| Objective1.To observe the effects of varicocele on testicular organ index,semen quality,testicular tissue morphology and microstructure of spermatogenic cells,oxidative stress level and the degree of sperm DNA damage by establishing a male Wistar rat model of varicocele.2.To investigate the protective effect of L-carnitine on the reproductive ability of male Wistar rats with varicocele using L-carnitine intervention in the male Wistar rat model of varicocele.The mechanism of action of L-carnitine was also investigated based on the FoxO signaling pathway.Methods1.40 8-week-old male Wistar rats were randomly divided into group A（control group）,group B（sham-operated group）,group C（model group）,and group D（L-carnitine group）,10rats in each group.Group A was a blank control without any manipulation,group B rats were only separated from the fascia around the left renal vein to expose the left renal vein without ligation,and groups C and D used incomplete ligation of the left renal vein to establish a rat model of varicocele,and after successful modeling,group D was given intraperitoneal injection of L-carnitine(100mg×kg-1×d-1),and groups B and C were given intraperitoneal injection of saline in equal volume once a day.On the 28th day,after judging the successful construction of the model,the rats in the above four groups were weighed,and the specimens of left epididymis and left testis were collected under anesthesia and then executed by the method of neck breaking.2.Take the left testis of the rats,weigh and calculate the left testicular organ index.3.Take the left epididymis and weigh it,prepare sperm suspension,and perform sperm count under the microscope,calculate the relative sperm count,and observe the forward motility and total motility of sperm.4.The left testicular tissue was taken,and the pathomorphological observation of the left testicular tissue of each group of rats was performed by HE staining;the testicular spermatogenic cells and supporting cells were observed under transmission electron microscopy;the homogenate was prepared from the remaining left testicular tissue,and the content of malondialdehyde（MDA）,8-hydroxydeoxyguanosine acid（8-OHd G）,reactive oxygen species（ROS）and superoxide dismutase（SOD）activity were detected in the left testicular tissue homogenate,responding to the level of oxidative stress within the testicular tissue.5.The DNA damage test kit was used to perform comet tail dragging assay,and comet tail length（TL）and comet tail moment（OTM）were measured and analyzed to detect the extent of DNA damage in testicular cells.6.FoxO3a,catalase（CAT）,SOD2 protein expression in the testis tissue of rats in each group were analyzed by protein immunoblotting method.Results1.Comparison of testicular organ indices of male Wistar rats in each group:compared with the control group,the testicular organ indices of rats in the varicocele model group were significantly lower（P<0.001）;while the intervention with L-carnitine could effectively improve the testicular organ indices of rats（P<0.001）.2.Comparison of semen parameters among the male Wistar rats:the relative sperm count,forward motion rate and total sperm viability of semen in the model group and levocarnitine group showed a significant decrease compared with the control group（P<0.01）,while the forward motion rate and total sperm viability increased after the intervention with L-carnitine（P<0.001）,and the relative sperm count also increased significantly（P<0.05）.3.HE staining of the left testis sections of rats in each group showed that:（1）The testicular tissue of rats in the control group had intact basal membranes of the germinal tubules,neatly and closely arranged supporting cells and germinal cells,closely arranged germinal cells at all levels,about 5-7 layers,and aggregation of mature spermatozoa in the center of the tubular lumen,with Johnsen score（8.7±0.900）;（2）No significant differences were seen in the histomorphology of the testes under light microscopy in the sham-operated group compared with the control group,and the difference in Johnsen score（8.5±0.806）was not statistically significant（P>0.05）;（3）The number of mature spermatozoa in the testicular spermatogenic cell level and canal lumen of rats in the model group was significantly reduced compared with that in the control group,with disorganized spermatogenic cells at all levels and a large number of spermatogenic cells shed,and the Johnsen score（4.7±0.900）was reduced compared with that in the control group（P<0.001）;（4）In the L-carnitine group,the testicular tissue of spermatogenic cells were clearly arranged and more compact,with mature spermatozoa visible in the center of the lumen,and the Johnsen score（7.1±1.136）was lower（P<0.05）and higher（P<0.001）than that of the control group.4.Transmission electron microscopic observations of the left testis of each group of rats:（1）The mitochondrial cristae were in the shape of tubular vesicles and evenly distributed in the mitochondria,and the rough endoplasmic reticulum and Golgi apparatus were not hypertrophied and expanded;（2）In the model group,the left testicular cells of rats were obviously edematous,the mitochondria were heavily swollen,the matrix was shallow and locally dissolved,some mitochondria were severely vacuolated or even ruptured,the mitochondrial cristae were heavily fractured and missing,and the rough endoplasmic reticulum and Golgi apparatus were moderately dilated;（3）In the L-carnitine group,the left testicular cell bodies were clear and intact,some mitochondria were mildly swollen,the mitochondrial cristae were clearly structured,the rough endoplasmic reticulum and Golgi apparatus were regular in shape,and no edema expansion was observed.5.Comparison of MDA,8-OHd G,ROS content and SOD activity in the left testis of rats in each group:（1）MDA content:Compared with the control group,the MDA level in the left testis of rats in both the model group and the levocarnitine group was increased（P<0.05）;intervention with levocarnitine could effectively reduce the MDA level in the testis of rats in the model group（P<0.001）;（2）8-OHd G content:compared with the control group,the 8-OHd G content in the left testis of rats in both the model group and the L-carnitine group was increased（P<0.05）;the intervention with L-carnitine could effectively reduce the 8-OHd G content in the testis of rats in the model group（P<0.05）;（3）ROS content:Compared with the control group,the ROS content in the testis tissue of rats in the model group was increased（P<0.001）;the ROS content in the testis tissue of rats in the L-carnitine group was significantly reduced compared with that in the model group（P<0.001）;（4）SOD activity:compared with the control group,the SOD activity in the testis tissue of rats in both the model group and the levocarnitine group was reduced（P<0.05）;while L-carnitine intervention could effectively increase the SOD activity in rats induced by varicocele model（P<0.001）.6.Comparison of the results of comet electrophoresis test in rats of each group:compared with the control group,the TL and OTM values of testicular cell DNA of rats in the model group and levocarnitine group were increased（P<0.01）;compared with the model group,the TL value of testicular cell DNA of rats in the levocarnitine group was changed without statistical significance（P>0.05）,while the OTM value was significantly decreased（P<0.01）.7.Compared with the control group,the expression levels of FoxO3a protein in the left testicular tissue of rats in the model group were increased（P<0.001）,while the expression levels of CAT protein and SOD2 protein were significantly decreased（P<0.01）,and the expression levels of FoxO3a protein,CAT protein and SOD2 protein in the testicular tissue of rats in the levocarnitine group were increased（P<0.01）;and the use of levocarnitine increased the expression levels of FoxO3a protein,CAT protein and SOD2 protein in the testicular tissue of rats in the model group（P<0.01）.Levocarnitine intervention increased the expression of FoxO3a protein,CAT protein and SOD2 protein in testicular tissues of rats in the varicocele model（P<0.01）.Conclusion1.Varicocele model can reduce testicular index,decrease total sperm viability,forward motion rate and relative sperm count in Wistar male rats,increase the level of oxidative stress in the testis,weaken the antioxidant capacity,and cause morphological damage to testicular tissues and structural damage to organelles within supporting cells and spermatogenic cells.2.Levocarnitine can increase CAT and SOD2 expression in testicular tissues by mediating the activation of FoxO signaling pathway,enhance the antioxidant capacity of rat testes,reduce oxidative damage in testes of Wistar male rats with varicocele,and thus protect the reproductive function of rats. |
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