ultrastructural and immunohistochemical study of basal lamina of the testis in adolescent varicocele

Ultrastructural and immunohistochemicalstudy of basal lamina of the testis inadolescent varicocele

Giuseppe Santoro, M.D.,* Carmelo Romeo, M.D.,† Pietro Impellizzeri, M.D.†

Carmelo Gentile, M.D.,† Giuseppe Anastasi, M.D.,* and Agatino Santoro, M.D.*

Faculty of Medicine and Surgery, University of Messina, Messina, Italy

Objective: To evaluate a possible involvement of the extracellular matrix (ECM) in the testes of adolescentswith varicocele.

Design: Cross-sectional study.

Setting: University-based medical center.

Patient(s): Twenty-four adolescents aged between 13 and 18 years underwent surgical treatment for repairof left idiopathic varicocele.

Intervention: A testis biopsy was performed at time of surgery.

Main Outcome Measure(s): Transmission electron microscopy study of basal lamina and immunofluores-cence studies of collagen type IV and laminin, two major components of basal lamina.

Result(s): Transmission electron microscopy observations showed an uneven profile of the basal lamina witha variable thickness. Immunofluorescence studies showed an irregular immunofluorescent line that appearedinterrupted in some observations. Collagen type IV showed some areas of strong immunostaining with otherareas with reduced immunoreactivity.

Conclusion(s): Our ultrastructural and immunohistochemical observations highlight focal damage at thelevel of peritubular basal lamina, but this damage is not as severe as that described in adult varicocele. Initialinvolvement of basal lamina could represent one of the mechanisms responsible for varicocele-inducedhistologic alterations of the testes. (Fertil Sterilt 2000;73:699–705. ©2000 by American Society for Repro-ductive Medicine.)

Key Words: Adolescent varicocele, basal lamina, collagen type IV, laminin, physiopathology, immunohis-tochemistry

Varicocele is a common pathologic condi-tion in adolescents, with an incidence varyingbetween 9% and 25.8% (1). Different studieshave described numerous changes in testicularhistology (2–5), and the mechanisms that reg-ulate these alterations have not been com-pletely elucidated. Little attention has beengiven to the role played by the extracellularmatrix (ECM).

The ECM creates a network of locally se-creted polysaccharides and proteins that inter-mingles with the cell population of differenttissues. At the level of the interface betweenthe epithelium and connective tissue, the ECMis organized in a thin but strong layer knownas basal lamina, which plays a key role inregulating cell activity (6); any alteration of the

basal lamina can lead to tissue dysfunction. Inthe testes in particular, cells cooperate in ECMcomponent deposition (7) and interact witheach other through the ECM (8). Furthermore,a well-functioning epithelium depends on cell-matrix interactions (9); interactions among Ser-toli cells, peritubular myofibroblasts, Leydigcells, and germ cells are also essential for sper-matogenesis (8).

To evaluate a possible role of ECM in ado-lescent varicocele, we performed an ultra-structural study of basal lamina, with use ofa transmission electron microscope, and animmunohistochemical study of the two majorcomponents of basal lamina, laminin and col-lagen type IV, with use of a confocal laser-scanning microscope. Laminin is secreted by

Received July 1, 1999;revised and acceptedSeptember 16, 1999.Reprint requests: GiuseppeSantoro, Department ofBiomorphology, TorreBiologica 1st Floor,Policlinico Universitario,Viale Gazzi, Messina, Italy98125 (FAX: 39-090-692449;E-mail: [email protected]).* Department ofBiomorphology.† Institute of PaediatricSurgery.

FERTILITY AND STERILITY tVOL. 73, NO. 4, APRIL 2000Copyright ©2000 American Society for Reproductive MedicinePublished by Elsevier Science Inc.Printed on acid-free paper in U.S.A.

0015-0282/00/$20.00PII S0015-0282(99)00611-1

699

Sertoli cells (10) and to a lesser degree by peritubular cells(11, 12) and localized at the level of the lamina rara of thebasal lamina. Its main function is regulating the processes ofadhesion, proliferation, and cell differentiation (6). Collagentype IV, secreted by myofibroblasts and Sertoli cells (7, 13),is mainly localized at the level of the lamina densa and is aubiquitous component of basal lamina in different tissues(6). The results are discussed in relation to those obtained byother investigators in normal testes (14).

We hypothesize that modifications of ECM componentscould be already present in adolescent varicocele and play arole in varicocele-induced morphological changes.

MATERIALS AND METHODS

Twenty-four testicular biopsy specimens were obtained,with prior informed consent, from adolescents aged from 13to 18 years (mean age, 16.2), affected by left idiopathicvaricocele. Patients with varicocele were diagnosed after

physical examination, echocolor Doppler, and phlebographicstudies. A grade II or III varicocele (after Horner) was anindication for treatment. Surgical treatment was undertakenwith selective microsurgical ligation of ectatic intrafunicolarand extrafunicolar vessels, after delivery of testis (accordingto Goldstein) (15), and spermaticoepigastric microsurgicalderivation (a microsurgical vascular anastomosis betweenthe spermatic vein and the inferior epigastric vein) (accord-ing to the Belgrano technique) (16). Further information onthe patients they treated as well as the main histologicobservations are reported in Table 1.

Biopsy specimens were processed for transmission elec-tron microscopy and for immunofluorescent studies usingdifferent protocols.

In transmission electron microscopy the specimens wereimmediately fixed for 3 hours in 4% glutaraldehyde in 0.2 Mphosphate buffer, pH 7.4, at 4°C. After rinsing in the samebuffer, the specimens were postfixed in 1% OsO4 in 0.2 M

T A B L E 1

Study patients summarized by: age, pubertal stage (Tanner), varicocele degree (Horner), testicular volume (sonography),hormonal parameters, and main histological features (pathology reports). Dx 5 right. Sn 5 left.

PatientAge(y)

Pubertalstage Grade Testicular volume

Hormones(FSH, LH, SHBG) Histologic findings

1 18 V II Dx 5 523 33; Sn5 503 32. Within normal ranges Intertubular edema; peritubular fibrosis2 17 V II Within normal ranges Interstitial edema; peritubular fibrosis3 16 II II Dx 5 483 193 34; Sn5 473 183 35. Within normal ranges Interperitubular fibrosis; tubular invaginations4 18 IV II Dx 5 543 333 42; Sn 533 303 41. Within normal ranges Interstitial edema; peritubular fibrosis5 16 IV III Dx 5 423 28; Sn5 413 28. Within normal ranges Interperitubular fibrosis; incomplete spermato-

genesis6 15 IV III Dx 5 393 203 27; Sn5 383 213 27. Within normal ranges Peritubular fibrosis; incomplete germinal

epithelium7 17 IV II Interstitial edema; fibrosis and metaarteriovenular

stasis8 13 I I Atrophy Dx Consistency Sn1 Within normal ranges Interstitial edema; fibrosis9 17 IV II Dx 5 433 163 28; Sn5 413 173 28. Within normal ranges Interperitubular edema; interperitubular fibrosis;

blocked spermatogenesis in many tubules10 16 IV II Within normal ranges Interstitial edema; peritubular fibrosis; blocked

acromosomogenesis11 17 IV II Consistency Sn2 Within normal ranges Intertubular edema; peritubular fibrosis12 17 IV III Dx 5 453 183 29; Sn5 443 203 29. Within normal ranges Interperitubular fibrosis; peritubular edema;

blocked spermatogenesis in many tubules13 13 I II Consistency Sn2 FSH1; LH 1; SHBG1 Peritubular fibrosis; intertubular edema14 16 IV II Dx 5 553 34; Sn5 233 09. Within normal ranges Intertubular edema; fibrosis; germinal line

disarranged15 16 IV II Within normal ranges Interstitial edema; peritubular fibrosis16 17 IV III Dx 5 453 303 22; Sn5 433 293 23. Within normal ranges Interstitial edema; peritubular fibrosis17 17 IV III Intertubular edema; interstitial fibrosis18 15 III II Dx 5 433 243 21; Sn5 433 213 24. Intertubular edema; peritubular fibrosis19 15 IV II20 16 IV II Dx 5 203 343 26; Sn5 173 323 25. Within normal ranges Interperitubular fibrosis; intertubular edema;

blocked spermatogenesis in many tubules21 15 II II Within normal ranges Interstitial edema; intertubular fibrosis22 18 V II Dx 5 413 293 20; Sn5 403 273 19. Within normal ranges Peritubular fibrosis; intertubular edema23 17 IV III Dx 5 443 283 19; Sn5 373 193 22.24 16 III II Dx 5 383 173 20; Sn5 303 133 19. Within normal ranges Interstitial edema; fibrosis

Santoro. Basal lamina in varicocele. Fertil Steril 2000.

700 Santoro et al. Basal lamina in adolescent varicocele Vol. 73, No. 4, April 2000

phosphate buffer, pH 7.2–7.4. They were then dehydrated ingraded alcohol and acetone and flat embedded in Durcupan(Wallac Oy, Turku, Finland). The ultrathin sections were cuton an LKB Ultratome V ultramicrotome, stained with uranylacetate and lead citrate, and photographed with a transmis-sion electron microscope Philips CM10.

For immunofluorescence studies, the biopsy specimenswere fixed for 4 hours in 4% paraformaldehyde in 0.2 Mphosphate buffer (pH 7.4) at 4°C. After repeated rinsing in0.2 M phosphate buffer and in phosphate-buffered saline(PBS), the biopsy specimens were infused in 12% and 18%saccharose and then frozen in liquid nitrogen and sectionedwith use of the Bright cryostat. The 20-mm serial sections(40–50 sections per sample) were placed on gelatin-coatedglass slides and preincubated with a solution containing 0.6mL of sheep serum, 1 mL of 45 mM NaCl, and 1 mL of 20mM phosphate buffer for 15 minutes to avoid unspecificbackground staining. The sections were incubated for 15minutes with a solution containing PBS, bovine serum albu-min (BSA) 1%, and Triton X-100 0.3%. This solution wasalso used as dilution and rinsing buffer.

The primary antibodies used were as follows: anticolla-gen type IV (mouse ascites fluid, Clone COL-94; SigmaChemical Co., St. Louis, MO) at a dilution of 1:500, andantilaminin (mouse ascites fluid, Clone LAM-89; SigmaChemical Co.) at a dilution of 1:2,000. As a secondary anti-body we used a sheep anti-mouse IgG biotinylated (Amer-sham International, Little Chalfont, Buckinghamshire, UnitedKingdom) at a dilution of 1:500 and as fluorochrome Texas-red streptavidin (Amersham International) at a dilution of1:100. Negative controls were performed either by omittingthe primary antibody or by replacing the primary antibodywith nonimmune rabbit or mouse serum. Positive controlswere performed with use of antisera on normal human skin,for which data for laminin and collagen type IV are alreadyavailable (17).

The sections were observed and photographed with aLeica TCS 4D upright confocal microscope (Leica, Heidel-berg, Germany) with immersion objectives. It is equippedwith an argon krypton laser (lambda 568 nm, 100 mW;lambda 488 nm, 100 mW; and lambda 647 nm, 100 mW).Collected images were digitized at a resolution of 8 bits intoan array of 5123 512 pixels. The software controlling themicroscope and the processing of the images was providedby the manufacturer.

Optical sections of fluorescence specimens were obtainedwith a laser line (568 nm) and a tetramethylrhodamineiso-thiocyanate (TRIC) set of filters at 1-second scanning speedwith an average of up to 8. The stacks of images obtainedwere processed with different software functions: [1] singlesection, [2] 3D rotate 0° single view (overlay): overlappingthe images obtained from the single optical sections (eitherthe whole stack or only a part) to obtain a reconstruction ofthe sample throughout its thickness, [3] rotation: rotation at

different angles of the images of the whole sample or of itsparts, [4] shadow forming process: it enhances cell profilethrough the creation of different shadow effects.

The ultrastructural aspect of a normal basal lamina andthe immunohistochemical distribution of laminin and colla-gen type IV in a normal testis, reproduced with the permis-sion of Archives of Histology and Cytology (14), were usedas a control testis.

RESULTS

Transmission Electron MicroscopyLamina propria always appeared thickened because of an

increased deposition of collagen fibrils, which was princi-pally localized in the first extracellular layer. The increasedthickness was responsible for the formation of the observeddeep invaginations facing the germinal epithelium. Basallamina appeared around the seminiferous tubule; it wasformed by two different layers, one electron-dense and theother electron-transparent, displaying an uneven profile witha variable thickness; myofibroblasts were partly surroundedby basal lamina (Fig. 1).

F I G U R E 1

Basal lamina, facing the germinal epithelium, is formed by anelectron-dense and an electron-transparent layer displayinga wavy profile with a variable thickness (original magnifica-tion, 314,050).


FERTILITY & STERILITY t 701

ImmunofluorescenceLaminin immunostaining displayed an irregular immuno-

fluorescent line with a wavy profile that in some placesappeared to be interrupted at the peritubular level. Outsidethis line of fluorescence, other areas of positivity were seenbut were less intense and more irregular; this finding wasprobably due to the interrupted presence of the basal laminaof myofibroblasts (Fig. 2). The 3D reconstruction and the20° rotation of the entire thickness of the section displayed awider surface of the peritubular basal lamina. This projectionhighlighted areas of irregular immunostaining that createwide empty spaces in the thickness of the peritubular basallamina (Fig. 3).

Collagen type IV immunostaining was mainly localized atthe level of the basal lamina facing the germinal epithelium,surrounding blood vessels, and to a lesser degree inside thelamina propria (Fig. 4). The 3D reconstruction and the 20°rotation of the sample displayed a peculiar deposition ofcollagen at the peritubular level, which seemed to be orga-nized in areas of annular thickness with a wavy profile, alter-nating between areas of strong immunopositivity and areasof reduced immunoreactivity (Fig. 5). Use of the shadow-forming process highlighted the peculiar aspect of the wavydistribution of the peritubular basal lamina and a differentimmunopositivity of collagen type IV (Fig. 6).

DISCUSSION

The presence of a varicocele influences spermatogenesis,and early treatment during childhood or adolescence couldhave a beneficial effect (18). This recommendation has beenderived from clinical and histologic observations. Clini-cally, varicocele in adolescents have been associated witha smaller, softer testissimilar to that observed in adult varico-cele. Histologically, morphological alterations observed inadolescents are similar to those observed in adult varicocele.Previous studies on testicular damage have focused attentionon the intratubular and interstitial components (2, 4, 5, 19,20). Moreover, these alterations are progressive‘ (19, 21).Little attention has been focused on ECM, in particular in thebasal lamina.

In normal human testes, as reported by Holstein et al.(14), it is possible to observe a basal lamina (Fig. 7) localizedaround the seminiferous tubules, that divides tubules fromlamina propria, and a basal lamina that surrounds the myo-fibroblasts in part. The peritubular basal lamina has a regularprofile and constitutes two different thin layers: one electron-transparent, known as lamina lucida or rara, close to theplasma membrane of germinal epithelium, and the otherelectron-dense, known as lamina densa, in close relationshipto the first extracellular layer of lamina propria (6).

Basal lamina is synthesized by surrounding cells (7, 10–

F I G U R E 2

Immune reaction with antilaminin shows an interrupted pro-tein distribution at the level of the basal lamina facing thegerminal epithelium (single section; original magnification,3920).


F I G U R E 3

Immune reaction with antilaminin shows areas of interruptedfluorescence in the thickness of the peritubular basal lamina(3D reconstruction and 20° rotation; original magnification,3930).



13), and its major components are collagen type IV andlaminin; it is formed by a strong layer of collagen type IVconnected to other specific molecules, such as laminin,which are necessary for connection to adjacent cells.

Collagen type IV has been identified as a continuous anduniform line in normal human testes (14), with immunohis-tochemical reactions in both layers of the peritubular basallamina and in all the peritubular layers but with a reducedintensity. It is also present in the connective tissue surround-ing blood vessels with a strong immunoreaction (Fig. 8).

Moreover, laminin immunolocalization was identified, bythe same investigators, at the level of lamina lucida, as acontinuous and uniform line, and in the extracellular matrixof some external layers (14) (Fig. 9). Laminin plays animportant role in binding epithelial cells, via specific mem-bers of the integrin family (22), and connecting other ECMcomponents such as collagen type IV (23).

These two molecules play a fundamental role in theregulation of major biological cellular functions (24–26).Through the relationship with integrins, messages are trans-mitted from the ECM to the nuclear compartment (throughthe cytoskeleton), thus controlling the processes of differen-tiation, proliferation, adhesion, migration, and gene expres-sion (9, 27). It seems clear that quantitative and qualitativealterations in the basal lamina components and/or in theirgenetic regulation could be the basis of an acquired orinherited pathology.

In the present study, adolescents with varicocele dis-played an irregular and sometimes interrupted immunolocal-ization of laminin at the level of basal lamina facing germi-nal epithelium. This finding could be interpreted as a sign ofdistress of the basal lamina itself. Because an intact basallamina regulates cell polarity and cellular metabolism aswell as induces cellular migration and differentiation (6), andbecause ECM is fundamental for spermatogenesis (8), it ispossible to hypothesize that the morphological alterationsreported in varicocele could also depend, at least in part, onmodifications of the relationship among cells of the laminapropria, ECM, and germinal epithelium.

Collagen type IV in adolescents with varicocele displayedareas of annular thickening of the immunoreaction alternat-ing with areas of interrupted and reduced immunopositivityat the peritubular level. Moreover, distribution of the immu-noreaction appeared as irregular and wavy, following themorphology of the peritubular basal lamina observed with atransmission electron microscope along the deep invagina-tions; a similar ultrastructural morphology has been ob-served in adult varicocele and in other testicular pathologies(28, 29). The deep invaginations can be a consequence of an

F I G U R E 4

Immune reaction with type IV anticollagen shows fluores-cence at the basal lamina, peritubular, and vessel level (singlesection; original magnification, 3460).

Santoro. Basal lamina in varicocele. Fertil Steril 2000. F I G U R E 5

Immune reaction with type IV anticollagen shows areas withannular type thickening of the peritubular basal lamina (three-dimensional reconstruction and 20° rotation; original magni-fication, 3460).



increasing deposition of ECM components (30). The in-creased deposition of collagen fibrils starts from the firstextracellular layer and successively involves the other extra-cellular layers. Because of this finding, the overall thicknessof the lamina propria is increased but without signs ofsclerosis, as is observed in adult varicocele (31).

In the present study, there was a high distribution ofcollagen type IV around blood vessels; its deposition in the

lamina propria could depend on both Sertoli cells and myo-fibroblasts secreting activity. In particular, cultured Sertolicells have a higher proportional synthesis than myofibro-blasts. In vitro cocultures of Sertoli cells and myofibroblastsdisplay a higher synthesis rate, relative to total synthesis, thanmonoculture alone, indicating an in vitro biosynthetic coop-eration between these two cell types (13). The altered distri-bution of collagen type IV, observed in our study in the peri-tubular basal lamina, could result in unfavorable conditionsfor cooperation between myofibroblasts and Sertoli cells.

In conclusion, our ultrastructural and immunohistochem-ical observations highlight focal damage principally local-ized at the level of the peritubular basal lamina, but this

F I G U R E 6

Immune reaction with type IV anticollagen: the shadow form-ing process highlights the wavy distribution at the basallamina level (original magnification, 3460).


F I G U R E 7

Control testis: cross section of the peritubular basal laminashowing a regular profile and thickness. Basal part of germi-nal epithelium (1), basal lamina (2), and first extracellular layerof collagen fibrils of lamina propria (3) (original magnification,311,500) (reprinted with permission of Archives of Histologyand Cytology).


F I G U R E 8

Control testis: laminin immunoreactivity is distributed as acontinuous and uniform line in the peritubular basal laminaand in the extracellular matrix of some external layers (orig-inal magnification, 3630) (reprinted with permission of Ar-chives of Histology and Cytology).


F I G U R E 9

Control testis: collagen type IV immunoreactivity in two ad-jacent seminiferous tubules. The reaction precipitate is lo-cated in the peritubular basal lamina as a continuous anduniform line, and in the extracellular matrix of most layers,but with a reduced intensity (original magnification, 3630)(reprinted with permission of Archives of Histology andCytology).



damage is not as severe as that already described in adultvaricocele (31). Although the clinical implications of thisstudy are difficult to interpret because of the young age of thepatients, we believe that these observations could contributeto an understanding of the natural history of adolescentvaricocele. Because varicocele should be regarded as a dy-namic lesion that does not disappear spontaneously in adult-hood (32) and histologic changes reported seem to be pro-gressive (19, 21), it is possible to speculate that the initialdamage to the basal lamina observed in this study could onlyworsen if the pathophysiological mechanisms responsiblefor varicocele persist.

Acknowledgment:The authors thank Daniele Liberatore, M.D., (Leica) forhis expert technical assistance.

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ultrastructural and immunohistochemical study of basal lamina of the testis in adolescent varicocele

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