Varicocele is the pathological dilation of the venous pampiniform plexus in the spermatic cord (Naughton et al., 2001). Varicocele might be associated with male infertility. With diagnostic techniques such as scrotal ultrasonography and color Doppler imaging, varicocele has been demonstrated in up to 91% of subfertile human cases (Gonzalez et al., 1985; Resim et al., 1999) The pathophysiologies of the testicular damage in varicocele are not completely understood; however, gross testicular damages due to varicocele are well documented. The effect of the varicocele varies but may often result in a generalized failure of sperm production, characterized by abnormal sperm quality, ranging from oligozoospermia to complete nonobstructive azoospermia (Sandlow 2004). The varicocele affects not only the normal function and the fertilizing capacity of the sperm, but also the reproductive potential of the haploid male gamete (Redmon et al., 2002).
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Several studies have been carried out to give explanation for the altered spermatogenesis associated with varicocele (Jarow 2001; Salama et al., 2003; Sheehan et al., 2014). Varicoceles may have multiple mechanisms by which they negatively affect spermatogenesis. Impaired temperature regulation and ROS production could lead to DNA damage and progressive apoptosis of testis cells (Hamada et al., 2012; Gurdal et al., 2008; Wang et al., 2012; Saalu et al., 2013). Research at cellular and molecular level, while still in its infancy, may provide additional insights into the varicocele puzzle (Sheehan et al., 2014).
KIT signaling is widely known for its ability to promote cell survival, proliferation and differentiation. The receptor, KIT, and its ligand, KIT ligand (KITLG) also known as Stem cell factor, have been studied extensively (Rossi et al., 2000). The KIT receptor is a transmembrane protein with tyrosine kinase activity. It is an important member of type III receptor tyrosine kinase family. Binding of KITLG to KIT leads to activation of multiple pathways, including Src kinase, phospholipase C (PLC)-γ, Janus kinase (JAK)/Signal Transducers and Activators of Transcription (STAT), mitogen activated protein (MAP) kinase and phosphatidyl-inositol-3 (PI3)-kinase pathways (Roskoski 2005; Kazi et al., 2013). Dysfunction of KIT signaling results in an array of developmental defects in melanogenesis, hematopoiesis, gametogenesis and spermatogenesis (Reber et al., 2006; Liang et al., 2013; Roskoski 2005).
Several studies reported that KIT expression is regulated by various proinflammatory signals (Reber et al., 2006; Mithraprabhu & Loveland 2009). Differential effects are induced by some cytokines depending on the type of cell system. Cytokines are small soluble protein molecules with a crucial role in the regulation of inflammatory responses as well as in the induction of immunity to pathogens. Also, they transmit signals to surrounding cells for the regulation of cell growth and differentiation. In fact, they could trigger complex intracellular signaling events that regulate gene expression required for the cellular response (Callard et al., 1999). Among cytokines, interleukin 1 (IL-1), tumour necrosis factor (TNF), IL-4, granulocyte–macrophage CSF, fibroblast growth factor (FGF) and IL-10 have been reported to change KIT synthesis (Ronnstrand 2004; Sahin et al., 2006).
The heat shock proteins (HSPs) are well known as a family of endogenous, protective proteins. These molecules are located in the cytoplasm and nucleus (e.g. HSP70 and HSP90) to maintain normal cellular function. Signals such as reactive oxygen species (ROS), cytotoxic lysosomal enzymes and cytoskeletal alterations could activate HSP expression in the cell. HSPs suppress pro-inflammatory cytokines, reduce oxidative bursts, repair ion channels, protect from the toxic effect of nitric oxide, modulate immune-mediated injuries and prevent apoptosis (Afiyani et al., 2014 ; Ferlin et al., 2010). In the present study we aimed to investigate histopathologic changes in the varicocele testis, and whether non-varicocele testis is also influenced. Variation of HSP90, HSP70, IL-4, TNF, KITLG and KIT-Receptor gene expression and thus their contribution to possible infertility caused by varicocele is discussed.
Materials and Methods
Six adult male crossbred dogs (2–4 years old) with normal quality and approximately 30kg weight were used in this experiment. They were cared for Faculty of Shahrekord Veterinary Medicine and housed in pens with ample runes. Commercial dry dog food was provided twice a day, and the dogs were given access to water ad libitum. All animals were treated by anti-parasitic drugs (Mebendozole, 22 mg/kg, orally for 5 days and Praziquantel, 5 mg/kg, orally once). All animals were maintained according to the guidelines of Animal Care and Use Committee of the Faculty of Shahrekord Veterinary Medicine.
Induction of experimental varicocele in dog
To induce experimental varicocele by surgery, inguinal canal region of dogs was prepared aseptically for operation. Animals sedated with 2% acepromazine (0.2 mg/kg) and anesthesia was induced by ketamine and then maintained with 2% halothane. Dorsal recumbency position was chosen, then a skin incision was made in the skin of inguinal canal region. Spermatic cord was exposed and tunica vaginalis was incised to expose pampiniform plexus. To make a partial occlusion and congestion in pampiniform plexus, a piece of silicone tube (INWAY® Suprapubic Catheter, pfm Medical Co., Germany) as long as 1 cm was cut and longitudinally incised and was opened, then proximal part of pampiniform plexus was cited in it and to prevent the movement of the tube, three interrupted sutures was applied by 2.0 absorbable suture material and the skin was sutured by non-absorbable suture material. Dogs were kept for 2 weeks and the diameter of the testes examined and all data were recorded. At 15th postoperative day the animals were anesthetized, then spermatic cord was incised and 2 milliliters of iohexol contrast media (iodixanol, Visipaque 320; GE Healthcare, Canada) was injected in the testicular vein and radiographs was taken immediately in the injected area. This venography was done to confirm congestion and dilation of the venous pampiniform plexus in the spermatic cord of varicocele-induced testis. Finally, non-varicocele (left) and varicocele-induced (right) testes were dissected by castration of dogs. Half of each testis were immediately frozen in liquid nitrogen and stored at -70°C for subsequent RNA analysis. Another half was fixed in formalin solution followed by embedding in glycol methacrylate for histopathologic evaluation.
Histopathologic evaluation of induced varicocele model was carried out by Hematoxylin and Eosin staining in the non-varicocele and varicocele-induced testes. To evaluate spermatogenic activity, the spermatogenesis was categorized by using the Johnson’s score (Dohle et al., 2005). According to the following criteria, a grade from 1 to 10 for each tubule cross section was provided: 1 = no germ cells and no Sertoli cells present; 2 = no germ cells but only Sertoli cells present; 3 = only spermatogonia present; 4 = only a few spermatocytes present; 5 = no spermatozoa or spermatids but many spermatocytes present; 6 = only a few spermatids present; 7 = no spermatozoa but many spermatids present; 8 = only a few spermatozoa present; 9 = many spermatozoa present and disorganized spermatogenesis and 10 = complete spermatogenesis and perfect tubules.
RNA extraction and cDNA synthesis
Total RNA from left (non-varicocele) and right (varicocele) testes was extracted using Rimazol reagent (Sinaclon Bioscience, Tehran, Iran). Homogenized (Sinaclon Bioscience, Karaj, Iran); the RNA was then measured and qualified by spectrophotometry. Only RNA with an absorbance ratio (A260/280) of ≥1.9 was used for synthesis of cDNA (Muller, 2008). Gel agarose (2%) electrophoresis (stained with ethidium bromide) was applied to analyze RNA. The cDNA was produced from total RNA using M-MLV reverse transcriptase (Sinaclon Bioscience, Karaj, Iran) according to previous study (Hassanpour et al., 2009). To denature residual RNA in the cDNA mix, temperature of 75°C for 15 min was provided and then cDNA was stored at –20°C.
Quantitative real time PCR Analysis
The levels of HSP90, HSP70, IL-4, TNF, KITLG, KIT-Receptor and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) transcripts were determined by real-time reverse transcriptase (RT)-PCR using Eva-Green chemistry (Sinaclon Bioscience, Karaj, Iran). To normalise the input load of cDNA between samples, GAPDH was used as an endogenous standard. Specific primers of IL-4, TNF, KITLG, KIT-Receptor and GAPDH were designed based on mRNA sequences with Primer-Blast (www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC = BlastHome). Primers are listed in Supplementary Table S1. PCRs were carried out in a real-time PCR cycler (Rotor Gene Q 6000, Qiagen, USA) in three replicates for each sample of testis. 1 µl cDNA was added to the Titan Hot Taq Eva-Green Ready Mix (Bioatlas, Tartu, Estonia) (0.5 µM of each specific primer and 4 µl of Titan Hot Taq Eva-Green Ready Mix) in a total volume of 20 µl. The thermal profile was 95°C for 15 min, 35 cycles of 94°C for 40 s, 60°C for 35 s and 72°C for 32 s. At the end of each stage, the measurement of fluorescence emission was measured and applied for quantitative objectives. Gene expression data were normalized to GAPDH. Data were analyzed using LinRegPCR software version 2012.0 (Amsterdam, Netherland), to give the threshold cycle number and reaction efficiency (Ruijter et al., 2009). Relative transcript levels and fold changes in transcript abundance were calculated using efficiency adjusted Pfaffl methodology (Dorak 2006).
Data are represented as mean ± SE. Comparisons were made by using sample t-test between each non-varicocele and varicocele-induced testis. For the results that parametric test assumptions were not available for some variables, so the comparisons of two dependent groups were performed by Wilcoxon test. All statistical analyses were performed with the Statistical Package for Social Sciences software version 17 (SPSS Inc., Chicago, IL, USA). P values less than 0.05 were considered significant.
As right testicular venogram shows in figure 1, dilatation and toruosity of veins of the pampiniform plexus secondary to retrograde flow are observable.
Gross pathologic changes of varicocele-induced testes were congestion, edema and enlargement. Microscopic changes were evaluated after Hematoxylin and Eosin staining of different sections of testes and were compared between non-varicocele and varicocele-induced testes. The histopathologic changes were consist of testicular degeneration as spermatogenic arrest at the spermatocyte stage and formation of multinucleated spermatid as a result of failure of spermatid to separate (Fig. 2A); Coagulative necrosis in the seminiferous epithelium and the presence of eosinophilic material in the seminiferous tubules and hemorrhage in the interstitium (Fig. 2B); Testicular atrophy as complete absence of spermatogenesis but the sertoli cells were normal and shrinkage of some seminiferous tubules (Fig. 2C); Epididimyal atrophy as prominent dilation of epididimyal tubules with pressure atrophy of their columnar epithelium (Fig. 2D); Severe congestion and dilation of the spermatic cord vessels with inter vascular fibrosis (Fig. 2E); Epididymal squamous metaplasia and intertubular fibrosis (Fig. 2F).
Johnson’s score in the varicocele-induced and non-varicocele testes was 4 (1-8) and 9.6 (9-10) respectively. This parameter between non-varicocele and varicocele-induced testes was significant (P < 0.05).
HSP90, HSP70, KITLG, KIT-Receptor, TNF and IL-4 genes expression
Expression of HSP90, HSP70, KITLG, KIT-Receptor, TNF and IL-4 genes was studied using quantitative RT-PCR in the non-varicocele and varicocele-induced testes. Real time PCR results are shown in Table 1. Expression of GAPDH was detected in all testes and was not different in non-varicocele and varicocele-induced testes. Expression of HSP90, KITLG, KIT-receptor and TNF genes in varicocele-induced testes was significantly (P < 0.05) lower than non-varicocele testes that was 0.62, 0.83, 0.71 and 0.76 folds respectively while expression of HSP70 gene was significantly higher 2.9 fold in varicocele-induced testes than non-varicocele testes. Expression of IL-4 gene did not changed in varicocele-induced testes compared to control (P > 0.05).
This research was designed to induce an experimental varicocele model by a simple surgery in dog with minimum invasion and to investigate the expression of many genes involved in the varicocele-induced infertility. There are many limitations in the study of varicocele pathophysiology in human, and most of the studies in this aspect are non-invasive. On the other hand, there are alternative factors such as the status of the varicocele, patient age and level of fertility in the subject population. Thus, these parameters limited research in the human varicocele. Because of these limitations, induction of varicocele has been provided in several species as animal models (Salama et al., 2003). The most widely used animal model of varicocele involves partially occluding the left renal vein medial to the insertion of the left internal spermatic vein. Increased venous pressure proximal to the partial occlusion creates increased pressure in the left internal spermatic vein, resulting in dilatation of the left internal spermatic vein and pampiniform venous plexus. In all models, a midline abdominal incision must be made from xyphoid to pubis to expose the renal and pelvic vasculature (Najari et al., 2014). In the present study, the approach of surgery was only inguinal canal region and in contrast to other studies, abdominal incision was not made that it could be an advantage of this surgery. This route was also preferred by Animal Care Committee and approved. The histopthologic and venographic evaluations of manipulated testes, confirmed the induction of varicocele and subsequent infertility (caused by azoospermia), although the non-varicocele testis is slightly influenced as Jonson score showed (9-10). Then, it is possible that a transient inflammation occurred in the non-varicocele testis.
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Some studies suggest a relationship between cytokine levels and subfertility. It has been found that concentrations of interleukins such as IL-1, IL-6, TNF were significantly increased in semen of infertile patients (Moretti et al., 2014). In varicocele, it has been also suggested that expression of IL-1α and IL-1β as proinï¬‚ammatory cytokines were increased. These cytokines in varicocele shifted the balance in favor of inï¬‚ammatory and immune responses and causes harmful effects in testis tissue, which may cause male infertility. In the present study, the expression of IL-4 and TNF were evaluated in varicocele. The expression of TNF was decreased while IL-4 was increased (in a nonsignificant manner). It has been confirmed that IL-4 and TNF play as roles of anti-inflammatory and pro-inflammatory cytokines respectively (Chowdhury et al., 2006). The previous studies indicated that TNF level or TNF-related apoptosis-inducing ligand did not change in the varicocele (Moretti et al., 2014), although expression of receptors of TNF-related apoptosis-inducing ligand were different (Celik et al., 2013). However, these reports evaluated TNF or its receptors at levels of protein by ELISA determination, immunohistochemical and Western blotting techniques, while in our study, gene expression was evaluated by real time-PCR. Apparently, post-transcriptional and post-translational factors affecting gene, protein and activity of TNF, react at different pathways in varicocele. Another possibility suggests that the levels and subsequently effects of many cytokines would likely alter with duration of the varicocele. These changes could be partially related to interaction of anti-inflammatory (e.g., IL-4) and pro-inflammatory (e.g., TNF) cytokines in time-dependent. It must be noted that non-varicocele testis had probably sparse inflammation which could increase pro-inflammatory cytokines such as TNF.
In the current study, the expression of KITLG and KIT-Receptor were evaluated. The results for the first time demonstrated a reduction of these transcripts in varicocele-induced testis. Based on the different reports, KITLG/KIT-receptor appeared to represent one of the key regulators of testicular formation, development and function. Impairment of this system involved in the gonadal pathologies, including testicular developmental defects, infertility and testicular cancers. A reduction in the expression of KITLG/KIT-receptor has been evidenced in oligozoospermia / azoospermia associated with an increase in the germ cell apoptosis process (Mauduit et al., 1999; Zhang et al., 2011). Together, reduction in the expression of KITLG/KIT-receptor as reported in the present study could be critical factors in the infertility due to varicocele. It has been provided evidence that KIT expression is influenced by various cytokines in the inflammation depending on the model or type of cell system used for investigation (Mithraprabhu & Loveland 2009). However, in the present study, it appears that reduction of KITLG/KIT-receptor transcript was not mainly influenced by variation in expression of mentioned cytokines (i.e., TNF and IL-4) in the experimental varicocele of dogs.
It has been confirmed that the HSP family as a molecular chaperone is present in spermatocytes during meiosis, participating as an element of the synaptonemal complex, and during the maturation stage of spermiogenesis. Several functions have been postulated for HSP family, including folding, transport, and protein assembly in the cytoplasm, mitochondria, and endoplasmic reticulum (Afiyani et al., 2014). We observed a significant increase in HSP70 mRNA abundance in the testis with varicocele. In agreement with these results, it has been reported an increase of HSP protein and heat-shock factor expression in sperm from oligozoospermic and varicocele individuals (Ferlin et al., 2010). This response probably is an attempt to repair spermatogenic and germ-cell damage due to heat stress. Apparently, the gene expression of all HSP members could not increase during damage of varicocele to protect the testis cells as we observed in the result of HSP90 mRNA or Lima et al. (2006) found in the evaluation of HSP2A mRNA. Probably, in a time stage of varicocele, gene expression apparatus for some of HSP members would be the victim of damage itself. On the other hand, this situation could exacerbate the damage of varicocele in a positive feedback.
In conclusion, our data show that partial occlusion of proximal part of pampiniform plexus could induce varicocele in the testis of dogs. The gene expression of HSP90, TNF, KITLG and KIT-receptor were considerably decreased in varicocele-induced testes while HSP70 was increased. IL-4 did not changed. It is probably that these changes could involve in the pathophysiology of varicocele and related subfertility.
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