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From the Division of Urology, Department of Organ Therapeutics, Faculty of Medicine, Kobe University Graduate School of Medicine, Kobe, Japan.
| Correspondence to: Dr Tomomoto Ishikawa, Division of Urology, Department of Organ Therapeutics, Faculty of Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chuo-Ku, Kobe 650-0017, Japan (e-mail: iskwtmmt{at}med.kobe-u.ac.jp). |
| Received for publication May 3, 2007; accepted for publication August 15, 2007. |
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Abstract |
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Key words: Fertility, infertility, spermatogenesis, male reproductive tract
The testes of patients with idiopathic male infertility show
histopathologically evident thickening of the BM of the testicular tubules and
increases in the amount of interstitial connective tissue
(Martin and Timpl, 1987). The
BM contains several proteins, including laminin, type IV collagen, various
heparin sulfate proteoglycans, and ectatin/nidogen. In patients with
varicocele, Kleinfelter syndrome, crypto-orchidism, and Sertoli
cell–only syndrome (SCO), the BM is thickened, apparently due to
increased deposition of collagen fibrils in the extracellular space
(Virtanen et al, 1997). The
increased thickness of BM is associated with an increase in extracellular
matrix components and the irregular configuration of myofibroblasts facing the
germinal epithelium (Pollanen et al,
1985). Each laminin molecule is a heterotrimer assembled from
, β, and 
chains. There are 5 forms of chains
(1–5), 4 of β chains (β1–4), and 3 of
chains (1–3; Virtanen et al,
1997). Fifteen laminin trimers have been identified
(Nquyen and Senior, 2006).
Although the distribution of laminin chains has been described in the testes
of mice, the distribution of the 15 known laminin timers in human testes is
unknown.
To examine the expression of laminin chains within the human testis and the association with spermatogenesis, we determined the distribution of laminin trimers in testicular tissue from azoospermic men.
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Materials and Methods |
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Sample Preparation
Testis biopsy materials were fixed for about 12 hours in Bouin solution at
room temperature before embedding in paraffin. Serial paraffin sections (5
µm thick) were mounted on glass slides for hematoxylin-eosin staining and
immunohistochemistry. The histologic status of the specimens was evaluated on
sections stained with hematoxylin-eosin. These sections were measured for BM
thickness and the diameter of testicular tubules with an eyepiece scale
(Olympus, Tokyo, Japan). The Johnsen score (JS) was also determined, as
previously described (Johnsen,
1970). To calculate the JS, the sum of all scores was divided by
the total number of testicular tubular sections.
Immunohistochemistry
For immunohistochemical detection of laminin trimers, sections were stained
with the labeled streptavidin-biotin method using a DAKO LSAB2 kit/horseradish
peroxidase (HRP; DAKO, Carpinteria, Calif). Rabbit antilaminin polyclonal
antibodies, 1–5, β1–3, and 1–3 chains,
were used (Table 1). These were
raised against amino acids at the C-terminus of each laminin chain of human
origin, were specific for each isoform, and did not cross-react with other
isoforms. Sections were incubated in 6 M urea in 0.01 M glycine-HCl buffer (pH
3.5) at room temperature for 20 minutes, followed by three rinses in
phosphate-buffered saline (PBS; 140 mM NaCl, 8 mM
Na2HPO4, and 2 mM NaH2PO4 at pH
7.2). Endogenous peroxidase was blocked by incubating sections in fresh 3%
H2O2 in methanol for 5 minutes. After rinsing three
times in PBS, sections were incubated with primary antibodies at 1:100
dilution for 60 minutes. Biotinylated goat anti-rabbit -globulin was
used as the secondary antibody for 30 minutes. After incubation with
streptavidin-biotin–peroxidase complex for 10 minutes, peroxidase
activity was detected by diaminobenzidine peroxidase. Sections were
counterstained with methyl green, dehydrated in butanol, and mounted in Eukitt
(Osler, Berlin, Germany).
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Laminin Index
At least 20 testicular tubules per section were examined with an Olympus
BX50 microscope and photographed with an Olympus C35DX camera and PM30 film.
As a negative control, the primary antibodies were omitted. The intensity of
laminin immunoreactivity was determined as follows. Each section was viewed
under a microscope with an attached video camera. The camera was linked to a
Photograb-300 unit (Fuji Photo Film, Tokyo, Japan) within a Macintosh Power
Mac G3 microcomputer (Apple Computers, Cupertino, Calif). Each image was
imported directly to the Photoshop 4.0J program (Adobe Systems, San Jose,
Calif) and then converted to grayscale images, abandoning the red and green
channels. Laminin chains, stained brown by diaminobenzidine peroxidase, were
extinguished to a greater extent than other areas. Processed images were
exported to a freeware image analysis program, National Institutes of Health
Image 1.62f. To calculate the fraction of the area occupied by laminin
immunoreactive tissue, a threshold was applied to each image at a contrast
level that distinguished between laminin immunostaining (rendered black) and
the remaining area (rendered white). Black pixels detected in the BM of
testicular tubules, as opposed to blood vessels, were regarded as laminin
expression in the testicular tubule. More than 20 testicular tubular sections
per testis were analyzed. The total area of laminin expression in testicular
tubules was divided by that of testicular tubule profiles to calculate the
laminin index.
Hormone Assay and ELISA
Serum follicle-stimulating hormone (FSH), lutenizing hormone (LH), and
testosterone (T) concentrations were determined by chemiluminescence assays
(Architect; Abbott Japan, Matsudo, Japan). Blood samples were drawn between
0900 and 1000 hours. Laminin was estimated in the seminal plasma by using the
double-antibody enzyme immunoassay (Chemicon, Temecula, Calif), which is in
the format of a competitive inhibition enzyme-linked immunosorbent assay
(ELISA). Standards and samples are diluted and preincubated with polyclonal
rabbit antibody to human laminin. The polyclonal antibody binds to laminin in
the standards and samples. The mixture is then transferred to the human
laminin-coated plate. Free rabbit anti-human laminin binds to the laminin on
the plate. Goat anti-rabbit immunoglobulin G HRP conjugate reacts with bound
rabbit anti-laminin. This color was quantitated using an ELISA reader; the
intensity of the color is inversely proportional to the concentration of
laminin in the original sample. A standard curve is constructed, and sample
values are interpolated. The test sensitivity is 30 to 70 ng/mL. The assay
performance and interassay and intraassay variations were within the
manufacturer's limits.
Statistics
Statistical analysis was performed using the nonparametric Mann-Whitney
U test to detect differences between patient groups. Correlations
were analyzed by Spearman correlation coefficient. Statistical significance of
difference was evaluated using analysis of variance (ANOVA). A value of
P < .05 was considered significant.
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Results |
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BM thickness was significantly correlated with testicular tubule diameter (r = –0.49, P = .004, negatively; Figure 1A) and FSH (r = 0.52, P = .008, positively; Figure 1B) but did not correlate with the concentrations of JS (r = –0.48, P = .10), LH, and T (data not shown).
Immunohistochemical Analysis
Testicular tissue from a representative patient with OA was immunostained
using anti–laminin β2 chain
(Figure 2A). The β2 chain
of laminin was seen in the inner BM of testicular tubules as a strongly
stained, irregular, wavy line. The 1 and 1 chains were slightly
detected, whereas the 2, 3, 4, and 5; β1 and
β3; and 2 and 3 chains showed little staining intensity
(data not shown). In MA testes, the β2 chain was more intensely stained
than OA testes (Figure 2B), and
the 1 and 1 chains were slightly detected. In testes with SCO,
the β2 chain was detected more intensely than in other groups
(Figure 2C). In the SCO testis,
the epithelial BM and the first myoid cell layer were separated by a wide
homogenous layer negative for all laminin chains, resulting in the appearance
of 2 concentric rings around the tubular lumen.
Relationship Between Laminin Index and Clinical Parameters
The laminin index for the β2 chain in patients with SCO (23.2%
± 6.8%) is significantly higher than in OA (6.9% ± 4.1%;
P < .0001) and MA (17.8% ± 5.9%; P = .0002)
patients (Figure 3A).
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The laminin index for 1 in patients with OA, MA, and SCO was 8.2%
± 4.3%, 11.4% ± 5.3%, and 14.6% ± 5.5%, respectively.
Significant differences were found between OA and SCO groups (P =
.03) but not between OA and MA groups
(Figure 3B). No significant
differences were found in the laminin index for 1 in patients among OA
(14.7% ± 6.1%), MA (15.2% ± 5.3%), and SCO (11.3% ± 5.7%)
groups.
Seminal Laminin Levels in Infertile Patients
Comparison among mean seminal plasma levels demonstrated significant
differences between OA and SCO (P < .001;
Fig. 4). There was also a
significant difference between mean seminal laminin levels in the OA and those
in the MA group (P < .03; Fig.
4). Seminal plasma laminin concentration in infertile patients
demonstrated a nonsignificant correlation with age and T (data not shown).
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Comments |
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TopAbstractMaterials and MethodsResultsCommentsConclusionReferences |
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Hadley and Dym (1987) showed by immunocytochemistry at the ultrastructural level that laminin was present in the BM adjacent to myoid cells. Davis et al (1990) indicated the presence of laminin in myoid cells in vitro by both immunofluorescent staining and Western blot. In our study, laminin chains were present only in the inner epithelial BM in the SCO testis. Interestingly, in addition, in the SCO testis the epithelial BM and the first myoid cell layer were separated by a wide homogenous layer negative for all laminin chains, resulting in the appearance of two concentric rings around the tubular lumen. Kleinman et al (1987) pointed to this control over cellular functions by mediating adhesion to anchorage-dependent cells, providing signals for direct migration and controlling important cellular functions. Davis et al (1990) also demonstrated that testicular dysfunction can affect laminin contribution and that laminin can bind soluble growth factors, for example, fibroblast growth factor, transforming growth factor, and interferon gamma, concentrating peptides for subsequent interaction with cells.
It is generally agreed that Sertoli cells produce laminin and collagen IV (Borland et al, 1986); thus, the lack of immunoreactivity inside tubules suggests that laminin protein is not translated in Sertoli cells to a sufficiently high level to be detectable by immunohistochemistry.
Geipel et al (1993) showed that seminal laminin is significantly elevated in fertile men compared with postvasectomy men, suggesting that some seminal laminin is derived from the vas deferens. El-Dakhly et al (2007) demonstrated that seminal plasma laminin levels in fertile cases had a significantly higher mean level than azoospermic cases (NOA, OA, and congenital bilateral absence of the vas deferens [CBAVD]).
The significant decrease of seminal laminin in SCO could be explained by failure of the intact spermatogenic process with disrupted testicular tissue architecture to reach sufficient levels of laminin due to increased deposition of collagen fibrils in the extracellular space. Therefore, we conclude that assessing seminal laminin levels could lead to prediction of the pathology of male infertility patients. Simultaneous measurement of many extracellular matrix components might also provide a clue to different interrelations.
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Conclusion |
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TopAbstractMaterials and MethodsResultsCommentsConclusionReferences |
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1 and 1 chains were slightly dominant in the BM of seminiferous
tubules in the human testes. Overabundance of the β2 chain of laminin is
associated with an abnormally thickened BM and is possibly related to
spermatogenic dysfunction. |
References |
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