Characterization and distribution of hyaluronan and the proteoglycans decorin, biglycan and perlecan in the developing embryonic mouse gonad

Abstract

The morphogenesis of tissues and organs requires dynamic changes in cells and in extracellular matrix components. It is known that various extracellular matrix molecules are of fundamental importance for gonad differentiation and growth. In the adult testis, the extracellular matrix represents an important component of the interstitium, participating in the transport of biologically active substances needed for the communication between different cellular components, as well as for the regulation of spermatogenesis and hormone production. The present study was designed in order to identify the proteoglycans biglycan, decorin and perlecan, as well as the glycosaminoglycan hyaluronan, during testis development in mouse embryos. Our data profile the chronology of testis differentiation, as well as the distribution of these extracellular matrix components during testis development in mice. We show that these extracellular matrix molecules are present early in the development of the gonads, suggesting that they play a role in gonad development. In addition, we found no decorin in the testicular cords. Furthermore, of the proteoglycans analysed, only biglycan was seen surrounding immature Sertoli cells and Leydig cell precursors in the testicular cords. This indicates that specific sets of extracellular matrix molecules are required in the various compartments of the developing gonad.

Introduction

Mammalian gonadal development and sexual differentiation are complex processes that require the coordinated expression of specific sets of genes in a strictly spatiotemporal manner (Viger et al. 1998). Gonadal differentiation initiates when the primordial germ cells (PGCs) arrive at and populate the genital ridges. The differentiation of a testis or an ovary is what holds the key to sexual development, and is under control of the SRY gene present on the Y-chromosome (Gubbay et al. 1990; Sinclair et al. 1990; Koopman et al. 1991; Mackay, 2000; Koopman, 2001; Veitia et al. 2001). In the male, supporting cell lineages in the genital ridges are induced to differentiate as Sertoli cells. Immature Sertoli cells form clusters enveloping the PGCs, and both are confined to a common compartment designated the testicular cords (Koopman et al. 1990; Palmer & Burgoyne, 1991), which, in all mammals, will form the seminiferous tubules and epididymis in adult testis.

The PGCs constitute an embryonic cell line that gives rise to gametes in vertebrates. At a very early stage of embryonic development, PGCs originate outside the embryo proper and migrate by a well-defined extracellular matrix (ECM)-rich pathway to the genital ridges (Fujimoto et al. 1985; Garcia-Castro et al. 1997; Soto-Suazo et al. 2002).

The morphogenesis of tissues and organs requires dynamic changes in the ECM. In fact, it is known that the ECM plays a fundamental role in the differentiation of the testis (Pelliniemi et al. 1984). In the adult testis, the ECM is an interstitial component that is important for the transport of the biologically active substances needed for the communication between different cellular components, as well as for the regulation of spermatogenesis and hormone production (Ungefroren et al. 1995). During the postnatal development of rat testis, laminin, fibronectin and entactin-1 play important roles in the morphogenesis process (Weber et al. 2002). The different tissue compartments in the testis (the interstitial space, the blood vessel walls and the basement membrane of the seminiferous tubules) are particularly rich in ECM molecules, such as proteoglycans (Takaba et al. 1991).

The differentiating gonads consist of somatic cells that constitute the coelomic epithelium, mesenchymal tissue, mesonephric tissue and PGCs. In the mouse, the differentiation of the testis initiates at 10.5 days post-conception (dpc), the time at which the majority of PGCs are arriving at the genital ridges (Ginsburg et al. 1990; Gomperts et al. 1994; Molyneaux et al. 2001). At this time, PGCs acquire an elongated shape, exhibit pseudopodia and present an irregular outline under the electron microscope (Soto-Suazo & Zorn, 2005; Pereda et al. 2006). Histochemical detection of alkaline phosphatase activity can be used to identify PGCs (Clark & Eddy, 1975). However, the most precise method of identifying PGCs involves the use of monoclonal antibodies, such as the carbohydrate stage-specific embryonic antigens (SSEAs) α-SSEA-1, α-SSEA-3 and SSEA-4 (Fox et al. 1981; Shevinsky et al. 1982). At 12.5 dpc, the XY gonad presents clear morphological changes that are not apparent in the XX gonad (McLaren, 1983; Schmahl et al. 2000). At this stage the mouse testis presents large peripheral blood vessels and a newly forming testicular cords. As early as 13 dpc, the differentiation of testicular cords occurs rapidly. Over the following days (14 and 15 dpc), the testicular cords as well the surrounding basement membrane enlarge significantly. Embryonic testis development requires the morphogenesis of cords and the growth of their cell populations to allow organ formation. It is well accepted that the coordination of growth and differentiation of various cell types requires locally produced ECM components (Pelliniemi et al. 1984; Hadley et al. 1985, 1988; Zanchetta & Munari, 1987; Gelly et al. 1989; Santamaria et al. 1990; eeviewed by Luck, 1994; Mackay, 2000).

In early reports, structural components of the ECM were detected at different developmental stages in the differentiating rat gonad: fibronectin was detected in embryos from embryonic day (E)12 to E15 by Paranko et al. (1983); fibronectin and laminin were detected in embryos from E13 to E17 by Agelopoulou & Magre (1987); entactin-1 was detected in young adult male rats by Weber et al. (2002); type I and III collagen were detected in testis from the undifferentiated stage E12 until birth by Paranko (1987); type IV collagen was detected in rat gonads from 12 dpc to 20 dpc by Gelly et al. (1989); fibroblast growth factors and receptors have been detected in the gonads of postpartum rats (Cancilla et al. 2000); and transforming growth factor-alpha was detected in cultivated mature gonads of rats from 13 dpc until birth by Levine et al. (2000).

Proteoglycans and glycosaminoglycans are important for organogenesis and ECM assembly, creating distinctive profiles that affect many of the characteristics of the extracellular microenvironment in a variety of development systems (Toole, 1981, 1991). Due to their ability to bind growth factors, decorin and biglycan have been correlated with cell proliferation and differentiation (Yamaguchi et al. 1990; Ruoslahti & Yamaguchi, 1991). The glycosaminoglycan hyaluronan occupies large domains of the extracellular spaces. Because it is highly polyanionic and is hydrophilic in nature, hyaluronan can expand the extracellular space and create ‘paths of least resistance’ for cell migration (Toole & Trelstad, 1971; Browder et al. 1991; Toole, 1991). In addition, hyaluronan is believed to play an important role in controlling cell growth and differentiation during embryonic development (Toole, 2001). The proteoglycan versican, a member of the hyalectin family of molecules, binds to hyaluronan and to ECM compounds in a wide variety of tissues, creating loose, hydrated matrices during key events in development and disease. Versican also regulates cell adhesion, survival, proliferation and migration, as well as ECM assembly and determination of the cell phenotype (Wight, 2002).

Considering the importance of ECM molecules in organogenesis, the aim of the present study was to identify the proteoglycans biglycan, decorin and perlecan, as well as the glycosaminoglycan hyaluronan, during testis development. We report the chronology of testis differentiation, as well as the expression and distribution of these ECM components during testis development of mice.

Materials and methods

Tissue collection

Swiss female mice aged 2–3 months were used. The animals were housed in a light- and temperature-controlled environment (12-h light/dark cycle, 22 °C), with food and water available ad libitum. Females were caged overnight with breeding males and examined the next morning to check for a vaginal plug, which would indicate that copulation had occurred. The day on which a plug was identified was considered 0 dpc (Fujimoto et al. 1985). At least three animals per day were used at 12, 13, 14 and 15 dpc. The animals were deeply anaesthetized with single intraperitoneal injections of tribromoethanol (Avertin®; 0.025 mL/g body weight) and killed by cervical dislocation, after which the uteri were removed. On each day post-conception, five embryos were dissected from the uterus under a stereomicroscope and were then cut into two transverse sections. Embryos were fixed by immersion for 4 h in methacarn (absolute methanol/chloroform/glacial acetic acid; 6 : 3 : 1) with gentle stirring and were then washed in ethanol, also with gentle stirring, after which they were dehydrated and embedded in Paraplast (Oxford Labware, St. Louis, MO, USA) at 60 °C. National guidelines for laboratory animal care were followed, and all experiments were approved by the Institute of Biomedical Sciences Animal Ethics Committee (authorization number, 144/2002).

Immunoperoxidase staining

Embryos were cut on a microtome (Micron HM-200) into 5-µm sections, adhered to glass slides using 0.1% poly-l-lysine (Sigma, St Louis, MO, USA) and then dried at room temperature. Each of the succeeding steps was followed by thorough rinsing with phosphate-buffered saline (PBS).

Sections were treated with 3% H₂O₂ in PBS for 30 min to block endogenous peroxidase activity. All steps were performed in a humidified chamber, and care was taken to avoid drying out of sections. Non-specific staining was blocked by incubating the sections for 30 min with normal goat serum diluted 1 : 1 in PBS–10% bovine serum albumin. For each antibody, non-specific reactions were blocked by incubating the sections for 2 h with undiluted SuperBlock (Pierce, Rockford, IL, USA) blocking buffer.

For decorin and biglycan immunoreaction, sections were incubated for 1 h at 37 °C in 20 mm Tris-HCl, pH 6.0, containing 0.2 U/mL of chondroitinase ABC (Seikagaku Corp., Tokyo, Japan). For heparan sulfate proteoglycan (HSPG) immunoreaction, the sections were incubated for 1 h at 37 °C with 300 U mL⁻¹ solution of bovine testis hyaluronidase (Sigma) in 0.1 m sodium acetate buffer.

Rabbit polyclonal antibodies against murine decorin (LF-113) and biglycan (LF-106) (Fisher et al. 1995), which recognize an epitope in the core protein of each of these macromolecules, were diluted to 1 : 2000 and 1 : 1000, respectively.

The rat anti-HSPG monoclonal antibody (A7L6; Chemicon International, Temecula, CA, USA), which reacts with core protein domain 4 of the perlecan proteoglycans (Folkvord et al. 1989), was diluted to 1 : 100 in PBS–0.3% Tween 20.

To avoid background staining, monoclonal antibodies were preconjugated with biotin as described by Soto-Suazo et al. (2002).

For hyaluronan detection, the sections were incubated with a biotinylated probe (kindly provided by Dr H. Nader, UNIFESP, São Paulo, Brazil) consisting of the link protein-like domain of the aggrecan core protein (Tengblad, 1980). The probe was diluted to 1 : 80 in PBS–0.3% Tween 20 in PBS–0.3% Tween 20. After 1 h at 37 °C, the probe was incubated overnight at 4 °C. For the detection of perlecan, slides were incubated with secondary antibodies (directly) with the hyaluronan probe or with rat heparan-sulfate monoclonal antibody, after which they were rinsed three times in PBS and incubated for 1 h at room temperature in the streptavidin/peroxidase complex (Vector). For the detection of decorin and biglycan, the sections were washed thoroughly with PBS followed by incubation for 1 h at room temperature with biotin-conjugated goat anti-rabbit IgG (Vector) diluted 1 : 1000 in PBS.

Peroxidase was visualized using 0.03% 3,3′-diaminobenzidine (Sigma) in PBS with 0.03% H₂O₂. The sections were counterstained with Mayer's haematoxylin. The specificity of immunolabelling was tested by omitting the primary antibody. The diversity of tissues present in each embryo was also very useful for evaluating the specificity of the antibodies. The samples were examined with a Nikon Eclipse E600 microscope. Images were captured using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA).

Results

Morphological analysis

The developing gonad of the E12 embryos consists of loose mesenchymal tissue supported by the developing mesonephric tissue and covered by coelomic epithelium, which surrounds a mass of mesenchymal tissue and PGCs within the developing gonad (Schmahl et al. 2000). As development progresses, testicular cords are separated from the epithelium. Simultaneously, the epithelium and the outermost testicular cords become separated by mesenchymal tissue, which differentiates into the developing tunica albuginea. After E13, the testis rapidly increases its volume and becomes flattened. We found the gonad to contain germ and somatic cell lines arranged in cords that were connected to the mesonephros on the basal side of the gonad (Fig. 1a–d).

Fig. 1.

Morphological stages of testis development: (a) E12 embryo presenting morphologically undifferentiated gonads; (b) E13 embryo showing the developing testis; (c,d) E14 and E15 embryos presenting differentiated testicular cords. H&E staining. CC, coelomic cavity; G, gonad; GR, gonadal ridges; MT, mesonephric tubule; MM, mesonephric mesenchyme; M, mesenchyme of testis; TC, testicular cords. Scale bar, 50 µm.

At E14 and E15, the cells among the testicular cords were organized as an anastomosed network, forming structures in the shape of simple arches that were aligned generally perpendicular to the long axis of the testis. These arches were connected to the rete testis (Fig. 1c–d).

Immunohistochemical and histochemical analysis

Biglycan, decorin, perlecan and hyaluronan were detected in the mesonephric–gonadal complex of mouse fetuses, as well as in growing testis, from E12 to E15 (Figs 2 and 3). During that same period, however, the immunoreaction for decorin was restricted to the basement membrane of the mesonephric tubule and the surrounding mesenchyme (Fig. 2a–d).

Fig. 2.

Immunoperoxidase for decorin: (a,b) E12 and E13 embryos, respectively, presenting immunoreaction in the basement membrane of the mesonephric tubule (arrow); (b) E13 embryo. Decorin is observed in the mesenchyme surrounding the mesonephric tubule (arrowhead); (c) E14 embryo presenting no immunoreaction for decorin in the gonad; (d) E15 embryo presenting immunoreactivity in the basement membrane of the coelomic epithelium (arrow) as well as in the mesenchyme of the mesonephros (MM). (e) Control section for decorin. (f–j) Immunoperoxidase for biglycan: (f) E12 embryo presenting immunoreaction in the mesenchyme of the genital ridges (arrowhead) and in the basement membrane of the mesonephric tubules (arrow); (g) gonad of an E13 embryo. Biglycan is present in the mesenchyme among the testicular cords (arrowhead) and in the basement membrane of the testicular cords (arrow); (h) E14 embryo. The immunoreaction is observed in the mesenchyme of the developing gonad (arrowhead); (i) E15 embryos. Strong immunoreactivity is seen in the cytoplasm of immature Sertoli cells and Leydig cell precursors of the testicular cords (small arrows, as well as within the mesenchymal cells (arrowhead). (j) Control section for biglycan. G, gonad; GR, gonadal ridges; MT, mesonephric tubule; M, mesenchyme of testis; TC, testicular cords; MM, mesonephric mesenchyme. Scale bar, 50 µm.

Fig. 3.

Reaction for hyaluronan: (a,b) E12 and E13 embryos, respectively. Hyaluronan is seen in the mesenchyme of the mesonephros (arrows) and in the region of the basement membrane of the mesonephric duct (small arrow). Note the reaction in the basement membrane of the coelomic epithelium (asterisk) and in the mesenchyme of the developing gonad (large arrowhead). Note that the immunoreaction is stronger at E13 than at E12; (c,d) E14 and E15 embryos, respectively. Hyaluronan is present in the basement membrane region (arrow) and in the mesenchyme of the testicular cords (arrowhead); (e) control section for hyaluronan. (f–j) Immunoperoxidase for perlecan: (f) gonads of E12 embryos. Perlecan is observed in the basement membranes of the mesonephric duct (arrow) and coelomic epithelium (arrowhead); (g–i) E13, E14 and E15 embryos, respectively. Perlecan is observed in the regions of the basement membranes of the testicular cords (arrows), blood vessels (arrowhead) and coelomic epithelium (asterisk). (j) Control section for perlecan. BV, blood vessel; G, gonad; GR, gonadal ridges; MT, mesonephric tubule; MM, mesonephric mesenchyme; M, mesenchyme of testis; TC, testicular cords; CC, coelomic cavity. Scale bar, 50 µm.

Morphologically undifferentiated gonads

In E12 mouse fetuses, the developing gonad consisted of the coelomic epithelium and the underlying mesenchyme, containing only a few mesonephric tubules. Biglycan, perlecam and hyaluronan were immunodetected in the basement membrane regions of the mesonephric–gonadal complex, whereas hyaluronan and biglycan were immunodetected in the mesenchyme of the gonadal ridge and mesonephros (Fig. 2f–l and Fig. 3a and 3f).

Developing testis

In E13 mouse fetuses, the gonadal ridge had thickened, and some mesonephric tubules were associated with the growing region. The testicular cords seemed to be connected to the surface epithelium. Perlecan, biglycan and hyaluronan were observed surrounding the differentiating testicular cords, the coelomic epithelium and the blood vessels, whereas biglycan and hyaluronan were observed in the mesenchyme lying between the testicular cords (Figs 2g and 3b,d,g). The pattern of immunoreaction was similar at E14 and E15, being especially pronounced at E15. Both testes contained elongated arch-like cords, which appeared as semicircular structures (Fig. 1c,d). The testicular cord basement membrane was immunoreactive to perlecam and biglycan antibodies, as well as presenting histochemical staining for hyaluronan (Figs 2h and 3c,d,h,i). The mesenchymes surrounding the testicular cords were immunoreactive for biglycan and histochemically reactive for hyaluronan (Figs 2f–I and 3b–d). Biglycan was observed in the testicular cords, surrounding the immature Sertoli cells and Leydig cell precursors, chiefly at E15. In E15 embryos in particular, the immunoreaction for biglycan was observed very close to the nuclei of germinal and mesenchymal cells, suggesting intracytoplasmic localization of biglycan in these cells (Fig. 3i).

Control sections were created by omitting the incubation with the primary antibodies. The control sections presented no immunoreactivity.

Discussion

The present study describes, for the first time, the distribution of decorin, biglycan, perlecan and hyaluronan in the developing gonads of mice. Importantly, although all of these molecules were present at all stages studied, the distribution of each varies among the different structures of the mesonephric–gonadal complex. Decorin was present exclusively in the basement membrane regions of the coelomic epithelium and mesonephric tubules, whereas perlecan, biglycan and hyaluronan were present in the basement membrane regions of the developing testicular cords. In addition, hyaluronan and biglycan were present in the mesenchyme surrounding the testicular cords. It is noteworthy that, of the three proteoglycans analysed, only biglycan was observed in the proximity of the testicular somatic cells, immature Sertoli cells and Leydig cell precursors.

To elucidate the complex relationships that form during gonadal development, variations in the ECM molecule profiles, such as those observed in our study, need to be correlated with the various morphological phases. At E12.5, the mouse testis is first distinguishable from the indifferent gonad due to the presence of a large peripheral blood vessel and newly forming testicular cords (McLaren, 1983). These cords consist of centrally organized PGCs surrounded by a layer of pre-Sertoli cells and an outer sheath of peritubular myoid cells, the Leydig cell precursors lying among them (McLaren, 1985). The tunica albuginea is fully formed by E15 (Mackay & Smith, 1989). A similar sequence of events in early testicular development is seen in rats (Magre & Jost, 1980) and rabbits (Wartenberg et al. 1991). The first event leading to testicular differentiation seems to be that PGCs cluster with certain somatic cells, the future Sertoli cells, thereby forming testicular cords (Byskov & Hoyer, 1994).

Epithelial–mesenchymal interactions clearly have a significant influence on the differentiation of testicular cords (Cunha et al. 1980, 1981; Bernfield et al. 1984). Virtually all basement membranes contain, as an intrinsic component, the multifaceted proteoglycan perlecan (Couchman & Ljubimov, 1989; Murdoch et al. 1994). We found that concentrations of perlecan in the basement membranes increased progressively during testicular cord differentiation. From E12 to E13, there is rapid cell proliferation, and this phenomenon requires the disruption of the coelomic basement membrane restricted to the gonadal ridges (Gelly et al. 1989). Discontinuity of the basement membrane surrounding the differentiating testicular cords occurs at E13. Interactions among mesenchymal cells, presumptive Sertoli cells and gonocytes are responsible for the synthesis of newly discontinuous basement membrane. It has been shown that, in vitro, the deposition of a complex matrix requires the presence of differentiated Sertoli and peritubular cells (Skinner et al. 1985). Soto-Suazo et al. (2002) suggested that, during testicular cord development, perlecan acts as a barrier that prevents the PGCs escaping the developing gonads, simultaneously facilitating PGC migration.

The fibroblast growth factor (FGF) family has various biological functions. Members of the FGF family can influence cell growth, cell differentiation and cell migration. In addition, basic FGF (bFGF) plays a role in angiogenesis and in tissue repair (Brucato et al. 2002). In rat testes, bFGF affects Leydig and Sertoli cell steroidogenesis (Laslett et al. 1995; Schteingart et al. 1999). It is known that the biological activity of bFGF is mediated by interaction with the high-affinity cell surface bFGF receptor. Moreover, interaction between bFGF and HSPGs such as perlecan and syndecan modulates FGF activity, as well as facilitating receptor binding and activation (Yayon et al. 1991; Iozzo, 1998).

It has been shown that HSPGs are present on the surface of immature rat Sertoli cells (Mounis et al. 1991; Brucato et al. 2001), and that these cells express syndecan1–4 mRNA (Brucato et al. 2000). During testicular development, the physiology of Sertoli cells changes dramatically. Cell proliferation first decreases and then ceases, allowing the establishment of the blood–testis barrier at approximately day 20 postpartum (Brucato et al. 2002). Oestradiol synthesis by the Sertoli cells in postpartum testes is stimulated by follicle-stimulating hormone under the regulation of FGF and HSPG molecules (Brucato et al. 2002). We therefore suggest that bFGF, syndecan and perlecan also stimulate the growth and differentiation of Sertoli cells and gonocytes during intrauterine testis development.

Decorin and biglycan belong to the family of the leucine-rich small proteoglycans. In the present study, decorin was not detected in the developing testicular cords, being seen exclusively in the basement membrane of coelomic epithelium and mesonephric tubules and only during their early development. By contrast, biglycan was detected in the basement membrane of developing testicular cords, as well as surrounding the immature Sertoli cells and Leydig cell precursors. This finding is in agreement with those of previous studies showing that decorin is absent from the gonads of E12 mouse embryos (Soto-Suazo et al. 2002). In addition, it indicates that biglycan plays a role in gonad development, whereas decorin does not.

In addition to playing structural roles (mainly in collagen fibrillogenesis), decorin and biglycan also bind transforming growth factor beta (TGF-β), thereby participating in the regulation of cell proliferation (Yamaguchi & Ruoslahti, 1988; Hildebrand et al. 1994). There are indications that the interaction between decorin and TGF-β is competitively inhibited by biglycan (Hocking et al. 1999). Decorin binding is thought to neutralize TGF-β biological activity. However, due to the reversibility of this interaction, decorin also acts as a local reservoir for TGF-β in tissues (Ruoslahti & Yamaguchi, 1991).

Studies involving rat Sertoli cells have demonstrated in vivo TGF-β immunoreactivity (Teerds & Dorrington, 1993) and in vitro TGF-β production (Skinner & Moses, 1989; Mullaney & Skinner, 1993). The strategic presence of biglycan in the basement membrane and in the pericellular region might facilitate the binding of the majority of the produced growth factors, preventing them from being diffused out of the testis cells (Ungefroren et al. 1995). In addition, the presence of biglycan in the ECM might be related to collagen fibrillogenesis, as has been observed in other models (Vogel et al. 1984; Vogel & Trotter, 1987; Uldbjerg & Danielsen, 1988; Hocking et al. 1998).

During organogenesis, a complex and intricate ECM network fills the space between the cells and provides a unique histoarchitecture for the pathway along which PGCs move. The ECM complex also provides strength and physical support for the tissues (Pereda et al. 2006). Therefore, migratory cells might utilize the space created by the special arrangement and composition of the ECM molecules, which offer less resistance to cell movement (Erickson, 1990; Browder et al. 1991). The genesis of these extracellular spaces has been associated with the accumulation of hyaluronan (Toole, 1981). In the hyaluronan-rich matrices, the stimulation of cell proliferation (Brecht et al. 1986) and migration (Toole, 1981) depends upon the matrix being hydrated and offering low resistance, thereby shielding cells from contact inhibition. The pericellular matrix expansion also requires interaction of versican, hyaluronan and CD44 (Wight, 2002). This macromolecular complex increases the viscoelastic nature of the pericellular matrix, creating a highly malleable extracellular environment that supports the cell shape change necessary for cell proliferation and migration (Toole, 1982, 2001; Lee et al. 1993).

According to Toole (2001), the characteristics of the ECM might be important for keeping cells separate and mobile, discouraging premature aggregation, adhesion and differentiation. In addition, hyaluronan interacts with the cell surface receptor CD44, thereby creating a similar extracellular environment during critical morphogenetic events such as cell movement and proliferation (Knudson & Knudson, 1993; Knudson et al. 1993). Furthermore, versican also participates in cell proliferation and contributes to the pericellular ECM expansion that is required for the proliferation and migration of cells (Evanko et al. 1999, 2001).

Considering these previous findings, we can conclude that the presence of hyaluronan in the interstitium and basement membrane regions of testicular cords plays a role in organ growth, which requires cell proliferation and migration for the establishment of a mature gonad.

The mechanism involved in the formation and differentiation of the testes and testicular cords remains unclear. However, structural studies have shown that the formation of a basement membrane and the presence of ECM molecules might be important for morphogenetic events and organ differentiation. It is known that mesenchymes condense around the testicular cords and that progressive differentiation of the basement membrane occurs after the fetal testosterone peak (Gondos, 1980). The ECM plays a crucial role in tissue formation during organogenesis and creates a barrier among androgen production by interstitial Leydig cells, intertubular Sertoli cells and PGCs (Weber et al. 2002). Within the lamina propria of the seminiferous tubules, the production of the ECM results from the cooperation of somatic Sertoli and peritubular cells (for a review, see Skinner, 1991). The presence of perlecan, biglycan and hyaluronan surrounding the Sertoli cells during all of the developmental stages observed in the present study suggests that these molecules play a role in the morphogenesis of the testis.

In summary, our results suggest that the presence of perlecan, biglycan and hyaluronan plays a role in gonadal development. In addition, we showed that decorin is absent from the testicular cords, whereas, of the proteoglycans analysed, only biglycan was observed surrounding the cells of the testicular cords. These data suggest that specific ECM molecules are required for testis development. Further studies involving knockout animals are needed in order to test this hypothesis.