| Part 1:Molecular mechanism of immune orchitis and spermatogenesis impairment induced by glycosylation loss in Asn297 region of IgG Fc region[Objective]The molecular mechanism of immunological orchitis caused by the abnormal glycosylation of the Asn297 site of the IgG fragment and the products of excessive ROS in sperm,especially the white blood cells,can inhibit the defense mechanism of antioxidants on sperm and sperm plasma membrane were explored.[Methods]This study collected serum samples of patients with 127 immune orchitis,Single-molecule real-time(SMRT)Circular consensus sequencing(CCS)used for the analysis of the sequence data of the Fc region Asn297 site protein of IgG and finding the mutation sites and Sanger sequencing to verify the mutation position of the Asn297 gene.The correctness of the point.The chemiluminescence method was used to detect the content of seminal plasma ROS and malondial dehyde(MDA)in seminal fluid and liquid chromatography-mass spectrometry,and the results of biochemical examination,histopathology and immunohistochemistiy were used to analyze the molecular pathogeny of the pathogenesis of immune orchitis.[Results]The serum IgG concentration of patients was 2231.4 ± 291.5mg/dl,higher than the normal range 80?1400mg/dl,while 38 cases of control group were 386.3 ±182.6mg/dl.The concentration of 50%hemolytic unit complement(CH50)was 128.32±61.51 U/ml,which was significantly higher than the normal value(23?46 U/ml),while the corresponding concentration in the control group was 31.43 ± 8.37 U/ml.The serum complement C3 concentration was 5.23 ± 2.36 g/L,which was significantly higher than the normal value(0.9?1.8 g/L),while the complement C3 concentration in the control group was 1.2?0.3 g/L.The t test of the experimental group and the control group showed that there was statistical difference between the two groups(P<0.05).The concentrations of reactive oxygen species in the semen were significantly higher than those in the control group in the concentrations of O2-,H2O2,and OH.The IgG Fc region(294?300)of the patient was higher in the total diversity of amino acids than in the control group,indicating that the 7 amino acids around Asn297 in the patient's gene sequence changed greatly,while the 297 site amino acids in the control group were fixed at high frequency of asparagine.In 36 patients,the mutation at Asn297 locus(CAC)was(AAC).The mutation of codon 891 of exon sixth is the substitution of A to C,which results in the conversion of amino acids from asparagine to other amino acids.96 people(96/127,75.59%)had Asn297 mutation and glycosylation.The 892 codon in exon sixth of the 31 patients had a pathological mutation,that is,the transformation of asparagine(AAC)to serine(AGC).29 cases of IgG gene insertion or deletion of the code shift mutation.31 cases showed no evidence of mutations in the Asn297 gene.They may have other loci gene mutations that have not yet been detected.On the contrary,no mutation was detected in the IgG gene of 38 healthy men.According to the results published in GenBank,asparagine mutation at Asn297 locus is very low in healthy people.Immunohistochemical staining showed that there were immune complex deposits and a large number of lymphocytic infiltration in the testis,epididymis and tunica vaginalis,the thickening of the wall of the small vessels,a large number of neutrophils in the endovascular,the fibrosis of the basement membrane of the fine tubule,the only supporting cells in the cavity,the thinning of the spermatogenic epithelium,the vacuolization of the spermatogenic epithelium,and the row of spermatogenic cells.[Conclusions]The abnormal glycosylation of IgG Asn297 loci can lead to immune orchitis;the increase of active oxygen cluster in the seminiferous tubules causes DNA damage of spermatogonial cells and causes spermatogenesis disorder.Part 2:Interaction mechanism between insulin synthesis and hypothalamus-pituitary-gonad axis function[Objective]The transcription and expression of kisspeptin,an upstream regulator of hypothalamus pituitary gonadal axis regulated by insulin.To observe the changes in the expression of kisspeptin in the hypothalamus and the changes of reproductive function in the hypothalamus kisspeptin neuron specific gonadal receptor knockout mice,and to reveal the molecular role of insulin through the central regulation of the reproductive function.[Methods]Using microfluidic chip and other advanced technologies,through the three-dimensional culture of Gnv-3 cells,the analysis of cell connotations,the effects of insulin on the tracer,extraction,capture,fragmentation,separation,and liquid phase mass spectrometry of kisspeptin translation,with the aid of various animal intervention and gene knockout models.Insulin(10U/mL and 20U/mL)and Dex(10-6 mol/L and 10-7 mol/L)were used to intervene 72 h,and the changes of kispepetine mRNA and protein expression after intervention of 6 h,24 h,48 h and 72 h were evaluated.Insulin(3U/Kg)and Dex(1mg/Kg)were used to intervene in the castrated male mice for 4 weeks.The changes of LH level and kisspeptin immunohistochemical staining in the preoptic region of the hypothalamus were detected before and after the intervention.[Results]In the study of abnormal pancreatic model induced by streptozotocin(STZ)in male SD rats,the total testosterone of 10 rats in the normal group was 56.2 ± 6.3 ng/dl,and the total testosterone of the abnormal group of pancreas(n=13)rats was 37.8 + 7.4 ng/dl and P<0.05.The LH of the normal group was 5.3 ± 1.2 ng/dl,and the LH value of the pancreas abnormal group was 2.1 ± 0.9 ng/dl,P<0.05.The results of immunohistochemistry showed that the number of kisspeptin immunoreactive cells in the hypothalamic arcuate nucleus of rats with abnormal insulin secretion was significantly lower than that of the control group.The number of kiss-ir cells in the normal group was 428 ± 46 ng/dl,and the kiss-ir cells in the abnormal group of pancreas were 216 ± 24 ng/dl and P<0.05.Insulin and RAPA intervention regulate the expression level and subcellular distribution of mTOR protein in Gnv-3 cells.Control is the control.Gnv-3 cells use insulin(0.2,2.OU)and RAPA(300ng/ml)incubated for 24 hours to extract cytoplasmic protein/nucleoprotein and total protein.Western Blot method is used to detect the expression level of SP1 protein.RT-PCR confirms the cell Expression of IR receptor.The kissl-ir cells of hypothalamic arcuate nucleus(kissl-ir)in the hypothalamus of normal and ovariectomized,ovariectomized and insulin injection groups were measured and analyzed.The results showed that the kissl-ir cells in the hypothalamus of the ovariectomized SD rats were significantly lower than those of the other two groups,but there was no significant difference between the nomal group and the ovariectomized group and the insulin injection group.The level of LH in ovariectomized rats increased gradually,and LH reached(5.78 ± 2.84)ng/mL after 4 weeks of castration,which was significantly higher than that before castration(0.90 ±0.26)ng/mL,FSH reached(76.32 ± 22.46)mIU/mL,and was significantly higher than that before castration(22.90 ± 8.26)mIU/mL,P ? 0.01.Low dose insulin(0.2 IU/kg)interfered with LH level(5.29 ± 2.14)ng/mL after ovariectomized rats for 4 weeks.Medium dose of insulin(2 IU/kg)intervention after 4 weeks of castrated rats,LH level(3.21 ± 1.86)ng/mL,compared with the control group significantly decreased,P ? 0.05;large dose(10 IU/kg)intervention for 4 weeks after the castrated rats,LH level(7.21 ±2.92)ng/mL,significantly higher than the control group,P<0.05.Compared with the control group,the expression of kisspeptin immunohistochemical staining in the preoptic region of the hypothalamus increased slightly after the intervention of the small dose of insulin(0.1 IU/kg)for 4 weeks in the ovariectomized rats.The expression of kisspeptin immuno histochemical staining in the preoptic region of the hypothalamus increased significantly after 4 weeks in the rats with moderate dose of insulin(1 IU/kg),P<0.05.The significant difference in the expression of kisspeptin immuno histochemical staining in the preoptic region of the hypothalamus of rats after the intervention of the large dose(10 IU/kg)in the preoptic area of the hypothalamus of the rat was observed,P ? 0.05.[Conclusions]Insulin can be involved in the function of the gonadal axis of the male rats by increasing the expression of Kisspeptin in the preoptic region of the hypothalamus.In the ovariectomized model of female rats,insulin regulates the Kisspeptin of the hypothalamus through the mTOR pathway to improve the reproductive function. |
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