Comparative Expression Analysis of HSP70, HSP90,
IL-4, TNF, KITLG and KIT-receptor Gene between
Varicocele-Induced and Non-Varicocele Testes of Dog
This study was designed to create an experimental varicocele model by a
simple surgical procedure in dog with minimum invasion and to investigate the effect of
varicocele-induced infertility on the expression of six related genes (
Materials and Methods
In this experimental study, the proximal part of the pampini-form plexus of dog testes was partially occluded without abdominal incision which was confirmed by venographic examination. To evaluate varicocele in its acute form, dogs were castrated after 15 days and testes were dissected. Histopathologic evaluation was undertaken and the relative expression of the six genes was assessed by quantitative realtime polymerase chain reaction (PCR).
Microscopic changes showed tubule degeneration. The Johnson score was significantly decreased in the varicocele testes when compared with non-varicocele testes.
We conclude that partial occlusion of the proximal part of the pampiniform plexus induces varicocele in the testis of dog. Differential expression of the mentioned genes may be responsible for the pathophysiology of varicocele and related subfertility.
Varicocele is a pathologic dilation of the venous pampiniform plexus in the spermatic cord (1) and is thought to be associated with male infertility. Diagnostic techniques such as scrotal ultrasonography and color Doppler imaging have demonstrated that varicocele may be the cause of 91% of subfertile human cases (2, 3). The pathophysiology of testicular damage in varicocele is not completely understood, however, histopathologic testicular damages due to varicocele are well documented. The effect of varicocele varies, but may often result in a generalized failure of sperm production (from oligozoospermia to complete nonobstructive azoospermia) (4). Varicocele not only affects the normal function and the fertilizing capacity of the sperm, but it also affects the reproductive potential of the haploid male gamete (5). Several studies have suggested varicocele-mediated mechanisms to explain impaired spermatogenesis (6-8). Impaired temperature regulation and reactive oxygen species (ROS) production may lead to DNA damage and progressive apoptosis of testicular cells (9-12). Research at cellular and molecular level, while still in its infancy, may provide additional insights into the varicocele puzzle (8).
The signaling of KIT is well-known for its ability to potentiate cell survival, proliferation and differentiation. The KIT receptor and its ligand, KIT ligand (KITLG), have been widely studied (13). The KIT receptor is a transmembrane protein with tyrosine kinase activity and a member of the type III receptor tyrosine kinase family. Binding of KITLG to KIT leads to the 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 (14, 15). Dysfunction of KIT signaling thus results in an array of developmental defects in melanogenesis, hematopoiesis, gametogenesis and spermatogenesis (14, 16, 17).
Cytokines are small soluble proteins with a crucial role in the regulation of inflammatory responses. Also, they transmit signals to surrounding cells for the regulation of cell growth and differentiation. They could trigger complex intracellular signaling events that regulate gene expression required for the cellular response (18). A number of studies have reported that KIT expression is regulated by various proinflammatory signals (16, 19). Differential effects are induced by some cytokines depending on the type of the cell system. Among cytokines, interleukin 1 (IL-1), tumour necrosis factor (TNF), IL-4, granulocyte- macrophage colony stimulating factor fibroblast growth factor (FGF) and IL-10 have been reported to change KIT synthesis (20, 21). However, the effects of cytokines and KIT signaling in the inflammatory process of varicocele are predicatable.
The heat shock proteins (HSPs), a family of endogenous,
protective proteins, are located in the
cytoplasm and nucleus (e.g. HSP70 and HSP90
respectively) to maintain normal cellular function.
ROS, cytotoxic lysosomal enzymes and cytoskeletal
alterations are able to activate HSP expression.
HSPs, in turn, suppress pro-inflammatory cytokines,
reduce oxidative bursts, repair ion channels, protect
against the toxic effect of nitric oxide, modulate
immune-mediated injuries and prevent apoptosis.
The function of HSP and its dependant factors in
inflammation provides a basis for its possible involvement
in the pathophysiology of varicocele.
Indeed, the presence of many HSPs in varicocele
has been confirmed by previous studies (22, 23). In
the present study, we therefore aimed to investigate
histopathologic changes in the varicocele testis and
whether same changes can be identified in non-varicocele
testis. Variation in the expression of
Materials and Methods
In this experimental study, six adult male crossbred dogs (2-4 years old) with normal quality and approximately 30 kg weight were used in this experiment. They were cared for in the Faculty of Shahrekord Veterinary Medicine and housed in pens with ample run. Commercial food was provided twice a day and the dogs had free access to water. Anti-parasitic drugs were administrated to all dogs (mebendozole, 22 mg/kg, orally for 6 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.
Experimental varicocele induction in dog
To induce experimental varicocele by surgery, the inguinal canal region of dogs was prepared aseptically for operation. Dogs were sedated with 2% acepromazine (0.2 mg/kg) and anesthetized by ketamine and then maintained with 2% halothane. An incision was made in the skin of the inguinal canal region while animals were in the dorsal recumbent position. Spermatic cord was exposed and tunica vaginalis was incised to expose the pampiniform plexus. To make a partial occlusion and congestion in the pampiniform plexus, a piece of silicone tube (INWAY® Suprapubic Catheter, pfm Medical Co., Germany) of 1 cm long was longitudinally incised and opened, and then proximal part of the pampiniform plexus was cited in it. To prevent the movement of the tube, three interrupted sutures were 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 were examined and recorded. On the 15th postoperative day, the animals were anesthetized, their 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 were taken immediately from 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 varicoceleinduced (right) testes were dissected by castration of dogs. This was undertaken after two weeks to evaluate varicocele in its acute form (short time) as observed in many adult men (24). Half of each testis was immediately frozen in liquid nitrogen and stored at -70°C for subsequent RNA and expression analyses. Another half was fixed in formalin solution followed by embedding in glycol methacrylate for histopathologic evaluation.
Histopathologic evaluation of the induced varicocele model was carried out by hematoxylin and eosin staining in the non-varicocele and varicoceleinduced testes. To examine spermatogenic activity, spermatogenesis was categorized by using the Johnson score (25). A grade from 1 to 10 for each tubule cross section was provided according to the following criteria: i. No germ cells and no Sertoli cells present, ii. No germ cells but only Sertoli cells present, iii. Only spermatogonia present, iv. Only a few spermatocytes present, v. No spermatozoa or spermatids but many spermatocytes present, vi. Only a few spermatids present, vii. No spermatozoa but many spermatids present, viii. Only a few spermatozoa present, ix. Many spermatozoa present and disorganized spermatogenesis, and x. Complete spermatogenesis and perfect tubules.
RNA extraction and cDNA synthesis
Total RNA from left (non-varicocele) and right (varicocele) testes was extracted using the Rimazol reagent (Sinaclon Bioscience, Iran) and then homogenized (Sinaclon Bioscience, Iran). The quantity of extracted RNA was then measured by spectrophotometry. Only RNA samples with an absorbance ratio (A260/280) of ≥1.9 was used for synthesis of cDNA (26). Gel agarose (2%) electrophoresis (stained with ethidium bromide) was applied to analyze the quality of extracted RNA. The cDNA was produced from total RNA using M-MLV reverse transcriptase (Sinaclon Bioscience, Iran) according to the protocol of a previous study (27). To denature residual RNA in the cDNA mix, the sample was heated at 75°C for 15 minutes and subsequently stored at -20°C.
Quantitative real time polymerase chain reaction analysis
The expression levels of
Data are represented as mean ± SE. Differential expression was assessed statistically by using paired t test between the non-varicocele and varicoceleinduced testis pair. When the assumptions behind a parametric test were violated, comparisons were made by the Wilcoxon test. All statistical analyses were performed with the Statistical Package for Social Sciences software version 17 (SPSS Inc., Chicago, IL, USA). When paired t test was done, differences between paired values were consistent and P<0.05 were considered statistically significant.
As observed in the right testicular venogram (Fig .1,), dilatation and toruosity of veins of the pampiniform plexus, secondary to retrograde flow, were apparent.
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 then compared between non-varicocele and varicocele-induced testes. The histopathologic changes consisted of testicular degeneration as well as spermatogenic arrest at the spermatocyte stage and formation of multinucleated spermatid due to failure in spermatid separation (Fig.2A,). In addition, coagulative necrosis in the seminiferous epithelium and the presence of eosinophilic material in the seminiferous tubules along with hemorrhage in the interstitium were induced (Fig.2B,). Testicular atrophy was also present in the form of complete absence of spermatogenesis (but with normla Sertoli cells) and shrinkage of some seminiferous tubules (Fig.2C,). Furthermore, epididimyal atrophy as a prominent dilation of epididimyal tubules with pressure atrophy of their columnar epithelia (Fig.2D,), severe congestion and dilation of the spermatic cord vessels with inter-vascular fibrosis (Fig.2E,), and epididymal squamous metaplasia and intertubular fibrosis (Fig.2F,) were also among the induced histopathologic changes.
|Target||Sequencing primer (5ˊ-3ˊ)||PCR product (bp)||Accession no.|
|Gene||Relative gene expression||Ratio (varicocele/non-varicocele)||Pooled SD||P value|
|Non-varicocele testis||Varicocele testis|
Expression analysis of the six related genes
Expression level changes of all six genes were
quantified using real time quantitative PCR (RTqPCR)
and are shown in Table 2. Expression level
of GAPDH was not different in non-varicocele and
varicocele-induced testes. Expression of
This study was designed to induce an experimental varicocele model by a simple surgical procedure in dog with minimum invasion and to also investigate the expression of a number of genes involved in varicocele-induced infertility. There are many limitations in the study of varicocele pathophysiology in humans with most studies being non-invasive. In addition, there are other factors such as the status of the varicocele, patient age and level of fertility in the subject population that further hinder the identification of its pathologic basis, thus limiting research on varicocele in humans. Because of these limitations, varicocele has been induced in several species as animal models (7). The induction of varicocele in most animal models involves partially occluding the left renal vein medial near to the kidney. Increased venous pressure proximal to the partial occlusion creates the increased pressure in the left internal spermatic vein, thus resulting in dilatation of the left internal spermatic vein and the pampiniform venous plexus. In all models, a midline abdominal incision must be made from xyphoid to pubis to expose the renal and pelvic vasculature (30). In the present study, the surgical approach was only in the inguinal canal region and contrary to other studies abdominal incision was not made, rendering this method more advantageous. This route was also preferred by the Animal Care Committee and was therefore approved. The histopathologic and venographic evaluations of manipulated testes confirmed the induction of varicocele and subsequent infertility (caused by azoospermia), while the nonvaricocele testis was shown to be slightly influenced as the Jonson score showed values ranging 9-10. This may be due to a transient inflammation in the non-varicocele testis.
Some studies have suggested a relationship between
cytokine levels and subfertility. It has been
found that concentrations of interleukins such as
IL-1, IL-6 and TNF were significantly increased
in semen of infertile patients (31). In varicocele,
it has been also suggested that expression of IL-
1α and IL-1β, as proinﬂammatory cytokines, were
increased. These cytokines in varicocele shift the
balance in favor of inﬂammation and immune responses
and therefore result in harmful effects in
testicular tissue, which may lead to male infertility.
In the present study, the expression of IL-4
Another possibility is that the levels and the subsequent effects of many cytokines alters with varicocele duration. These changes could be to some extent related to the interaction of anti-inflammatory (e.g., IL-4) and pro-inflammatory (e.g., TNF) cytokines in a time-dependent manner. It must be, however, noted that non-varicocele testes probably had a slight inflammation, resulting in an increase in pro-inflammatory cytokines such as TNF.
In the current study, the expression of
The HSPs are present in spermatocytes during
meiosis, participating as an element of the synaptonemal
complex, and during the maturation stage
of spermiogenesis. We observed a significant increase
Our data show that partial occlusion of the
proximal part of the pampiniform plexus induces
varicocele in the testis of dog. The expression of
This work was financially supported by the Applied Research Centre, Vice Chancellor for Research of Shahrekord University. All authors declare that they have no conflicts of interest.