The Experimental Study Of The Varicocele Effect On The Ultrastructure Of Testis, Sperm Morphology And Motility

Objective: Varicocele is common and frequently-occurring disease in Young men, the expansion and tortuosity of spermatic vein is caused by the spermatic venous blood obstruction or venous valve failure. The left spermatic vein return to the left renal vein with the right angles and the right spermatic vein return to the inferior vena cava with the oblique angle, Therefore, varicocele always occurs in the left. Varicocele is considered as the first cause of male infertility in young adults by the World Health Organization. The current study show that the cause of varicocele causes male infertility are immune, endocrine, apoptosis, anti-blood flow, temperature and infection etc. But, there is no one can fully explain the pathogenesis. We aim to investigate the mechanism of varicocele cause male infertility through observe the ultrastructure of sertoli cells, leydig cells and spermatogenic cells of testis with transmission electron microscopy after the creation of experimental varicocele model in rats, detect the sperm motility and morphology with the computer-aided semen analysis (CASA) system. It can provide reference to the treatment and research of the clinical patients of varicocele.Methods:1 Animal group: Forty health adult male Sprayue-Dawley rats(50±10d)with weight from 240 to 290 grams were divided randomly into two groups. Varicocele group (Experimental group n =20), Sham group (control group n=20). All rats were kept under the same conditions for 12 weeks.2 Model: Varicocele animal model were established by the method according to Turner: First, all rats were anesthetized through intraperitoneal injection with 10% chloral hydrate. The upper abdominal quadrant was through a midline laparotomy incision. The abdominal content were packed to the right in order to visualize the left kidney, left adrenal vein. We inserted a needle of a syringe 8 (0.8mm Diameter) between left adrenal vein and inferior vena cava after completely remove the tissue around the left renal vein. a 3-0 silk suture was used to ligate the inferior vena cava and needled at the position of the intersection of left adrenal vein and inferior vena cava. Like this, we can reduce half of the left renal vein diameter. Then we can see a left renal vein of dilatation and congestion after pull out the needle. At last, we sutured incision with 4-0 silk after washing the peritoneal. The rats of control group was not narrowed the left renal vein, the other procedures were as the same as the experimental group. then we injected penicillin (20 million units, 1/d) to the abdominal last three days after operation. We killed the rats at the 12 weeks after operation. If the left spermatic vein diameter was greater than 1mm and the weight of left and right kidneys were no significant difference demonstrate the animal model was successful.3 The collection of testicular specimen and observation with the electron microscope: First, we cut off the left testis of the two group rats, Immediately, put 1mm3 size of the testicular tissue in 1% paraformaldehyde and 2% glutaraldehyde mixture about 24 h, rinsed about 12h with PH 7.4 PBS. Then, we put the specimens in the 1% osmium tetroxide acid about 2h. After rinsing the specimens with PH 7.4 PBS, we dehydrated it with the graded ethanol and embedded in epoxy resin. After making the specimens into thin slices and staining with uranyl acetate and lead citrate, we observed the ultrastructure of the left testis with TEM about HITACHI-H7500.4 Semen specimen collection and testing: After rats were sacrificed off, we immediately cut off the epididymis of both groups and removed the fatty tissue around the epididymis. We cut off the epididymal tail after washing it with a PBS solution. Then, we cut three longitudinal cracks at the end of the epididymis with the scissors of ophthalmology, put the tissue in the 2ml centrifuge tube containing PBS. At last, we put the centrifuge tube in the water bath at 37℃,our aim was to make the sperm free swim. We took out it after about 20 min. 4.1 We drawed 10μl of the sperm suspension with pipette. We detected the sperm motility using CASA system. Detecting parameters: Sperm density(ρ), MOT, Rectilinear motion count, Rectilinear motion viability, VAP, a, b, VCL, VSL, BCF, MAD, LIN, WOB, ALH.4.2 We drawed 40μl sperm suspension with the adjustable pipette and droped the semen on glass slides. Then, we pushed the semen, air dried, fixed 10 min with methanol, air dried, put it in the staining cylinder containing 1% eosin staining dye. We took off the slides after a half-hour and washed it, when it became dry, each animal semen was produced 2 copies. At last,we observed the sperm morphology under light microscopy and calculated the rate of abnormal sperm.All data are statistically analyzed by using X±s. Every groups are compared by Wilcocon test with SPSS 13.0 software for statistical analysis. Testing standards at a=0.05 or a=0.01.Result:1 The results of electron microscopy: Experimental group compared to control group , the basement membrane of sertoli cell was edema, most of cell fusion, cell junction of Sertoli-Sertoli were blurred or broken, the number of mitochondria and the smooth endoplasmic reticulum of cytoplasm were significantly reduced and there were a lot of lysosomal granules in the cytoplasm of Sertoli cells; The number of mitochondria and smooth endoplasmic reticulum of cytoplasm and the microvilli of leydig cells were significantly reduced. But, there were a lot of lysosomal granules; large vacuoles, apoptotic cells, chromatin condensation in the cytoplasm of leydig cells; The nuclei of sperm was abnormal, there was a lot of large vacuoles and chromatin condensation in the nuclei of sperm, The nuclear membrane and acrosome were disappeared or smaller in the part of the sperm cells.2 The results of semen analysis: Experimental group compared to control group, the value of MOT and STR was reduced and the difference was significant between two groups(P<0.05). The value ofρ, rectilinear motion count, rectilinear motion viability, VAP, a, b, VCL, VSL and ALH was significantly reduced, but, the rate of abnormal sperm was significant increased, The difference was significant between two groups(P<0.01). The Value of MAD, LIN, WPB and BCF was not significant change and the difference was not statistically significance(P>0.05).Conclusion:1 Varicocele can reduce the number of mitochondria and smooth endoplasmic reticulum, increase the number of lysosomes and break the cell junction in sertoli cell.2 Varicocele can reduce the number of mitochondria and smooth endoplasmic reticulum, increase the number of lysosomes , apoptotic cells and chromatin condensation in leydig cell.3 Varicocele can lead to nuclei of sperm was abnormal, nuclear membrane and acrosome were disappeared and chromatin was condensation.4 Varicocele can reduce the fertility by reducing the sperm motility and increasing the rate of abnormal sperm.