturb MII spindle assembly and damage the maturation quality of oocytes.2.The effects of DOX on mouse ovarian function and transcriptome sequencing.(1)4-week-old female Kunming mice were injected with DOX at a dose of 10 mg/kg by single intraperitoneal injection,and the estrous cycle of mice was detected after 21 days.The results showed that the diestrus stage and the estrous cycle of mice in the DOX group were significantly longer than those in the control group(Diestrus: 1.67 ± 0.45 d vs control1.17 ± 0.39 d,P < 0.05;Estrous cycle: 5.58 ± 0.48 d vs control 4.25 ± 0.45 d,P < 0.01).On the 28 th day after DOX administration,the serum E2 and AMH levels of mice decreased significantly(P < 0.01),while FSH levels increased significantly(P < 0.01).After 28 days of administration,the reproductive performance of female mice was evaluated by mating with normal male mice.The results showed that the implantation sites of female mice in the DOX group were few and unevenly distributed at 10 ～ 12 days after mating,and the number of implanted embryos and subsequent litter size were significantly lower than those in the control group(P < 0.05 or P < 0.01),which indicated that DOX interfered the estrous cycle and hormone levels,affected the ovarian function,and reduced the fertility of female mice.(2)Ovarian histomorphological examination showed that mouse ovarian cortex became thinner,medulla enlarged,follicular atresia and growing follicles decreased after DOX treatment.On the 28 th day,the number of primary follicles,secondary follicles and corpus luteum decreased significantly(P < 0.01).Masson staining showed that the proportion of collagen area in the DOX group significantly increased(6.62 ± 0.98% vs control 3.88 ± 1.07%,P < 0.05).These results showed that DOX destroyed the structure of ovarian tissue,induced collagen deposition in ovarian tissue,and hindered the development of ovarian follicles.(3)Immunofluorescence staining showed that γ-H2 A.X bright spots or TUNEL positive fluorescence signals aggregated or clustered in granulosa cells of ovarian antral follicles after 24 h from DOX injection.The ultrastructure of ovarian tissue by transmission electron microscope showed that the mitochondria of ovarian granulosa cells swelled,vacuolated and the mitochondrial crista twisted after DOX injection for 28 d.These results indicated that DOX with certain concentration in vivo can seriously damage mitochondria of mouse ovarian granulosa cells,induce DNA damage and apoptosis of oocytes,and may lead to abnormal development and atresia of ovarian follicles.(4)RNA-Seq technique was used for transcriptome sequencing analysis.Compared with the control group,588 differentially expressed genes(361 up-regulated,227 downregulated)and 266 differentially expressed lnc RNA(166 up-regulated,100 down-regulated)were obtained in the ovarian tissues of mice after 28 d from DOX injection.The enrichment GO analysis of differentially expressed genes showed that the extracellular region,extracellular space and proteinaceous extracellular matrix were the highest significance among all GO items,and these regions were related to the structure and function of ovarian tissues and cells.The highest enrichment degree in the biological process was white fat cell differentiation,and the others involved negative regulation of synaptic transmission,acute inflammatory response,positive regulation of cell death.These processes were related to cell differentiation,organism protection and death.Molecular functions involved 2-acylglycerol O-acyltransferase activity,protein-arginine deiminase activity,Toll-like receptor 4 binding and calcium ion binding,suggesting that these enzymes and molecules play an important role in the regulation of ovarian function.KEGG analysis showed that the differential genes were significantly enriched in steroid hormone biosynthesis,ECMreceptor interaction,cell adhesion molecules,Protein/Fat digestion and absorption,and disease-related pathways such as type I diabetes and tumor.The analysis of target genes of differentially expressed lnc RNA showed that the TOP 30 GO terms included positive regulation of mitochondrial translation,mitochondrial small ribosome subunit,mitochondrial intermembrane space,mitochondrial ribosome binding,etc.These genes were significantly enriched in the structure and function of subcellular organelles such as mitochondria.These results indicated that DOX has an extensive influence on extracellular matrix,mitochondrial function,hormone secretion,substance metabolism,growth and differentiation of mouse ovarian tissue,and finally resulted in the decline of ovarian function.(5)By analyzing the differentially expressed genes and their protein interaction networks,the expression levels of collagen I,IV,V increased in the DOX group,and matrix metalloproteinases and their tissue inhibitors(MMPs/TIMPs)may play an important role in follicular development,extracellular matrix degradation and remodeling.The expression levels of CEBPA and adipokines(adiponectin,resistin and leptin)were up-regulated,suggesting that CEBPA may play an important role in ovarian cell metabolism and follicular development after DOX administration.The expression levels of some members of cytochrome P450 family(CYP1a1,CYP17a1,CYP19a1)increased,while the expression levels of other members(CYP2a5,CYP11b1),estrogen key synthase HSD17b7 and estrogen receptor ESR2 decreased,which indicated that ovarian cells had complex regulation in hormone synthesis and estrogen effect after DOX administration.The expression level of DNA damage-induced transcription factor 4(DDIT4)increased,suggesting that DOX may be involved in the interaction of intracellular protein molecules and the regulation of ovarian cell proliferation and apoptosis through DDIT4.The expression level and function analysis of these genes are helpful to understand the possible mechanism of DOX-induced ovarian damage and functional repair.To sum up,this study finds that DOX with certain concentration can cause serious DNA damage in mouse oocytes during mature culture,and induce meiosis MI arrest by mediating the continuous activation of spindle assembly checkpoint.DOX interferes with spindle assembly of MII oocyte and leads to the disorder of chromosome arrangement,thus reduce the quality of oocytes.Certain dose of DOX can cause DNA and mitochondria damage of ovarian granulosa cells in mice,destroy ovarian tissue structure,reduce ovarian reserve and damage reproductive function of female mice.Through RNA-seq screening of differentially expressed genes and functional analysis,it is speculated that MMPs/TIMPs,CEBPA,HSD17b7 and DDIT4 may play a key role in regulating ovarian extracellular matrix,cell metabolism and follicular development,steroid hormone synthesis after DOX treatment,and in inducing ovarian cell proliferation and apoptosis after DNA damage.The above research provides an important experimental basis for understanding how DOX leads to the failure of oocyte meiosis and maturation,the decline of oocyte quality,and the damage of ovarian function,and help to explore strategies to prevent the fertility loss caused by DOX for young female tumor patients who need chemotherapy.