Histochemical Study of the Interstitial Tissue in Scrotal and Abdominal Boar Testes

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The Veterinary Journal 2002, 163, 68±76 doi:10.1053/tvjl.2001.0630, available online at http://www.idealibrary.com on

Histochemical Study of the Interstitial Tissue in Scrotal and Abdominal Boar Testes E. PINART, S. BONET, M. BRIZ, L.M. PASTOR*, S. SANCHO, N. GARCIÂA, E. BADIA and J. BASSOLS Reproductive Biology Unit, Department of Biology, Faculty of Sciences, University of Girona, Campus de Montilivi, 17071 Girona, Spain and *Cellular Biology Unit, School of Medicine, University of Murcia, Campus de Espinardo, 30071 Murcia, Spain

SUMMARY The present study describes the glycosidic content of the interstitial tissue in testes from healthy boars and from unilateral and bilateral abdominal cryptorchid boars using lectin histochemistry. The Leydig cells of healthy boars contained glycans with fucosyl, mannosyl, glucosyl, neuraminic acid and galactosyl residues, which have structural and transport functions, and participate in androgen synthesis and in cell regulation. Unilateral cryptorchidism induced high glucosyl and low galactosyl content in the Leydig cells of scrotal testes, resulting in impaired androgen production. In abdominal testes, the Leydig cells exhibited increased amounts of glucosyl and reduced amounts of galactosyl and neuraminic acid residues, resulting in defective cell regulation and lack of androgen synthesis. In healthy boars, the extracellular glycans contained fucosyl, galactosyl, glucosyl and neuraminic acid residues, which confer viscoelasticity on the interstitial tissue and participate in substrate transport, hormome binding and cell±cell interaction. Unilateral cryptorchidism did not induce anomalies in extracellular glycans in scrotal testes, but unilateral and bilateral cryptorchidism resulted in an increased content of fucosyl and galactosyl, and a decreased content of glucosyl and neuraminic acid residues in abdominal testes, leading to reduced viscoelasticity and defective substrate transport across the extracellular matrix. # 2002 Harcourt Publishers Ltd

KEYWORDS: Abdominal cryptorchidism; Leydig cells; fibroblasts; extracellular matrix; Sus domesticus.

INTRODUCTION Cryptorchidism is the most frequent male sexual disorder in mammals, arising from a failure in the descent towards the scrotum of one testis (unilateral cryptorchidism) or both testes (bilateral cryptorchidism). The condition can be either total, where the testes remain in the abdominal cavity (abdominal cryptorchidism), or partial, with the testes lodged at different levels of the inguinal canal (inguinal cryptorchidism) (Pinart et al., 2000). The interstitial tissue of healthy post-pubertal boars occupies about 30±35% of the testicular parenchyma; it is composed of abundant Leydig cells, a few fibroblasts and mast cells, and scarce and Correspondence to: Dr. Elisabet Pinart. Reproductive Biology Unit, Department of Biology, Faculty of Sciences, University of Girona, Campus de Montilivi, 17071 Girona, Spain. Tel.: ‡34 972 41 83 66; Fax: ‡34 972 41 81 50; E-mail: [email protected] 1090-0233/02/010068 ‡ 09 $35.00/0

unevenly distributed blood capillaries (Pinart et al., 2001a, b). All these elements are immersed in an extracellular matrix of proteoglycans (Grudet et al., 1999), which are expressed not only in extracellular matrix, but also in interstitial cells (Grudet et al., 1999) and exert essential roles in testicular dynamics by participating in cell±cell and cell±extracellular matrix interactions, acting as anchore devices for hormones and growth factors, and regulating cell function (Arenas et al., 1998a; Grudet et al., 1999). In previous studies we found that, after puberty, right-sided unilateral abdominal cryptorchidism induces a significant development of the interstitial tissue in scrotal and abdominal testes of boars. This tissue occupies 50% of scrotal and 55% of abdominal testicular parenchyma (Pinart et al., 1999a). Bilateral abdominal cryptorchidism also leads to a significant development of the interstitial tissue of post-pubertal boars, comprising about 60% of # 2002 Harcourt Publishers Ltd

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testicular parenchyma in the left testis and 95% in the right one (Pinart et al., 1999a). Unilateral abdominal cryptorchidism does not affect the structural organization of the interstitial tissue in scrotal testes, but both unilateral and bilateral abdominal cryptorchidism induce severe structural and cytological anomalies of the interstitial tissue in ectopic testes, such as Leydig cell immaturity, fibroblast proliferation, collagenization of the extracellular matrix, angiogenesis, erythrocyte and leukocyte infiltration, and mastocytosis (Pinart et al., 2001a,b). In several tissues, mastocytosis, leukocyte extravasation, and fibroblast proliferation are associated with disturbances in the content and nature of extracellular proteoglycans, as well as in the glycosilation pathways of interstitial cells ( Jezek et al., 1999). The increasing use of lectin histochemistry in the testis has provided information about the sugar nature of glycoconjugates in the seminiferous epithelium, but little data exist about the glycan content of the interstitial tissue under normal and pathological conditions (Arenas et al., 1998a). The present work describes the lectin affinity of Leydig cells, fibroblasts, and extracellular matrix in the testicular interstitial tissue of healthy boars and of unilateral and bilateral abdominal cryptorchid boars. The study of cryptorchidism in boars is of great interest because the results can be extrapolated to humans (Pinart et al., 1999b; 2000). Moreover, recent data highlight the importance of studying post-pubertal cryptorchidism in order to understand better the pathogenesis of reduced fertility in

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treated and untreated adult cryptorchid males (Pinart et al., 2000; 2001b). MATERIAL AND METHODS The study was performed using nine Sus domesticus post-pubertal males: three healthy boars, three boars with spontaneous unilateral abdominal cryptorchidism on the right side, and three boars with spontaneous bilateral abdominal cryptorchidism. The animals were kept in a controlled environment with an average temperature of 18 C and fed a nutritious diet. The males were slaughtered at nine months of age, and the testes were removed immediately and processed for light microscopy. Testicular samples were fixed in Bouin's fluid, dehydrated in increasing ethanol series and embedded in paraffin at 56±58 C (Pinart et al., 1999a). Sections were cut at 5 mm and stained with thirteen lectins conjugated with peroxidase (PNA, SBA, WGA, LTA, Con A, DBA, UEA-I, HPA, RCA-I, LFA, AAA, SNA, and GNA). The procedure was as follows (Calvo et al., 2000): (1) in hydrated sections, endogenous peroxidase was blocked with 1% v/v H2O2 for 30 min; (2) after washing in TBS (Trisbuffered saline, pH 7.4), the slides were incubated for 1 h at room temperature in a moist chamber with horseradish peroxidase-conjugated lectins (Sigma); (3) after washing in TBS, the peroxidase was developed by immersing the slides in TBS with 0.05% v/v 3,3 0 -diaminobenzidine tetrahydrochloride and 0.3% v/v H2O2 for 30 min; and (4) the sections were

Table I Concentration and specificity of lectins Taxonomic name (abbreviation)

Concentration

Specificity*

Lotus tetragonolobus (LTA) Ulex europaeus (UEA-I) Auleria aurantia (AAA) Galantus nivalis (GNA) Canavalia ensiformis (Con A) Helix pomatia (HPA) Dolichos biflorus (DBA) Glycine max (SBA) Arachis hypogea (PNA) Ricinus communis (RCA-I) Limax flavus (LFA) Triticum vulgaris (WGA) Sambucus nigra (SNA)

25 mg/mL 25 mg/mL 15 mg/mL 15 mg/mL 20 mg/mL 15 mg/mL 15 mg/mL 15 mg/mL 15 mg/mL 10 mg/mL 25 mg/mL 10 mg/mL 15 mg/mL

-L-Fuc -L-Fuc (1 ! 6)Fuc -D-Man -D-Man > -D-Glc -D-GalNAc -D-GalNAc -/ -D-GalNAc -D-Gal > -D-Gal (1 ! 3)-D-GalNAc Gal- (1 ! 4)-GlcNAc NeuAc Gal- (1 ! 4)GlcNAc > D-GclNAc > Neu5Ac Neu2,6-Gal (1 ! 4)GlcNAc > Neu2,6-Gal (1 ! 4)GalNAc

*Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose; Neu, neuraminic acid.

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counterstained with Delafield's haematoxylin. The specificity and concentration of each lectin are summarized in Table I. For each testis, two different sections were stained with each lectin in order to assess both the reproducibility of staining and the variation in staining intensity within animal groups. To overcome subjectivity, all the samples were examined by the same experienced technician. RESULTS There was little individual variation among the three healthy boars, the three unilateral cryptorchid boars and the three bilateral cryptorchid boars in the lectin binding pattern of the testes. The lectin affinity of Leydig cells, fibroblasts, and extracellular matrix in the interstitial tissue of the left and right testes from healthy boars, the left (scrotal) and right (abdominal) testes from unilateral cryptorchid boars, and the left (abdominal) and right (abdominal) testes from bilateral cryptorchid boars are expressed in Table II. The Con A and SBA affinities of the interstitial tissue in scrotal and abdominal testes are illustrated in Figs 1 and 2, respectively. DISCUSSION The boar is considered an appropiate animal model to study human cryptorchidism (Hutson et al., 1998; Pinart et al., 1999a) due to marked similarities between humans and boars in sperm morphology, testicular structure, time of testicular descent etc. (Hutson et al., 1998). However, cryptorchidism in boars has been little studied due to the difficulties and expense of research using these animals. Besides, cryptorchidism manifests in different modalities, so it is difficult to find boars of the same age expressing the same type of cryptorchidism (Pinart et al., 1999a). Carbohydrates linked to proteins determine folding, conformation, function, turnover, solubility, targeting, and sorting of the molecule (ToÈpferPetersen, 1999). The oligosaccharides that link to proteins by a N-acetylgalactosamine residue to asparagine aminoacids are named N-linked glycans; these oligosaccharides exhibit high content of terminal mannose and/or neuraminic acid (Spicer & Schulte, 1992; Wheatley & Hawtin, 1999). Other oligosaccharides attach to serine or threonine aminoacids via a N-acetylgalactosamine; these O-linked glycans contain galactosyl, fucosyl,

N-acetylneuraminic, and N-acetylglucosamine residues (Wheatley & Hawtin, 1999). In O-linked glycans, D-N-acetylgalactosamine and D-N-acetylglucosamine complexes can contain a sulphate group, thus forming sulphated glycans (Salustri et al., 1999). Proteoglycans have generalized effects, but they also show specific functions depending upon the tissue (Spicer & Schulte, 1992). The Leydig cells of healthy boars had both Nlinked and O-linked glycans, as indicated by the presence of fucosyl, glucosyl, galactosyl, and neuraminic acid residues (Table II). Similar carbohydrate moieties were reported in glycans of human (Arenas et al., 1998a) and rodent (Grudet et al., 1999) Leydig cells. According to Grudet et al. (1999), O-linked glycans of Leydig cells are frequently sulphated. In general, sulphated residues participate in cellular uptake pathways (Salustri et al., 1999), but in Leydig cells they are also required for androgen synthesis (Grudet et al., 1999) and for autocrine regulation of cell activity (Cancilla et al., 2000). Fucoconjugates participate in transport of substrates throughout the cytoplasm (ToÈpfer-Peterson, 1999; Wheatley & Hawtin, 1999), and glucose and mannose residues participate in transport of ions (Spicer & Schulte, 1992). -D-N-acetylgalactosamine is important for the transport of fluids and ions (Spicer & Schulte, 1992) and in the control of membrane permeability (ToÈpfer-Petersen, 1999). Fucosyl, glucosyl, and galactosyl residues also have structural functions (Arenas et al., 1998b). Neuraminic acid complexes participate in cell protection against dehydration, in transport of metabolites and ions across cellular membranes, and in hormone binding to the plasma membrane (Arenas et al., 1998b). Cytoplasmic Nglycans have structural functions, but in Leydig cells they also regulate the folding of LH receptors (Zhang et al., 1995). In this study, Leydig cells of scrotal testis from unilateral cryptorchid boars exhibited anomalies in the sugar nature of O-linked glycans; therefore, as compared with healthy boars, they had altered contents of glucosyl and galactosyl residues (Table II). The changes in the pattern of glycosilation in Leydig cells are considered to be highly sensitive markers of altered function (Arenas et al., 1998a). Therefore, our results suggested that unilateral abdominal cryptorchidism induced disturbances in transport of fluids and ions and in membrane permeability, and also abnormal synthesis of androgens in Leydig cells of scrotal testis. Alterations in substrate transport and in membrane permeability of Leydig cells were described in ectopic testes of unilateral cryptorchid

Table II Lectin affinity of the interstitial tissue in the left and right testes of healthy boars, the left (scrotal) and right (abdominal) testes of unilateral cryptorchid boars, and the left (abdominal) and right (abdominal) testes of bilateral cryptorchid boars Boars

Interstitial tissue

Leydig cells Fibroblasts Extracellular Right testis Leydig cells Fibroblasts Extracellular Unilateral cryptorchid Left testis Leydig cells boars Fibroblasts Extracellular Right testis Leydig cells Fibroblasts Extracellular Bilateral cryptorchid Left testis Leydig cells boars Fibroblasts Extracellualr Right testis Leydig cells Fibroblasts Extracellular

Lectin affinity* LTA

UEA-I

AAA

GNA

Con A

ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ‡ ‡ ÿ ‡ ‡ ÿ ‡ ‡

ÿ ÿ/‡ ‡ ÿ ÿ/‡ ‡ ÿ ÿ/‡ ‡ ‡ ‡ ‡‡ ‡‡ ‡ ‡‡ ‡‡‡ ‡ ‡‡

‡ ‡ ‡‡ ‡ ‡ ‡‡ ‡ ‡ ‡‡ ÿ/‡ ÿ/‡ ‡ ÿ/‡ ÿ/‡ ‡ ÿ/‡ ÿ/‡ ‡

‡ ÿ ÿ ‡ ÿ ÿ ‡ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ

‡ ÿ/‡ ‡ ‡ ÿ/‡ ‡ ‡‡ ÿ/‡ ‡ ‡‡ ÿ/‡ ‡ ‡‡ ÿ/‡ ‡ ‡‡‡ ÿ ÿ

Left testis

matrix

matrix

matrix

matrix

matrix

matrix

HPA DBA ‡ ÿ ÿ ‡ ÿ ÿ ‡ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ

‡ ÿ ÿ ‡ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ

SBA

PNA

RCA-I

LFA

WGA

SNA

‡ ÿ/‡ ‡ ‡ ÿ/‡ ‡ ÿ ÿ/‡ ‡ ÿ ‡ ‡‡ ÿ ‡ ‡‡ ÿ ‡ ‡‡

‡‡ ‡ ‡‡ ‡‡ ‡ ‡‡ ‡ ‡ ‡‡ ÿ ÿ/‡ ‡ ÿ ÿ/‡ ‡ ÿ ÿ ÿ

ÿ ‡ ‡‡ ÿ ‡ ‡‡ ÿ ‡ ‡‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡

‡ ‡ ‡‡ ‡ ‡ ‡‡ ‡ ‡ ‡‡ ÿ ÿ/‡ ‡ ÿ ÿ/‡ ‡ ÿ ÿ/‡ ‡

ÿ/‡ ‡ ‡‡ ÿ/‡ ‡ ‡‡ ÿ/‡ ‡ ‡‡ ‡ ‡‡ ‡‡‡ ‡‡ ‡‡ ‡‡‡ ‡‡ ‡‡ ‡‡‡

‡ ÿ ÿ ‡ ÿ ÿ ‡ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ

HISTOCHEMISTRY OF THE TESTICULAR INTERSTITIUM

Healthy boars

Testes

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*Staining intensity: ÿ negative, ‡ weak, ‡‡ moderate, ‡‡‡ strong.

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Fig. 1. Con A affinity of the left testis (A) of healthy boars, the left (scrotal, B) and right (abdominal, C) testes of unilateral cryptorchid boars and the left (abdominal, D) testis of bilateral cryptorchid boars. The interstitial tissue of the right (abdominal) testis of unilateral cryptorchid boars contains abundant small mesenchymatic cells showing greater Con A affinity than Leydig cells. Note the weak affinity of the Leydig cells in healthy boars, which showed large central cytoplasmic regions poorly stained. BV, blood vessels; F, fibroblasts; L, Leydig cells; ST, seminiferous tubules. A: 55; B: 60; C: 60; D: 70.

males, but not in the contralateral scrotal testes (Tanigawa et al., 1990). On the other hand, divergent views exist about the effects of unilateral cryptorchidism on the steroidogenic activity of the scrotal testis; thus, where as some authors describe unaffected androgen synthesis, others report increased or even decreased testosterone production (Pinart et al., 2001b). In normal and pathological conditions, Leydig cell function depends on the presence of specific germ cell populations in the seminiferous tubules (Wu & Murono, 1996). Pachytene spermatocytes and spermatids modulate the secretion of specific paracrine factors by Sertoli cells, which are implicated in the regulation of Leydig cell activities ( JeÂgou & Sharpe, 1993; Wu & Murono, 1996).

In several disturbances, including varicocele, X-irradiation, vitamin A deficiency, efferent duct ligation or heat stress, the damage to spermatogenesis results in impaired steroid production (JeÂgou & Sharpe, 1993). In the scrotal testis of unilateral abdominal cryptorchid boars, the alterations in Leydig cells correlate with the partial spermatogenic arrest at spermatocyte and spermatid stages (Pinart et al., 1999a,b). In the present study, Leydig cells of abdominal testes from post-pubertal boars displayed an altered sugar nature of both N-linked and O-linked glycans (Table II), suggesting disturbances in transport of nutrients and ions, in membrane permeability, in endocrine and autocrine regulation, and in steroid

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Fig. 2. SBA affinity of the left (scrotal, A) and right (abdominal, B) testes of unilateral cryptorchid boars and the left (abdominal, C) and right (abdominal, D) testes of bilateral cryptorchid boars. Note the highest SBA affinity of the extracellular matrix among Leydig cells in the abdominal testes as compared with the scrotal testis. EM, extracellular matrix; F, fibroblasts; L, Leydig cells; ST, seminiferous tubules. A: 70; B: 60; C: 70; D: 60.

production (Spicer & Schulte, 1992; Grudet et al., 1999; Cancilla et al., 2000). The decrease in testosterone production of ectopic testes was extensively reported in unilateral and bilateral cryptorchid men (Regadera et al., 1991; Sheth et al., 1996), dogs (Kawakami et al., 1993), boars (Raeside et al., 1988; Pinart et al., 2001b) and stallions (al-Bagdadi et al., 1991), as well as in men with a history of prepubertal cryptorchidism (Mieusset et al., 1995). In the Leydig cells of adult cryptorchid rats, Tanigawa et al. (1990) also found anomalies in transport of ions and nutrients, and altered membrane permeability. A close relationship exists between plasma membrane fluidity and number, type, and activity of gonadotropine receptors in Leydig cells; thus, the alterations of membrane fluidity result in abnormalities

in the content and nature of LH and hCG receptors, and therefore in decreased testosterone production (Wu & Chan, 1999). On the other hand, the anomalies in organelle membrane permeability leads to a low cellular activity (Tanigawa et al., 1990). In abdominal testes, the decrease in androgen synthesis was also related to disturbances in aromatase (Raeside et al., 1988) and glycosilation (Pinart et al., 2001b) pathways. The anomalies in Leydig cells of abdominal testes from post-pubertal boars develop as a result of abnormal paracrine stimulation from immature Sertoli cells (Antich et al., 1995; Pinart et al., 1999a; 2000). In scrotal tests of healthy boars, the histochemical correspondence between fibroblasts and the extracellular matrix (Table II) indicated that the sugar

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residues were mainly synthesized and secreted by these cells. However, the low lectin affinity of fibroblasts showed a low rate of synthesis and turnover of extracellular components at post-pubertal age. We recently reported that fibroblasts of post-pubertal healthy boars exhibited poor development of cell organelles implicated in synthetic pathways, such as endoplasmic reticulum, Golgi complex and nucleolus (Pinart et al., 2001b). Low glycan production was also described in fibroblasts from post-pubertal and adult men (Grudet et al., 1999) and rodents (Cancilla et al., 2000). Grudet et al. (1999) found that the greatest extracellular matrix production occurs perinatally, and then decreases progressively throughout the postnatal life. In healthy adult males, fibroblasts are implicated in paracrine control of spermatogenesis and steroidogenesis under the stimulus of Sertoli cells (Schteingart et al., 1999). In healthy boars, the extracellular glycoconjugates of the interstitial tissue had low content of Nlinked residues and abundant O-linked residues (Table II). Extracellular -D-galactose, D-N-acetylgalactosamine, and L-fucose have structural functions (Parillo et al., 1998). D-galactose, L-fucose, and neuraminic acid residues participate in cell±cell interaction; besides, L-fucose and neuraminic acid regulate the substrate diffusion across extracellular matrix and the hormone binding (Parillo et al., 1998). Glucose residues facilitate the ion transport into the cells (Spicer & Schulte, 1992), and N-acetylglucosamine residues increase the muco-elasticity of the extracellular matrix (Salustri et al., 1999). Extracellular sulphated proteoglycans mediate cell± matrix interactions (Arenas et al., 1998b; Salustri et al., 1999); in the testes, they also have an essential role in the regulation of synthesis and secretion of testosterone, and also seem to be important for the maintenance of colesterol supply to Leydig cells (Grudet et al., 1999; Cancilla et al., 2000). Lack of neuraminic acid--2, 6-galactose complexes in the extracellular matrix could be explained by the fact that these sugars inhibit intermolecular and intercellular interactions (ToÈpfer-Petersen, 1999). Therefore, extracellular glycans of the testicular interstitium seem to confer viscoelastic properties but also structural cohesion, and to facilitate both cell±cell communication and substrate diffusion. A similar glycan nature and function was reported in the extracellular matrix of post-pubertal testes taken from rats (Cancilla et al., 2000) and men (Arenas et al., 1998a). The interstitial tissue of the scrotal testes from unilateral cryptorchid exhibited no anomalies either

in the nature or in the content of sugar residues of the extracellular matrix. These results suggest that, despite the partial spermatogenic arrest (Pinart et al., 1999b) and the defective Leydig cell steroidogenesis, unilateral abdominal cryptorchidism did not alter the extracellular matrix synthetic pathways of fibroblasts in the scrotal testis. Recently reported data also show a lack of anomalies in both fibroblast density and ultrastructure in scrotal testis from unilateral cryptorchid males (Pinart et al., 2001b). It has been suggested that the damage of the extracellular matrix and fibroblasts occurs in cases of severe alterations in Sertoli cells (Schteingart et al., 1999) and/or of defective testicular blood supply (Setchell et al., 1995). In abdominal testes of unilateral and bilateral cryptorchid boars, the interstitial tissue showed decreased content of N-glycans and altered sugar composition of O-glycans (Table II). Previous studies described high contents of collagen fibres, fibronectin and laminin, and low contents of elastin in the interstitium of abdominal testes from adult males (al-Bagdadi et al., 1991; Regadera et al., 1991; Pinart et al., 2001b). These abnormalities correlate with a high synthetic activity of fibroblasts (Pinart et al., 2001b), and probably lead to decreased viscoelasticity, altered cell±cell and cell±extracellular matrix interactions, impaired substrate diffusion across the extracellular matrix, and defective colesterol supply to Leydig cells. In post-pubertal cryptorchid testes, the alterations in fibroblast metabolism correlate with Sertoli cell immaturity (Antich et al., 1995; Pinart et al., 2000), mast cell proliferation (Jezek et al., 1999; Pinart et al., 2001b), and defective testicular perfusion (Setchell et al., 1995; Pinart et al., 2001a). In conclusion, Leydig cells from healthy boars contained both N-linked and O-linked glycans, which had structural and transport functions and participated in androgen synthesis and in cell regulation. The extracellular matrix surrounding Leydig cells was mainly constituted by O-linked glycans, proceeding from fibroblasts, which confered viscoelasticity and structural cohesion to the interstitial tissue, and also facilitated the substrate diffusion and the endocrine regulation of Leydig cells. Unilateral abdominal cryptorchidism did not affect the sugar nature of extracellular glycans of the scrotal testis from post-pubertal boars, but resulted in an abnormal sugar content of O-linked glycans in Leydig cells, leading to impaired androgen production. Unilateral and bilateral cryptorchidism resulted in anomalies in N-linked and O-linked glycans of

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both Leydig cells and extracellular matrix of abdominal testes, which lead to decreased viscoelasticity of the interstitial tissue, altered substrate transport across the extracellular matrix, impaired endocrine and autocrine regulation of Leydig cells, and lack of androgen synthesis. Further investigations are necessary to understand better the mechanisms implicated in the partial and total arrest of steroidogenesis in scrotal and abdominal testes of post-pubertal cryptorchid males. Moreover, in abdominal testes, the knowledge of the factors that influence the extracellular matrix production may allow for the design of specific inhibitors that can prevent the cell degeneration at an early adult age (Tanigawa et al., 1990; Regadera et al., 1991; Pinart et al., 2000; 2001b).

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doi: 10.1053/tvjl.2001.0591, available online at http://www.idealibrary.com on

Book Review Avian Medicine Tully, T., Dorrestein, M. & Lawton, M. Oxford, ButterworthHeinmann, 2000, 411pp. 55. (hard) ISBN 0750635983

The first impressions of this book are very favourable. It is nicely laid out with, for the most part, good black and white photographs, figures and tables to supplement the text, and a section of colour photographs to illustrate a variety of conditions. Unfortunately, I found that typographic errors were quite common. Most were irritating, but shouldn't cause confusion. For example most readers will realise that A. fumagatis (page 50) is A. fumigatus. The index is clearly laid out and extensive, but I did have to search to find the Bursa of Fabricius listed as the `Cloacal bursa of Fabricius'. I found the first five general chapters extremely useful, taking the reader through basic avian anatomy, physiology and nutrition, the physical examination, clinical tests, imaging techniques and nursing of the sick bird. There are many differences between the examination techniques, diagnostic tests and treatments used in mammals and in birds, and these chapters cover many of the unique features of birds that have a bearing on disease processes and their management. The remainder of the book comprises ten chapters dealing with bird groups (psittacine birds, passerines and exotic softbills etc.), a chapter on the management of a multi-species bird collection in a zoological park, and a final chapter on drug dosing. I found the chapters on the bird groups to be a mixed bag, but generally very useful, with concise information on

taxonomy and biology, captive husbandry, feeding, reproduction and important diseases and their treatment. Unfortunately, the chapter on bird collections in a zoo, which I was particularly interested in, repeated much of the information from the other chapters. In particular, disease of the ramphastids (toucans) were covered three times in the book; in this chapter, in their own chapter, and in the one on passerines and exotic softbills! In complete contrast, penguins don't get a mention despite being popular zoo birds and subject to several diseases, most notably avian malaria, for which up-to-date information is essential. The final `Quick reference for drug dosing' gives a list of metabolic dose-rates for birds weighing between 10 g and 1.5 kg, with a conversion table for basal metabolic rate of passerines and non-passerines. Unfortunately, this makes it not quite the `quick reference' of the title, compared to more traditional formularies based on mg/kg for a range of species. There are now several books on avian medicine, so the choice of which book, or books, to purchase will depend largely on the needs of the individual. Anyone seeing a lot of psittacines will have the BSAVA guide, and probably `Avian Medicine: Principles and Applications' by Ritchie, Harrison and Harrison, and will find little in this book to add to these. Likewise, those with a particular interest in raptors will probably use the BSAVA guide to Raptors, Pigeons and Waterfowl and the `Raptor Biomedicine' series more than this. However, for vets who see a few each of a range of birds, `Avian Medicine' should prove extremely useful, and a first source of information when treating an unfamiliar bird or condition. EDMUND FLACH