| Objectives To investigate the effects of vitamin D as an adjuvant therapy on oocyte mitochondrial function,insulin resistance,sex hormone secretion and embryonic development of rats with polycystic ovary syndrome(PCOS),and its role of PI3K/AKT/m TOR signaling pathway in it.Methods Immature female SD rats(three weeks-old,77.6±5.3g)were initially divided into control group(n=35)and PCOS group(n=135).Rats in PCOS group were intragastrically fed with letrozole(1mg/kg·d,in 0.5% carboxymethyl cellulose,CMC-Na)and high-fat diet were administered intragastrically,meanwhile,control rats were intragastric with CMC-Na and ordinary diet,for consecutive 28 days.After successful modeling,the PCOS rats were randomly divided into PCOS group,VD group,Metformin+Dacin-35 group(M+D,conventional treatment group),and VD+Metformin+Dacin-35 group(VD+M+D,adjuvant treatment group),30 rats in each group.In the morning,the rats in M+D and VD+M+D groups were given Diane-35(0.2035mg/kg·d)and Metformin(200mg/kg·d),the rats in other three groups were given the same volume of CMC-Na,while in the afternoon,the rats in VD and VD+M+D groups were given VD3(200IU/kg·d),the rats in other three groups were given the same volume of corn oil,ordinary diet.The blood of inner canthus and ovary were collected after 21 days of continuous administration.Vaginal cytology was used to detect estrous cycle;The ovarian morphology was observed by paraffin section and HE staining,and the number of all follicles was counted in light microscope;ELISA was used to determine serum 25-(OH)D,Testosterone(T),Luteinizing Hormone(LH),Follicle-stimulating Hormone(FSH),Fasting Blood Glucose(FPG),Fasting Insulin(FINS);The mitochondrial membrane potential(MMP),reactive oxygen species(ROS),permeability transformation pore(mt PTP)and mitochondrial distribution of oocytes were detected by fluorescence probes;Mitochondrial morphology,apoptotic bodies and autophagosomes of oocytes and granulosa cells were observed by transmission electron microscopy;Apoptosis of granulosa cells was detected by flow cytometry;Western blot was carried to detect mitochondrial fusion and fission proteins Mfn1,Mfn2,Drp1,autophagy related proteins LC3,Beclin1,p62,apoptosis-related proteins Bax,Cleaved-caspase3,Bcl-2,PCNA,and the expression of signal pathway proteins PI3 K,p-PI3 K,AKT,p-Akt,m TOR,p-MTOR,PTEN,p-PTEN in ovarian tissues.The expression of Bcl-2,Bax and PCNA in ovarian tissues was detected by immunohistochemistry.Fertilized ova were cultured in vitro and embryo development was observed.Results 1 The estrous cycle of rats in PCOS group was disordered and the diestrus period was prolonged,while the estrous cycle of rats in treatment groups was basically normal.2Compared with PCOS group,atretic follicles and cystic follicles decreased,mature follicles and luteum increased in treatment groups(P(27)0.05),and the effect of adjuvant treatment group was better than that of VD group and conventional treatment group(P(27)0.05).3 Serum T,LH and FINS levels in treatment group were lower than those in PCOS group,LH/FSH ratio and HOMA-IR value were lower than those in PCOS group,and the effect of adjuvant treatment group was better than that of VD group,the differences were statistically significant(P<0.05).4 The MMP in the treatment groups was higher than that in the PCOS group(P(27)0.05),and the rate of mitochondrial abnormality and ROS level were lower than those in PCOS group(P(27)0.05),and the openness of mt PTP was lower(P(27)0.05).Observations under electron microscope exhibited that mitochondria of PCOS group were swollen and degenerated,vacuole appeared,mitochondrial crest disappeared,rod-shaped mitochondria decreased,circular mitochondria increased,and mitochondrial morphology was improved in the treatment groups.5 Compared with PCOS group,Drp1 protein expression in the treatment groups decreased(P(27)0.05),while Mfn1 and Mfn2 protein increased(P(27)0.05).6 The level of LC3-II and Beclin1 protein was significantly lower,whereas the level of p62 was significantly higher in the treatment groups than those in PCOS group(P(27)0.05).Electron microscopic results showed that the number of autophagosomes in oocytes and granulosa cells of PCOS group increased,while that in treatment group decreased.7 Compared with PCOS group,the level of Bcl-2 and PCNA protein in the treatment groups increased(P(27)0.05),and the levels of Bax and Cleaved caspase-3 protein decreased(P(27)0.05);Flow cytometry results showed that the apoptotic rate of granulosa cells in the treatment groups was significantly lower than that in the PCOS group,but still higher than that in the control group(P(27)0.05),but no statistically significant differences between the treatment groups(P(29)0.05).Electron microscope showed that PCOS group is characterized of granular cell volume small,the nuclear membrane intact,most nuclear chromatin condensation,and several blocks,scattered in the nuclear mass,intact organelles and the plasma membrane,apoptosis has been obviously improved in the treatment groups,VD group of visible chromatin slight edge phenomenon,while the nucleus chromatin in routine treatment group and adjuvant treatment group evenly distributed in the nuclear mass.8 Compared with the PCOS group,the level of pPTEN protein in the treatment group decreased(P(27)0.05),while the level of p-PI3 K,p-Akt and p-MTOR protein significantly increased.9 Significantly more oocytes were harvested and cleavage stage embryos were produced in the treatment groups than in the PCOS group(P(27)0.05),but there was no significantly difference between the treatment groups(P(29)0.05).The percentage of transferable embryos and rate of blastocysts in the treatment groups were higher than those in the PCOS group,but not significantly(P(29)0.05).Conclusions 1 VD as an adjuvant therapy can improve insulin resistance,sex hormone disorder and oocyte mitochondrial dysfunction in PCOS rats.2 VD as an adjuvant therapy can alleviated apoptosis and autophagy of oocytes and follicles in PCOS rats.3 VD as an adjuvant therapy can improve the developmental quality of PCOS rat embryos.4 VD as an adjuvant therapy may regulate the apoptosis and autophagy of PCOS rat ovarian cells through PI3K/AKT/m TOR signaling pathway.Figure 28;Table 17;Reference 146... |
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