| Background and objectives:Varicocele is an abnormal dilation of the spermatic veins and is the most common known cause of male infertility, but it can be treated. In a WHO study,11.7%of infertile men with normal semen analyses and25.4%of infertile men with abnormal semen analyses had a varicocele. At present, the investigations on the effects of varicocele have mostly focused on the testis. As we all know, the epididymis is a part of the male reproductive system and is essential for the storage, maturation, and transport of sperm. Most of the blood supply of the testis and epididymis are the same. The testis and epididymis are located within the scrotum. When varicocele caused a series of pathophysiological changes in testis, epididymis can also suffer the same damage. Therefore, epididymal involvement also plays an important role in the male infertility caused by varicocele.Numerous reports showed that varicocele in some men is a progressive and not a static lesion. However, some scholars argued against the concept that varicocele is a progressive disease process. Therefore, whether varicocele is a progressive or static disease is still under debate. Varicocelectomy is the most popular treatment, and results in the improvement of semen parameters and an increase in postoperative pregnancy rates. The spermatic artery is the major source of the blood supply to the mammalian testis and epididymis. Animal studies have revealed that in rats atrophy and pathologic damage occurred in most testes after ligation of the spermatic artery. Clinical studies reported that subjects with artery-preserving varicocelectomy (APV) had better quality semen than subjects who had undergone artery-ligating varicocelectomy (ALV). However, contradictory results have been reported in some studies, which showed no significant difference between ALV and APV in the improvements in semen quality and postoperative pregnancy rate. Therefore, whether it is necessary to preserve the spermatic artery during varicocelectomy is still under debate.It has recently been reported that leptin, the product of the obese gene, also plays an important role in rodent and human reproductive system. Leptin and leptin receptors are present in testicular tissue, and their expressions, closely related to hypoxia, may also be related to varicocele. Leptin plays key role in sperm capacitation, which suggests that leptin may be associated with the epididymis. At present, no study report expression of leptin and leptin receptor in the epididymis, and relationship between leptin expression in the epididymis and varicocele.Because it would not be ethical to obtain epididymal biopsy specimens from normal or infertile men, it is essential to use animal testing in the study of varicocele. The rats have been the most popular varicocele model because of the low cost, the anatomic similarity between rats and humans. In view of this, we first established an experimental varicocele (EV) model in rats. And then, we investigated the effects on the epididymis during progression of varicocele in a rat model, and expression of leptin and its receptor in epididymis of varicocele-induced rats, and the effects of ALV and APV on the epididymis of varicocele-induced rats.Methods:1. Effects on the ipsilateral epididymis during progression of varicoceleSixty male Sprague-Dawley rats randomly divided into the following6groups:EV6,12, and18groups (12rats each), according to the varicocele duration (6,12, or18weeks), and control6,12, and18groups (8rats each). The EV was induced by partial ligation of the left renal vein. The rats in the control group underwent a sham operation that separated the spermatic vessels without ligation. Rats were executed in6,12,18weeks after surgery, respectively. The left epididymis of every group was weighted. HE stain was used to observe the microstructure changes in the epididymal tissue. Electron microscopy was used for observation of ultrastructural changes of the epididymis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method was used to detect the apoptosis of the epididymal epithelium. The contents of sialic acid were measured by spectrophotometry, and carnitine concentrations were measured by DTNB. 2. Effects of artery-ligating and artery-preserving varicocelectomy on the ipsilateral epididymis of varicocele-induced ratsFifty male Sprague-Dawley rats were divided at random into4groups:control group (n=8), EV without treatment (EV group, n=14), EV with ALV (ALV group, n=14), and EV with APV (APV group, n=14). The rats in the EV, ALV, and APV groups were induced to experimental varicocele models by partial ligation of the left renal vein. After6weeks of modeling, all rats underwent a second surgery. The varicocele-induced rats in the APV group underwent APV that ligated only the left internal spermatic vein. The varicocele-induced rats in the ALV group underwent ALV that totally ligated the left internal spermatic artery and vein. The rats in the EV group underwent a sham operation that separated the spermatic vessels without ligation. The rats in the control group underwent a sham operation each time. Only the left renal vein was separated without partial ligation at the first procedure, and the spermatic vessels were separated without ligation at the second. All rats were executed in6weeks after the second operation. The left epididymis of every group was weighted. HE stain was used to observe the microstructure changes in the epididymal tissue. Electron microscopy was used for observation of ultrastructural changes of the epididymis. TUNEL method was used to detect the apoptosis of the epididymal epithelium. The contents of sialic acid were measured by spectrophotometry, and carnitine concentrations were measured by DTNB.3. Expression of leptin and its receptor in the ipsilateral epididymis of varicocele-induced ratsForty male Sprague-Dawley rats randomly divided into the following4groups:EV4. and8groups (12rats each), according to the varicocele duration (4, or8weeks), and control4,8groups (8rats each). The EV was induced by partial ligation of the left renal vein. The rats in the control group underwent a sham operation that separated the spermatic vessels without ligation. Rats were executed in4,8weeks after surgery, respectively. Expression of leptin and its receptor mRNA and protein in rat epididymis is detected by real-time quantitative PCR and immunohistochemistry, respectively.Results:1. Effects on the ipsilateral epididymis during progression of varicoceleThere were microstructural and ultrastructural damage to different degree in the epididymis of the each EV group, which gradually became severe as the duration of varicocele gradually increased. Weight, sialic acid and carnitine concentration of the epididymidis in each EV group was significantly lower than that in the corresponding control group (P<0.05) and showed a significant decline as the duration of varicocele gradually increased. Apoptotic index (AT) of epididymal epithelium in each EV group was significantly higher than that in the corresponding control group (P<0.01) and showed a significant increase as the duration of varicocele gradually increased.2. Effects of artery-ligating and artery-preserving varicocelectomy on the ipsilateral epididymis of varicocele-induced ratsMicrostructural and ultrastructural abnormalities of the epididymis were observed in the EV group, and especially in the ALV group. Weight, sialic acid and carnitine concentration of the epididymis gradually decreased from the control group to the EV group then to the ALV group (P<0.01). However, the epithelial apoptotic index orderly increased for the control group, EV group, and ALV group (P<0.01). Furthermore, no significant difference was found between the control and APV groups for these parameters (P>0.05).3. Expression of leptin and its receptor in the ipsilateral epididymis of varicocele-induced ratsThe expression of leptin and leptin receptor of epididymis in EV4and EV8groups was significantly higher than those in control4and control8groups (P<0.01), respectively. There was no significant difference between EV4and EV8group in the expression of leptin and leptin receptor (P>0.05). The mRNA expression of leptin and leptin receptor of epididymis in EV4and EV8groups was significantly higher than those in control4and control8groups (P<0.01), respectively. There was no significant difference between EV4and EV8group in the mRNA expression of leptin and leptin receptor (P>0.05).Conclusions:We had successfully replicated experimental varicocele model in rats. Results of this study showed that experimental varicocele caused progressive impairment of structure and function of epididymis. Experimental varicocele induced an increase in the apoptosis of the epididymal epithelium, which then increased gradually with time. Therefore, varicocele is a progressive and not a static lesion. Varicocele caused lesions of the epididymis, and APV was able to repair the lesions, but ALV caused additional lesions. Therefore, it is necessary to preserve the spermatic artery during varicocelectomy. Expression of leptin and its receptor in epididymis of varicocele-induced rats significantly increased. Leptin may play an important role in the male infertility caused by varicocele. |
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