Research On The Effect And Mechanism Of Vitamin D Abnormality On Spermatogenesis

Objective:More and more studies have shown that vitamin D not only plays an important role in maintaining calcium and phosphorus homeostasis and regulating bone metabolism in the body,but also significantly affects the reproductive system.Some studies have suggested that Vitamin D deficiency is associated with reduced reproductive function and fertility in males,but the conclusions are inconsistent and even contradictory.Furthermore,other studies show that high blood vitamin D concentrations may affect sperm parameters,such as lower sperm motility and concentration.Spermatogonial stem cells are the seed cells of male reproductive function.However,no specific effect of vitamin D on spermatogonial stem cells has been reported so far.This study aims to investigate the effects and mechanisms of vitamin D abnormalitiy on cell fate,such as proliferation and apoptosis of spermatogonial stem cells,and investigate the effects and mechanisms of vitamin D deficiency on spermatogenesis in male rats.Methods:Cell experiment part:1.Mouse spermatogonial stem cells（C18-4）were cultured in vitro and identified by qRT-PCR and immunofluorescence staining.2.The effect of 1α,25-（OH）₂D₃treatment on the viability and prolifration of C18-4 cells was detected at different concentrations and action time by the CCK-8 method.3.The effect of1α,25-（OH）₂D₃treatment with the concentration of 10^-7M on the apoptosis of C18-4 cells was detected by Flow cytometry（Annexin V/PI staining）4.Effects of 1α,25-（OH）₂D₃treatment with the concentration of 10^-7M on the m RNA expression of Bcl-2,Bax,Vdr and Pcna genes in C18-4 cells were detected by qRT-PCR method.Animal experiment part:1.Animal model establishment:SD male rats were randomly divided into two groups:（1）low vitamin D（VDI）group（n=5）fed with low vitamin D diet for 14 weeks;（2）normal diet（VD）group（n=7）fed with a normal level of vitamin D diet for 14 weeks.The body weight of the rats was recorded once every two weeks on average,and they were all killed after feeding for 14 weeks.The body length,body weight and left and right testicular weight were recorded2.Detection of serum indicators in rats:Blood samples were obtained from abdominal aorta,and the serum was separated after collection.Vitamin D（25（OH）D）,serum calcium,serum alkaline phosphatase and serum phosphorus were determined by the automatic biochemical analyzer.3.Sperm density in rat epididymis:Sperm suspension was prepared from the epididymis of rats and recorded.4.Preparation and staining of rat testicular sections:The testicular tissues of rat were paraffin-embedded and sectioned for H&E staining and PCNA immunohistochemical stainingStatistical methods:using SPSS 20.0 statistical package to analyze.Data results were expressed as mean±standard deviation（Mean±SD）.The t-test was used for comparison between two groups,and one-way ANOVA was used for comparison between multiple groups.If the variances are equal,then use LSD method,and if the variances are not equal,then use Dunnett’s T3 method.p<0.05 is considered a statistically significant difference.Results:Cell experiment part:1.Cell identification results:C18-4 cells were identified as mouse spermatogonial stem cells.2.CCK-8 assay results:compared with the control group,the A₄₅₀value was significantly reduced after 10^-7M concentration1α,25-（OH）₂D₃treatment for 24h and 48h（P<0.05）.3.Flow cytometry results:compared with the control group,the percentage of early apoptosis of cells was significantly increased after10^-7M concentration 1α,25-（OH）₂D₃treatment for 48h（P<0.05）.4.Real-time PCR results:compared with the control group,the m RNA expression of Bax and Vdr was significantly up-regulated（P<0.05）,while the m RNA expression of Bcl-2 and Pcna was significantly down-regulated（P<0.05）after 48h treatment with 10^-7M concentration of 1α,25-（OH）₂D_3.Animal experiments part:1.Measurement results of physiological indexes in rats:There were no significant differences in body weight,length,and weight of left and right testes between VDI group and VD group（P<0.05）.2.Serological test results:Compared with the VD group,the serum25（OH）D level was significantly decreased in the VDI group（P<0.05）,and the serum alkaline phosphatase level was significantly decreased（P<0.01）.3.Results of epididymal sperm density detection:Compared with the VD group,the epididymal sperm density in the VDI group decreased,and there was no statistical difference（P<0.05）.4.Morphological analysis results of of testis by H&E staining:Compared with the VD group,there were a few vacuoles,cell abscission and absence of sperm in the testicular cell sections of the VDI group,and the difference between the two groups was not statistically significant（P<0.05）.5.Testicular PCNA staining results:There was no significant difference in PCNA protein expression between the two groups（P<0.05）.Conclusion:1.Higher concentration of 1ɑ,25-（OH）₂D₃can inhibit the proliferation of mouse spermatogonial stem cells by inhibiting the expression of Pcna and promote the apoptosis of mouse spermatogonial stem cells by up-regulation of Bax and down-regulation of Bcl gene expression.2.Vitamin D insufficient diet in rats can result in a decrease of epididymal sperm density in trend.