| Taurine is one of the most abundant free amino acids in mammals,and plays important physiological functions in animals.Taurine deficiency can lead to a variety of reproductive system-related diseases.Cysteine dioxygenase(CDO)is a key enzyme in the taurine biosynthetic pathway.At present,the study of taurine in the reproductive system is mainly focused on pregnancy and early embryonic development.Functions of taurine and CDO in ovary have not been reported.In this study,Cdo knockout mice were used as experimental animal models to study the functions of CDO and taurine in ovaries.The main research contents and results are as follows:First,we detected the expression of Cdo in ovay by real-time PCR and western blot.The results shows that the expression of Cdo in ovaries after birth was much higher than that before birth,and there is no significant change in the expression of Cdo during estrus cycle.Immunohistochemical results showed that CDO was maily expressed in the cytoplasm of follicular granulosa cells and oocytes,suggesting that it may play roles in the follicular growth and oocyte maturation.To explore this,we firstly determined the reliability of the Cdo knockout mouse by gene sequence analysis,real-time PCR,western blot and immunohistochemical analysis.Then,high performance liquid chromatography(HPLC)was used to detect the taurine levels in Cdo knockout mouse.And the results showed that the taurine levels in serum,liver and ovary tissues were significantly decreased.Further studies showed that the reproductive capacity of Cdo knockout mouse was significantly decreased(litter size:WT 10.667±2.2365 vs KO 5±1.2405).To explore the reason for this,the estrus cycle and steriods levels were analysed.The results showed that steriod levels were decreased and estrus cycles were disordered in Cdo knockout mouse.Further studies showed that the expression of steroid synthesis related enzymes in follicular granulosa cells decreased,the amount of hormone synthesis decreased and cell proliferation decreased when Cdo expression decreased.Then,the oocyte maturation of Cdo knockout mice was detected and we found that the oocyte meiosis recovery blocked in the knockout mouse(GVBD rate:58.22±0.26%).The oocytes of Cdo knockout mice were then isolated and cultured in vitro and an appropriate concentration of taurine was supplymented.It was found that taurine could eliminate the obstruction of meiosis recovery caused by Cdo knockout(81.88±0.20%vs 60.15±0.17%).However,the supplyment of taurine had no significant effect on meiosis of wild-type oocytes.In order to explain this phenomenon,we studied the expression of CDO and taurine in the process of oocyte maturation by immunocytochemistry and HPLC respectively.It was found that GV(germinal vesicle,GV)?GVBD(germinal vesicle breakdown,GVBD)?PB1(first polar body,PB1)oocytes had CDO expression and high levels of taurine(4?6 pmol/oocyte).That is,under normal circumstances wild-type oocytes can synthesize taurine through the CDO/CSAD(cysteine dioxygenase/cysteine sulfonate decarboxylase,CDO/CSAD)pathway to meet their own needs.When CDO expression was deleted,the taurine synthesis pathway is blocked,and oocyte maturation is affected.And this affection can be eliminated by supplementing with taurine.To further explore the effects of CDO and taurine on oocytes,we also isolated Cdo knockout mouse oocytes for in vitro fertilization experiments.The results showed that the 2-cell rate of Cdo knockout oocytes 24 hours after fertilization was significantly lower than the control group(KO 31.85±0.21%vs WT 62.57±0.29%),and supplement with taurine significantly increased its 2-cell rate(54.87±0.22%).Cdo knockout will affect the development of early embryos of oocytes after fertilization.That is,Cdo knockout in the oocytes will affect the development of early embryos after fertilization.In summary,the results of this paper showed that the mouse ovary can synthesize taurine by CDO/CSAD pathway,and that CDO and taurine play a positive role on ovarian steroid synthesis and oocyte maturation.Cdo knockout will cause taurine level decreased and reproductive capacity weakened in mice.These results provide experimental basis for elucidating the function of taurine in the ovary and have reference significance for the clinical and practical practice of oocyte maturation and early embryo culture in vitro. |
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