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From the * Department of Pediatric Surgery and
Department of Anatomy, University of
Heidelberg, Heidelberg, Germany, and First
Department of Surgery, University of Athens, Athens, Greece.
| Correspondence to: Dr Evagelia Gougoudi, Pediatric Surgeon, 21 Nikis str, Paradisos Amarousiou, 15233 Attica, Greece (e-mail: evagougoudi{at}yahoo.com). |
| Received for publication November 26, 2006; accepted for publication May 3, 2007. |
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Abstract |
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Key words: Cryptorchidism, orchiopexy, postoperative testicular atrophy, inhibin ßB, mast cells
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Materials and Methods |
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Anesthesia and Operative and Postoperative Procedures
All examinations and operative procedures were performed under general
anesthesia. This was achieved by intraperitoneal injection of 2.5% thiopental
sodium diluted in 0.9% sodium chloride at a dose of 0.1 mL/100 g body weight
after the animals were anesthetized by ether inhalation.
A 2-mm endoscope (0 degrees; 7210 AWA; Karl Storz, Tuttlingen, Germany) was inserted into the peritoneal cavity at the middistal of the xiphoid-pubic line. Carbon dioxide pneumoperitoneum was created by a venous catheter (22 G) inserted on the right side of the endoscope, and a 1.7-mm "working" cannula (Karl Storz) was inserted on its left side. The right spermatic vessels were identified, dissected, coagulated, and divided by means of a hook approximately 1 cm away from the testis avoiding any contact with the deferent duct. The instruments were removed, pneumoperitoneum deleted, and fascia and skin closed in layers. The animals were returned to their cages to recover. In the sham-operated animals, we followed identical procedures, but instead of coagulating, we simply manipulated the vessels for approximately 6 minutes, time comparable to that needed for the FS procedure.
Both testes of all animals were ultrasonographically assessed 8, 29, 69, and 89 days after the operation. The day after (ie, the ninth, 30th, 70th, and 90th postoperative day [POD]), perfusion-fixation of the testes was performed on the animals by injecting 2% polyvinylpyrrolidone, 0.2% procaine hydrochloride, 1.5% glutaraldehyde, and 1.5% paraformaldehyde directly into the aorta. Then the gonads were removed and examined histologically and immunohistochemically. In addition, just before the perfusion of the fixative solution, blood was sampled to determine the levels of serum inhibin ßB.
Ultrasonographic Examination
Ultrasonography was always performed by the same examiner in a standardized
manner to minimize interobserver variability. We examined the borders and
volume of each testis as well as the normality and echogenicity of testicular
parenchyma with the Elegra apparatus (Siemens AG, Munich, Germany). Doppler
sonography was also employed to determine testicular vascularity and blood
supply. Unfortunately, the occasional development of massive calcification
precluded accurate testicular volume assessment in some animals
(Figure 1). Therefore, the
volumetric data were considered unreliable and were disregarded.
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Histology and Immunohistochemistry
Pathology was assessed in paraffin- and epon-embedded sections stained with
Richardson stain. Briefly, slices were incubated in
3,3'-diaminobenzidine, treated with 1% osmium tetroxide, and stained
with 1% uranyl acetate. Then they were dehydrated, treated with propylenoxide
and epon, and embedded in epon. Richardson staining (azur II and methylene
blue mixture) was selected because mast cells are easily identified due to
metachromasia.
Immunohistochemistry was performed with a commercially available kit employing a monoclonal antibody against the inhibin ßB subunit (Oxford Bio Innovation DSL Ltd, Oxfordshire, United Kingdom) and a biotinylated universal antibody (Vectastain biotinylated universal equine antibody; Vector Laboratories, Burlingame, Calif) according to the kit provider's instructions. In brief, thin sections were deparaffinized, rehydrated in a xylene/ethanol sequence, pretreated in sodium citrate solution (pH 6.0), and incubated with 3% H2O2 in 100% methanol to inhibit endogenous peroxidase activity. Then the sections were incubated with normal goat serum with 0.1% azide to block nonspecific background. Subsequently, they were treated with the monoclonal antibody against inhibin ßB and afterwards with the universal antibody. Following incubation with avidin-biotin complex, the slides were stained with 3-amino-9-ethylcarbazole and counter-stained with Mayer hematoxylin. With the same procedure, we prepared negative controls using the secondary antibody alone.
Morphometry
Morphometric studies were conducted with Leica Quantimet 500c software
(Leica Microsystems GmbH, Wetzlar, Germany), a professional image analysis
system commonly used in research and used as a standard for the evaluation of
other systems (Brockmann et al,
2004) The mean seminiferous tubule area (STA) was determined
semiquantitatively in the paraffin sections stained for inhibin ßB. The
area of 50 tubules in 4 cross sections of each gonad was determined, and the
average of these 200 measurements was calculated for each testis. Tunica
albuginea was determined as the mean value of 50 different measurements in
each testis. The number of mast cells was calculated by counting the total
number of mast cells in each section.
Determination of Serum Inhibin ßB Levels
Prior to the perfusion-fixation procedure, we sampled blood from the
inferior vena cava. Samples were centrifuged, and the supernatant serum was
frozen and stored in liquid nitrogen at –130°C. Serum inhibin
ßB levels were determined by enzyme-linked immunosorbent assay.
Statistical Analysis
Nonparametric tests were used in this study. Mean values were compared with
the Mann-Whitney U test and Kruskal-Wallis analysis of variance.
Correlations were evaluated with Pearson's correlation coefficient. All
analyses were performed with SPSS 10.0 for Windows software (SPSS Inc,
Chicago, Ill).
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Results |
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Light Microscopy
The structures of both testes in the control animals and of the left testes
in the FS group were normal. The seminiferous tubules had distinct, patent
lumens and were densely packed in a hexagonal pattern. Numerous tubules were
intimately associated with the tunica albuginea. Mean STA doubled (P
< .01) at POD 30 and remained stable (P > .05) for the rest of
the experiment (Table 2).
Within the germinative epithelium, the spermatogonia and Sertoli cells formed
a discontinuous layer adjacent to the basement membrane. Sertoli cells
containing small lipid vacuoles surrounded the germ cells. The interstitial
space contained a dense capillary network, a few macrophages, and small groups
of Leydig cells clustered around the capillaries. They were spherical or
elongated and contained small lipid vacuoles. A few mast cells were confined
to the peripheral zone of the testis near the tunica albuginea. The number of
mast cells remained constant throughout the experiment, whereas the thickness
of tunica albuginea increased significantly (P < .01) between days
30 and 70.
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The ischemic changes seen in the right testes after the division of their arteries could be graded into mild and severe forms. In the mild form, the apoptotic processes were restricted to the germ cells of the central area only. The severe form included progressive apoptosis of all germ cells, Sertoli cells, and Leydig cells; massive microlithiasis in the central zone; and complete obstruction of the seminiferous lumen by cellular debris. Of interest, those testes with severe degenerative alterations had reduced vascularity on ultrasound. In contrast, the testes with sonographically homogenous parenchyma and intact vascularity displayed either mild degeneration or no degeneration at all. Therefore, the 2 grades of injury probably reflect the sufficiency of the collateral blood supply.
Nine days after the procedure, the seminiferous tubules in all 4 right testes were shrunken with significantly reduced STAs (P < .01) compared with the left or control testes. The lumen contained numerous apoptotic cells in all stages of spermatogenesis and spermatid formation. The disorganized germinative epithelium consisted of disarranged spermatogonia, spermatocytes, and spermatids without stratification. The interstitial space was occupied by Leydig cells and macrophages. When compared with the left or sham-operated testes, the number of mast cells was significantly higher (P < .01) and the tunica albuginea was significantly thicker (P < .01) containing fibroblasts with numerous lipid droplets.
Thirty days after the procedure, all 4 right testes had significantly thicker (P < .05) tunica albuginea with newly formed vessels. Three characteristic and distinct zones were seen in the parenchyma (Figure 2). First, adjacent to the tunica albuginea, there was a discontinuous, peripheral zone (Table 2, subgroup 30 per) in which the seminiferous tubules were large, atrophic, and partially calcified. In the noncalcified areas, the germinative epithelium consisted of apoptotic spermatogonia and a few Sertoli cells. Several layers of peritubular myofibroblasts, numerous intact blood or lymphatic capillaries, and a delicate network of thin, elongated Leydig cells and fibroblast-like cells surrounded the seminiferous tubules. This peripheral zone was characterized by an increased number of blood capillaries, abundance of mast cells, and focal proliferation of Leydig cells. Second, the intermediate zone had numerous small atrophic, noncalcified seminiferous tubules scattered in loose connective tissue and enclosed by a single layer of flat myofibroblasts. Large lipid-loaded macrophage-like cells marked the damaged tubules, and numerous typical macrophages were seen in the interstitial space. Accurate measurement of STA was impossible due to the degradation process and the irregularity of the tubule contours. Third, the central zone (Table 2, subgroup 30 cen) contained relatively large, hexagonally, and tightly packed seminiferous tubules filled with cellular debris and lined with a completely atrophic epithelium. No spermatogenic cells or Sertoli cells could be recognized. A few blood capillaries; several calcified branches of the testicular artery, and widespread microlithiasis were also seen. Mean STA of the peripheral zone was significantly smaller (P < .01) than that of the central zone, and both differed significantly (P < .01) from those of the left testes. Also, the number of mast cells in the peripheral area was significantly higher (P < .01) compared with the left or sham-operated testes.
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Ninety days after the FS procedure, the testes of the 2 animals with decreased vascularity on ultrasonography were severely damaged (Table 2, subgroup 90B). They possessed a thickened (P < .01) tunica albuginea with newly formed blood vessels. Again, the 3 distinct zone patterns of the parenchyma were prominent, but neither STA nor mast cell density changes reached statistical significance. Of the remaining 4 animals with normal testicular vascularities, the 3 that presented intermediate testicular damage (Table 2, subgroup 90A; clusters of atrophic seminiferous tubules with significantly [P < .01] reduced diameters) were scattered among almost normal ones. The germinative epithelium was short, lacked the normal multilayered structure, and frequently consisted exclusively of highly vacuolated Sertoli cells. The last animal (Table 2, subgroup 90C) had a right testis of normal morphology with well-developed seminiferous tubules, typical germinative epithelium and interstitial space.
Analysis revealed that the increase in the number of mast cells correlated positively with the thickening of tunica albuginea (Spearman's rs = .9; P < .01) and negatively with the decrease in STA in the severely atrophic tubules (Spearman's rs = .7; P < .01).
Immunohistochemistry
In the control animals only, the Sertoli cells presented intense
immunohistochemical staining for inhibin ßB within the germinative
epithelium. Leydig cells, macrophages, spermatogonia, spermatocytes, and
spermatids exhibited very weak or no reactions at all. Peritubular myoid
cells, blood, and lymphatic endothelial cells were completely negative. The
intertubular space occasionally displayed homogeneous weak to moderate
staining for inhibin ßB. Immunohistochemistry of all the left testes
yielded comparable results.
Nine days after the FS procedure, distinct inhibin ßB staining was easily identified in the rarely seen, flattened, alive Sertoli cells lining the atrophic tubules of the peripheral zone of the right testes. The remaining seminiferous tubules lacked inhibin ßB staining. In the later groups, the flattened and vacuolated Sertoli cells maintained intense staining for inhibin ßB in the mildly damaged testes, although this was slightly weaker compared with the sham-operated animals. In contrast, the severely damaged testes presented inhibin ßB staining patterns similar to the zones seen in light microscopy. Staining was positive only in the few atrophic tubules of the peripheral zone and specifically in the clusters of 2 to 4 intact Sertoli cells in the basement membrane. On the contrary, the intermediate zone was completely negative. Moderate to intense nonspecific staining was observed in the cellular debris inside the calcified atrophic tubules of the central zone. This could represent either cell remnants or viable Sertoli cells scattered in the cellular debris.
Serum Inhibin ßB Levels
Serum inhibin ßB levels in the sham-operated animals showed normal
age-dependent decreases (Table
1). In the FS-operated animals, serum inhibin ßB levels were
lower from those of the control animals after 9 and 30 days. Later the serum
levels varied according to the degree of testicular degeneration seen in
pathology or sonography. The animals with normal sonography and mild or no
degradation on histology presented normal levels. On the contrary, the animals
with severely degenerated testes exhibited less than half of the inhibin
ßB levels compared with controls (P < .05). This decrease in
the levels of inhibin ßB correlated negatively and significantly to both
the increase of mast cell density and the thickening of tunica albuginea
(Spearman's rs = .8 and rs = .5,
respectively, P < .01 for both), but it did not correlate to the
decrease in STA.
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Discussion |
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Ligation of testicular vessels decreases testicular capillary blood flow from the first hour (Erçöçen et al, 2004). Reduction of testicular blood flow initially impairs spermatogenesis through apoptosis (Bergh et al, 2001), perhaps through a nitric oxide-mediated mechanism (Taneli et al, 2005). After a period of time, parenchymal perfusion seems to be more severely affected than capsular (Huang et al, 1992), and additional testicular structures are probably injured. Ischemia due to the FS maneuver results in central coagulative necrosis and peripheral interstitial fibrosis (Erçöçen et al, 2004). The seminiferous tubule borders become indistinct and spermatogenesis impaired (Erçöçen et al, 2004). Spermatozoa and spermatid cells are affected as early as 1 week and tubular morphology becomes disrupted in 2 weeks after the operation (Huang et al, 1992). In the long run, atrophy and fibrosis become apparent with reduction of testicular weight, sperm density, seminiferous tubule diameter, and various scores assessing the different cell populations (Salman and Fonkalsrud, 1990; Huang et al, 1992; Kelly et al 1992; Sperling et al, 2000; Guler et al, 2004).
Our findings are consistent with the literature because we observed early decreases in the central testicular vascularity. This in time became more severe and extended toward the periphery in those testes that eventually degenerated. In contrast, in the animals in which testicular blood flow had been restored, milder or no testicular degeneration was present. On histology, the early effects of ischemia included decrease in STA, accumulation of mast cells, and thickening of tunica albuginea, all at statistically significant levels. The inhibin ßB-positive, alive Sertoli cells became rarer and were located in the periphery of the testes, whereas serum inhibin ßB dropped to approximately half of the control animal levels. Later the animals in which blood perfusion had been restored maintained homogenous testicular parenchyma on ultrasonography, whereas the mild ischemic changes were restricted to the germ cells on histology. Inhibin ßB-producing Sertoli cells seemed unaffected, and inhibin ßB serum levels were normal. In contrast, the animals in which perfusion derangement persisted were additionally characterized by sonographically heterogenous parenchyma, probably reflecting the more severe changes caused by ischemia. The histologic parameters remained significantly different than the contralateral testes and control animals. The 3-zone pattern we noticed, with the more severely apoptotic processes located centrally, perhaps mirrors the distribution of blood flow impairment. Furthermore, the few alive, inhibin ßB-positive Sertoli cells were mostly located in the peripheral, least degenerated zone. Serum levels of inhibin ßB in these animals were less than half of the corresponding controls, a decrease that correlated significantly to mast cell accumulation and tunica albuginea thickening but not to seminiferous tubule narrowing.
Serum inhibin B levels in the rats have been related to the number and activity of Sertoli cells (Buzzard et al, 2004). Inhibin B production and secretion is stimulated by the presence of specific germ cells (Pineau et al, 1990; Allenby et al, 1991); therefore, its levels may be regarded as markers of spermatogenesis. The levels decrease with testicular torsion (Ozkan et al, 2001) or by hemicastration (Klaij et al, 1994), although these procedures affect the whole testis and not the germinative epithelium selectively. In our study, we observed both damage of the germinative epithelium and restriction in the number and location of Sertoli cells, although the latter was not quantified. Inhibin B subunit expression and localization to interstitial and Sertoli cells in the adult rat are similar to human prepubertal testicles (Roberts et al, 1989; Majdic et al, 1997). However, the extension of these conclusions from rodents to children and vice versa requires caution. Inhibin B secretion requires functional interaction of germ cells with Sertoli cells in the adult human testis (Andersson et al, 1998; Andersson, 2000; Frydelund-Larsen et al, 2002) but not in the prepubertal one (Andersson et al, 1998; Petersen et al, 1999), which is why inhibin B is readily detectable in prepubertal boys with Sertoli cell only syndrome but not in men with the syndrome (Andersson et al, 1998). A similar germ cell-Sertoli cell interaction seems to be required in mature rats (Allenby et al, 1991) but it may be more complicated in prepubertal rats (Klaij et al, 1994). This discrepancy may account for the lack of correlation between the decrease in levels of serum inhibin B and the reduction in STA in our study, although we cannot ignore the limited power due to the anesthesia-related mortality.
Of interest is the significant negative correlation of the reduction in serum inhibin ßB levels with both the thickening of tunica albuginea and increase in the number of mast cells. Changes in tunica albuginea due to new vessel formation following the FS procedure in rats has been reported (Guler et al, 2004), but the increase in the number of mast cells represents new information to the best of our knowledge. Although this increase could be related to angiogenesis, it is also likely that it may be related to the process of testicular degeneration and fibrosis. In humans, the chymase-containing subtype of mast cells has been implicated in testicular fibrosis and infertility (Meineke et al, 2000; Yamanaka et al, 2000), and mast cell blockers have already been used as treatment (Yamamoto et al, 1995). Furthermore, the mast cell accumulation in human pathologic testicular tissue correlates significantly with inducible nitric oxide synthase scores (Sezer et al, 2005). In rodents, mast cells probably participate in the regulation of steroidogenesis (Mayerhofer et al, 1989; Aguilar et al, 1995), and their number increases with testicular injury (Gaytan et al, 1989; Gaytan et al, 1990). In rats, inhibin B secretion by Sertoli cells is inhibited by inflammatory mediators (Okuma et al, 2005), which may be derived from mast cells. This could, at least partially, explain the correlation between the decrease in inhibin B levels and the increase in the number of mast cells, and the latter could be related to the testicular tissue damage through a nitric oxide-dependent mechanism (Sezer et al, 2005; Taneli et al, 2005). Whether this hypothesis is valid and whether mast cells contribute to testicular degeneration following the FS operation remains to be elucidated in the future.
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