The Egyptian Journal of Histology:
A histological study of ipsilateral testis after experimentally induced varicocele in albino rats and the role of L-carnitine supplementation
Gawish, Magdy F.; Azmy, Abeer M.; Abd El-Haleem, Manal Reda
Department of Histology and Cell Biology, Faculty of Medicine, Zagazig University, Egypt
Correspondence to Manal Reda Abd El-Haleem, Department of Histology and Cell Biology, Faculty of Medicine, Zagazig University, Egypt Tel: +0126422694; fax: 002/0552310294 e-mail: firstname.lastname@example.org
Received April 20, 2010
Accepted January 20, 2011
Introduction: Varicocele pathophysiology and its role in male infertility are still unclear. Varicocelectomy is not an effective treatment in such cases as it failed to restore fertility after surgery in many patients.
Aims of the study: The aims of this study were to investigate the histological alterations that might occur in the structure of the ipsilateral testis after experimental varicocele, to determine whether this effect was time dependent, and to evaluate the possible role of l-carnitine on the varicocele.
Materials and methods: Thirty-six young adult albino rats were used. They were equally divided into three groups. Group 1 was the control. Group 2 was the experimentally induced varicocele (EV) that was subdivided into two subgroups, EV6 and EV18, according to varicocele duration. Group 3 (EV-carnitine) was the experimentally induced varicocele left for 18 weeks followed by an intraperitoneal injection of 200 mg/kg L-carnitine (three times/week) for another successive 8 weeks. At the end of the experiment, the ipsilateral testes were extirpated and processed for light and electron microscope examinations. Epithelial height was estimated morphometrically and statistically analyzed.
Results: Testes of EV6 showed many distorted seminiferous tubules with irregular outlines, wide lumina, and disorganized epithelium including separation, sloughing, and multinucleated cells. Some areas of interstitium were wide. Acidophilic hyaline material was present in most of the interstitial spaces. Most of the tubules of EV18 were markedly distorted and were mostly lined by sertoli cells with a few spermatogenic cells. The tubular basement membrane of EV6 was relatively thick, irregular, and highly infolded and these changes were extremely obvious in EV18. Myoid cells appeared with irregular heterochromatic nuclei in EV18. Almost all sperm mid pieces in EV6 were markedly affected and no sperms were detected in most of the seminiferous tubules of EV18. Leydig cells in both subgroups showed variable-size electron-dense granules and cytoplasmic processes, which were more obvious in EV18. EV-carnitine nearly regained the normal architecture but a few tubules had a disorganized epithelium, a few affected sperms, and acidophilic hyaline material between some tubules. Leydig cells contained numerous mitochondria, a few variable-size electron-dense granules, a few lipid droplets, and no cytoplasmic processes. Estimation of epithelial height, which was statistically analyzed, confirmed the results.
Conclusion: Varicocele led to a deleterious effect on the ipsilateral testis that increased progressively with time. L-Carnitine supplementation improved the structure of testis of long-duration varicocele.
Varicocele is a pathological dilatation of the venous pampiniform plexus of the spermatic cord that occurs more frequently on the left side. Clinical varicocele is obscured in 10–20% of the male population and in 19–41% of men presenting for infertility investigations .
Varicoceles are more common on the left side where the left spermatic vein enters perpendicular to the left renal vein. The right side has some protective effect because the right spermatic vein enters obliquely into the inferior vena cava. The retrograde flow into the internal spermatic vein results in dilatation and tortousity of the pampiniform plexus .
The pathophysiology of varicocele is difficult to study in humans because their study design must not be invasive. Therefore, availability of appropriate numbers of both control and varicocele patients of desired ages and durations of varicocele is limited. Hence, animal models of varicocele were developed .
To create a varicocele model in animals [experimentally induced varicocele(EV)], ligation of the left renal vein is carried out leading to dilatation of the internal spermatic vein. This will alter certain measurable parameters in testicular physiology, blood flow and temperature [4–7]. The most commonly used animal model is the rat, as its pathophysiological processes after EV are similar to those in humans [3,8].
Surgical correction (varicocelectomy) was the most popular treatment for varicocele. This correction improved semen parameters in 50–80% of patients and consequently pregnancy rates by 31–71%. This indicated that varicocelectomy failed to restore fertility after surgery in some patients . In addition, recurrence after varicocelectomy was common . Therefore, some investigators considered that varicocelectomy was not an effective treatment for infertility .
Carnitine is a nonessential amino acid. Approximately 75% of the carnitine that is present in humans is derived from diet. It is considered as an antioxidant that removes extracellular toxic acetyl-coenzyme A that is responsible for mitochondrial reactive oxygen species (ROS) [11,12]. Carnitine showed a wide range of biological activities including antiapoptotic  and anti-inflammatory properties . In addition, L-carnitine treatment had protective effects in experimental testicular torsion . Earlier investigators found that carnitine concentration was reduced in seminal fluid of infertile patients . Others used it in an attempt to treat such cases [17,18].
Therefore, the aims of this study was to investigate the histological alterations that might occur in the structure of the ipsilateral testis after experimental varicocele, to determine whether this effect was time dependent, and to evaluate the possible role of L-carnitine on the varicocele.
Materials and methods
Thirty-six healthy young adult albino rats (aged 6–7 weeks old) weighing 170±10 g were used in this study. They were housed in stainless steel cages and maintained at room temperature. They were allowed water ad libitum and were fed a standard diet. They were equally divided into three groups: 1, 2, and 3. Group I was the control (C) that was subdivided into two subgroups, C6 and C18, according to the duration of weeks they were left. Group 2 was the EV that was subdivided into two subgroups, EV6 and EV18, according to varicocele duration 6 and 18 weeks, respectively . Group 3 was the EV left for 18 weeks and followed by intraperitoneal injection of L-carnitine (200 mg/kg, three times per week)  for another successive 8 weeks  (EV-carnitine).
Rats in which experimental varicocele was induced were anaesthetized with an intraperitoneal injection of pentobarbital (40 mg/kg). A midline incision was used to expose the left renal vein. A 3–0 silk suture was tied around both the left renal vein and a 0.5 mm-diameter syringe needle at the point medial to the insertion of the adrenal and spermatic veins into the renal vein. The needle was removed and the vein was allowed to expand within the bounds of the ligature. This reduced the renal vein diameter by approximately one-half. The abdominal incisions were repaired. After 6 and 18 weeks of varicocele induction, the rats were anaesthetized and the diameter of the left internal spermatic vein was checked to ensure varicocele development. Dilatation of the vein more than double of that in the control was considered a varicocele. The rats that did not have a varicocele were excluded . The control group underwent similar procedures, but without occlusion of the left renal vein (sham operation).
After different periods of varicocele induction in the EV group (i.e. 6–18), the left testes with successful varicocele were extirpated and compared with those of the animals in the corresponding control group. In rats of the EV-carnitine group, the left testes were extirpated after 26 weeks (i.e 18+8 weeks). A longitudinal incision was made in each testis to process one-half for light microscope and the other half for electron microscope examinations.
Specimens for light microscope examination were fixed in 10% neutral formol saline for 24 h and processed to prepare 5 μm-thick paraffin sections for hematoxylin and eosin staining . Specimens for the electron microscope were immediately fixed in 2.5% glutaraldehyde buffered with 0.1 mol/l phosphate buffer at a pH of 7.4 for 2 h and then postfixed in 1% osmium tetroxide in the same buffer for 1 h. They were processed to prepare semithin sections and then ultrathin sections. Semithin sections (1 μm thick) were stained with 1% toluidine blue for light microscope examination [22,23]. Ultrathin sections were obtained using a Leica ultracut UCT (Germany) and stained with uranyl acetate and lead citrate . They were examined with a JEOL JEM 1010 electron microscope in the Electron Microscope Research Laboratory of the Histology and Cell Biology Department, Faculty of Medicine, Zagazig University, and with a JEOL JEM 1200 EXII electron microscope at the Faculty of Science, Ain Shams University, Egypt.
The image analyzer computer system Leica Qwin 500 in the Histology and Cell Biology Department, Faculty of Medicine, Cairo University, was used to evaluate the epithelial height of seminiferous tubules (STs) in micrometer using the interactive measure menu. The procedure was performed using hematoxylin and eosin-stained sections at a total magnification of ×400 using 50 STs from five sections of each rat in randomly chosen six rats of each group.
Data for all groups were expressed as mean±standard deviation (X¯±SD). The data obtained from the image analyzer were subjected to SPSS program version 15 (“http://www.spss.com”, Chicago, USA). Statistical analysis using a one-way analysis of variance test for comparison between different groups was carried out. The post-hoc test (least significant difference) was used to determine the statistical difference between groups. P values less than 0.05, less than 0.001 and more than 0.05 were considered significant, highly significant, and nonsignificant, respectively.
All 24 rats undergoing partial ligation of the left renal vein showed the objective dilatation of the left internal spermatic vein. No rats in the control group showed a similar dilatation of the same vein.
Group 1 (control group)
Light microscope examination of the sections of the testes of the control (C6 and C18) albino rats showed that testicular parenchyma consisted of densely packed STs with little interstitium (Fig. 1). The STs were lined with stratified germinal epithelium formed of spermatogonia and primary spermatocytes. Sertoli cells with their large pale nuclei were observed between these cells. The tubules were ensheathed by a layer of flat myoid cells and their lumina contained sperms. The interstitium contained clusters of interstitial cells and blood vessels (Fig. 2). Electron microscope examination of the ultrathin sections of the testes of the same groups showed that spermatogonia were observed resting on the basement membrane. Their nuclei were rounded and their cytoplasm contained mitochondria. Primary spermatocytes had large euchromatic nuclei with synaptonemal complexes and a thin rim of cytoplasm. Spermatids had euchromatic nuclei and their cytoplasm contained peripherally located mitochondria. Numerous spermatids had acrosomal cap. Sertoli cells were observed close to the basement membrane with ovoid euchromatic nuclei. Their cytoplasm contained mitochondria and electron-dense granules of variable sizes and densities (Fig. 3). Tight junctions (blood–testis barrier) were observed between the adjacent sertoli cells (Fig. 4). Transverse sections in different parts of sperms showed that mid, principal, and end pieces had a central axoneme formed of nine doublets of microtubules with two central singlets. In the mid pieces, this axoneme was surrounded by nine dense bundles of fibrous and mitochondrial sheaths. In the principal pieces, the axoneme was surrounded by the fibrous sheath only (Fig. 5). Leydig cells had irregular nuclei with a thin rim of peripheral heterochromatin. Their cytoplasm contained numerous mitochondria with tubular cristae and a few lipid droplets (Fig. 6).
Group 2 (experimentally induced varicocele)
Light microscope examination of sections of the testes of subgroup EV6 showed that testicular parenchyma was formed of many distorted STs with irregular outlines, disorganized epithelium, and wide lumina. Some areas of interstitium were wide. Acidophilic hyaline material was present in most of the interstitial spaces. Congested blood vessels were observed (Fig. 7). The disorganized epithelium showed separation (Fig. 8a), sloughing (Fig. 8b), and presence of multinucleated cells (Fig. 8c). Electron microscope examination of the ultrathin sections of the testes of the same subgroup showed that the STs were enveloped by a relatively thick, irregular, highly infolded basement membrane. Their lining epithelial cells contained small vacuoles with intercellular separations (Fig. 9a and b). Almost all transverse sections in the mid pieces of sperms showed markedly affected disarranged axoneme, fibrous, and mitochondrial sheaths (Fig. 10). Leydig cells with lipid droplets, variable-size electron-dense granules, and multiple cytoplasmic processes were observed (Fig. 11).
Light microscope examination of the sections of the testes of subgroup EV18 showed that most of the STs were markedly distorted with very wide lumina, no sperms, and increased reduction in the thickness of their epithelial lining. This epithelium was formed of a few spermatogenic cells with darkly stained nuclei. A few tubules had a relatively narrow lumina with a few sperms (Figs 12 and 13). Electron microscope examination of the ultrathin sections of the testes of the same subgroup showed that the STs were mostly lined by sertoli cells and a few spermatogenic cells. The sertoli cells had euchromatic nuclei and mitochondria. Extremely thick and irregular basement membranes were observed. Myoid cells appeared with irregular heterochromatic nuclei (Fig. 14). Leydig cells had variable-size electron-dense granules and extensive cytoplasmic processes interdigitating with the neighboring ones (Fig. 15).
Group 3 (experimentally induced varicocele left for 18 weeks and followed by L-carnitine for another successive 8 weeks)
Light microscope examination of sections of the testes of the EV-carnitine group showed that they regained nearly their normal general architecture. Most of their STs were packed together with regular outlines and narrow interstitium. Acidophilic hyaline material was still observed between some tubules. In addition, most of the tubules restored their normal epithelial stratification and sperms were observed inside their lumina. A few tubules still had disorganization in their germinal epithelium, areas of epithelial separation, and a few sperms in their lumina (Figs 16 and 17). Electron microscope examination of the ultrathin sections of the testes of the same group showed that spermatogonia, primary spermatocytes, spermatids, and sertoli cells showed their normal fine structure. However, intercellular separations were still present between some cells. The basement membrane was thin and regular (Fig. 18). Most of the transverse sections of the midpieces of sperms had normal structure except for a few of them that showed an affected axoneme (Fig. 19). Leydig cells contained numerous mitochondria, a few variable-size electron-dense granules, and a few lipid droplets. No cytoplasmic processes were observed (Fig. 20).
Morphometrical and statistical results
Statistical analysis of the epithelial height of the STs of group 2 showed a highly significant decrease in EV18 as compared with others. However, the EV-carnitine group showed a highly significant increase in their epithelial height as compared with the EV18 subgroup. F value of one way ANOVA test=13.07 (Table 1 and Histogram 1).
The exact relationship between varicocele and infertility remains to be explained. Clinical studies showed that unrelieved venous stasis, persistent hypoxia/ischemia, and overproduction of ROS that occur in varicocele lead to decreased testicular volume and poor semen quality. These changes were obvious if varicocele was presented early in puberty. If it was neglected, the testes became atrophic with oligospermia or even with an absence of spermatozoa in seminal fluid (azoospermia) [24–28].
In this study, after comparing two periods of EV, we found that testicular impairment was time dependent as it progressively increased with the period of induction. Examination of sections of the testes of subgroup EV6 of the EV group (group 2) showed that testicular parenchyma was formed of many distorted STs with irregular outlines, disorganized epithelium, and wide lumina. Some areas of interstitium were wide. The disorganized epithelium showed separation and sloughing. The epithelial cells contained small vacuoles. Examination of sections from the EV18 subgroup showed that most of the STs were markedly distorted with very wide lumina and increased reduction in the thickness of their epithelial lining. A few tubules had a relatively narrow lumina. The epithelium was formed of a few spermatogenic cells with darkly stained nuclei. Most of the tubules were mostly lined by sertoli cells that had euchromatic nuclei and mitochondria. Morphometrical and statistical analyses of the epithelial height of the STs confirmed these results as it showed a highly significant decrease in EV18 as compared with others. Earlier studies clarified that the pathophysiological processes of massive loss of germ cells in varicocele was apoptosis-mediated and it increased with time [19,29]. This could lead to germ cell depletion (sertoli-only-syndrome-like feature) that occurred in parts of STs . Although sertoli cells were extremely resistant to cell death, their metabolic and regulatory pathways could be disturbed, resulting in germ cell degeneration. If sertoli cells were permanently affected, the recovery of spermatogenesis might not be possible or at least would be incomplete .
Some STs in the EV6 subgroup showed multinucleated cells in their lumina. Some investigators clarified that most of these cells were spermatocytes or spermatids that failed to separate as a result of failure of breakdown of intercellular bridges in the early phases of spermiogenesis . Other investigators observed that they were formed from fusion of spermatids secondary to retraction of sertoli cell processes .
In this study, examination of the same group showed that acidophilic hyaline material was present in most of the interstitial spaces, and congested blood vessels were observed. The acidophilic hyaline material could be attributed to excess lymphatic exudates oozing from degenerative lymphatic vessels  or to an increase in vascular permeability .
Electron microscope examination of the EV6 subgroup showed that almost all transverse sections in the mid pieces of sperms showed markedly affected disarranged axoneme, fibrous, and mitochondrial sheaths. In most of the STs of the EV18 subgroup, no sperms were observed. It was reported that the ROS might cause lipid peroxidation of sperm cell membranes, hence damaging the midpiece, axonemal structure, or disrupting the capacitation and acrosomal reaction . Subsequently, increased sperm abnormality and decreased sperm count and motility were associated with decreased fertility [35,36].
This ultrastructure examination of the EV6 subgroup showed that the STs were enveloped by a relatively thick, irregular, highly infolded basement membrane. This membrane became extremely thick and irregular in the EV18 subgroup. Myoid cells appeared with irregular heterochromatic nuclei. It was documented by some investigators that altered peritubular myoid cells caused an enlargement of the collagen fibril layer that was normally produced by these cells , whereas others attributed this thickness to an increased production of glycosaminoglycans and proteoglycans in the basement membrane as a defense reaction against the damaging activity of free radicals . Contrarily, other investigators claimed that the initial involvement of basal lamina could represent one of the mechanisms responsible for varicocele-induced histological alterations of the testes .
In this study, the EV6 subgroup showed Leydig cells with lipid droplets, variable-size electron-dense granules, and multiple cytoplasmic processes. Those of subgroup EV18 had variable-size electron-dense granules and extensive cytoplasmic processes interdigitating with the neighboring ones. In this study, we suggested that the observed electron-dense granules could be lipid in nature. One of the theories that attempted to explain the changes induced by varicocele was the venous lesion that led to vasodilatation and interference with the vascular countercurrent heat exchange. Therefore, the blood flow increased and testes became warmer . According to this theory, some studies claimed that infertility with varicocele might be attributable to dysfunction of Leydig cells. Temperature higher than the body temperature might cause damage of Leydig cells. This damage was associated with an unusual accumulation of lipid droplets, which was considered as storage depots of the precursors used in its synthesis of androgens, indicating that less substrate was being utilized. As the degree of stabilization of fatty acids by osmium tetraoxide fixative depended on their extent of unsaturation, the lipid droplets differed in electron densities. Its different sizes and densities meant that Leydig cell activity was reduced and steroid precursors were accumulated [1,41–43]. Leydig cells with cytoplasmic processes of different length and a few secondary branches were observed in testes with reduced spermatogenesis. These cells were present in small or large cell groups and had desmosome-like contacts and gap junctions between these processes and neighboring cells. These junctions carried signals that regulated their secretory activity .
In this study, examination of sections of the testes of group 3 (EV-carnitine) showed that they nearly regained their normal general architecture. Most of their STs were packed together with regular outlines and narrow interstitium. Acidophilic hyaline material was still observed between some tubules. In addition, most of the tubules restored their normal epithelial stratification. A few tubules still had disorganization in their germinal epithelium. These results were confirmed by statistical analysis that clarified a highly significant increase in their epithelial height as compared with the EV18 subgroup. Some investigators tried to evaluate the impact of surgical treatment (varicocelectomy) in varicocele. They found that surgical repair of the short-term experimental varicocele could restore full recovery of bilateral testicular weight, epididymal sperm content, and sperm motility. However, only partial recovery of the semen parameters after repair of the long-term ones was observed. Therefore, they decided that surgical repair alone was not enough in such cases [44,45]. Meanwhile, other investigators tried to evaluate the role of L-carnitine in the reduction or prevention of the testicular damage that occurred as a result of other conditions such as drug toxicity (i.e. etoposide)  or γ irradiation . Carnitines act as anti-ROS drugs as they are capable of returning the ROS concentrations to their physiological level with an improvement in sperm parameters [18,47,48].
Ultrastructural study of the EV-carnitine group examination showed that spermatogonia, primary spermatocytes, spermatids, and sertoli cells showed their normal fine structure. However, intercellular separations were still present between some cells. The basement membrane was thin and regular. Leydig cells contained numerous mitochondria, a few variable-size electron-dense granules, and a few lipid droplets. No cytoplasmic processes were observed. Earlier studies had identified the sertoli cell as a possible testicular target of carnitine activity because the latter had a direct action on sertoli cell metabolism . It influenced fat and carbohydrate metabolism by increasing fatty acid oxidation, glucose uptake, and lactate/pyruvate secretion . Other investigators demonstrated that carnitine could act through carnitine/organic cation transporter in sertoli cell membrane. They added that, if these cells were injured, spermatogenesis was impaired . Other investigators claimed that carnitine could act on stem spermatogonia by minimizing the DNA damage. These cells had extensive DNA repairing mechanisms. Hence, the ability of the testis to recover spermatogenesis depended on the survival of some stem spermatogonia and their ability to repopulate the testis with differentiating cells [21,46,51].
In this work, examination of sections of the testes of the same group showed that sperms were observed in most of the tubules, although they were a few in some of them. Ultrastructurally, most of the transverse sections of the midpieces of sperm had a normal structure, except for a few of them that showed an affected axoneme. Some investigators reported that L-carnitine played a key role in sperm metabolism by providing readily available energy for use by spermatozoa [16,47,52]. Hence, several experimental and clinical studies used carnitine supplementation to treat oligoasthenospermia because carnitine therapy improved the sperm function, spermatozoa kinetics, fertilization capacity, and morphological characteristics [48,53–56].
From the results of this study, we concluded that varicocele led to a deleterious effect on the ipsilateral testis that increased progressively with time. In addition, L-carnitine administration improved the structure of testis of long-duration varicocele. Therefore, L-carnitine could be considered as a therapeutic hope for those with long-duration varicocele and for those who failed to become fertile after varicocelectomy, or who are not suitable for surgery. We recommend further studies using a combination of varicocelectomy and L-carnitine therapies for long-duration varicoceles.
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L-carnitine supplement; testis histology; ultrastructure; varicocele
© 2011 The Egyptian Journal of Histology
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