Differential Localization of Fibroblast Growth Factor Receptor-i,-2,-3 ...

Download PDF
13 downloads 0 Views 1MB Size Report
Comment
ABSTRACT. Fibroblast growth factors (FGFs) are essential for embryonic development and have been implicated in testis development and function. The effects ...
BIOLOGY OF REPRODUCTION 58, 1138-1145 (1998)

Differential Localization of Fibroblast Growth Factor Receptor-i, -2, -3, and -4 in Fetal, Immature, and Adult Rat Testes' Belinda Cancilla2 and Gail P. Risbridger Institute of Reproduction and Development, Monash University, Clayton, Victoria 3168, Australia ABSTRACT Fibroblast growth factors (FGFs) are essential for embryonic development and have been implicated in testis development and function. The effects of FGFs are mediated through four high-affinity receptors (FGFRs), which have different binding affinities for each of the ligands. We have used indirect avidinbiotin-horseradish peroxidase-enhanced immunohistochemistry to localize FGFR-1, -2, -3, and -4 in fetal, immature, and adult rat testes. In the fetal testis, immunoreactivity for FGFR-1 was seen in gonocytes, Sertoli cells, Leydig cells, and mesenchyme, and FGFR-3 was localized in gonocytes. In the immature testis, FGFR-1 was localized to spermatogonia, and all four FGFRs were localized in pachytene spermatocytes, immature adultlike Leydig cells, and peritubular cells. In the adult testis epithelium, Sertoli cells were immunoreactive for FGFR-4, and germ cells were immunoreactive for all four FGFRs, with specific receptors localized to specific stages of germ cell development. In the adult testis interstitium, FGFR-1, -2, and -4 were localized in Leydig cells, and FGFR-1 and -4 were also localized in peritubular cells. The discrete cell- and stage-specific localization of FGFRs in the fetal, immature, and adult rat testis suggests that FGFs exert specific roles through these receptors in spermatogenesis, Leydig cell function, and testicular development. INTRODUCTION Fibroblast growth factors (FGFs) are a family of heparinbinding polypeptides involved in an array of biological processes during development and adult life [1, 2]. The FGF family consists of at least 12 members [2]; FGF-1 and FGF2 were the first members of this family to be isolated and characterized, and the other members include FGF-3 (int2), FGF-4 (hst/ks), FGF-5, FGF-6, FGF-7 (KGF), FGF-8 (AIGF), FGF-9, FGF-10, FGF-11, and FGF-12. Heparan sulfate proteoglycans (HSPGs) are low-affinity binding sites for FGF ligands and are present in the extracellular matrix or as cell surface-bound molecules [1]. Four high-affinity FGF receptor (FGFR) genes have been characterized; these encode transmembrane protein tyrosine kinase receptors [2]. The four FGFR genes give rise to many receptor isoforms due to differential mRNA splicing. Seven functionally distinct variants have been characterized, and the affinities of these receptor variants for FGF-1 through FGF-9 have been determined [3]. FGF binding to FGFRs requires a tripartite interaction with heparin or HSPGs [4]. This interaction can involve two mechanisms: heparin or HSPGs can induce dimerization of FGF ligands, which then bind to FGFRs, thus enhancing efficient FGFR dimerization [5]; and HSPGs bind to both FGF liAccepted December 1, 1997. Received August 25, 1997. 'This work was supported by a grant from the National Health and Medical Research Council, Australia. 2Correspondence: B. Cancilla, Institute of Reproduction and Development, Monash Medical Centre, 246 Clayton Rd., Clayton, Vic. 3168, Australia. FAX: 61-3-9550-3584; e-mail: [email protected]

gands and FGFRs forming a ternary complex, thereby enhancing FGF-FGFR interaction [6]. FGFs are essential for embryonic and fetal development. This has been demonstrated through the generation of knockout mice for FGF-3, FGF-4, FGF-5, and FGF-7 and for FGFR-1 and FGFR-3. These transgenic mice have early neural defects, affected limb induction, disrupted organ development, skeletal dysgenesis, and specific defects in hair growth and structure [2]. The development and function of testes in these transgenic mice have not been described. In the male gonads, FGFs are implicated in spermatogonial and Sertoli cell mitosis and in Leydig cell function. Almost exclusively, these observations have focused on FGF-1, FGF-2, and FGFR-1. FGFR-1 has been immunolocalized in adult rat testis [7]; FGFR-1 mRNA has been detected in the immature rat testis, and levels were found to decrease during development [8]. FGF-2 ligand has been isolated from bovine testis [9], and a number of immunohistochemical studies have localized FGF-2 protein to embryonic, postnatal, and adult rat germ cells [7, 10, 11] and to spermatogonia of adult human testis [12]. FGF-2 protein was also detected in fetal [11] and adult Leydig cells [7]. The expression of FGF-2 mRNA was detected by Northern blots in whole pubertal rat testes and was found to have decreased with testicular development [13]; in pubertal testes, the mRNA was found in isolated peritubular, Sertoli, and Leydig cells. FGF-2 acts as a survival factor for Sertoli cells and a mitogenic factor for gonocytes in perinatal Sertoli cell-gonocyte cocultures [14]. Immature Leydig cells are able to bind FGF-1 and FGF2 in vitro through a specific FGFR [15, 16], and exogenously added FGF-2 is internalized by HSPG/FGFR interaction in cultured rat immature Leydig cells [17]. FGF-2 is involved in adult and immature Leydig cell function [18]. FGF-2 stimulates steroidogenesis by immature Leydig cells in the absence of LH; this activity is mediated through HSPGs [19]. HSPGs are also essential for the autocrine growth factor regulation of adult Leydig cell steroidogenesis [20]. We have recently reported differential effects of FGF-1 and FGF-2 on Leydig cells, where the effects are dependent on the specific stage of Leydig cell differentiation and development [21]; these effects may be mediated by different FGFRs. Collectively, these observations suggest that FGFs are produced by testicular cells and that they act through specific receptors in the testis. The autocrine/paracrine actions of these ligands occur within both the interstitial and epithelial compartments of the testis. To date, little attention has focused on the cell-specific production of FGF ligands or the cell-specific localization of the receptors through which these ligands exert their biological actions. The aim of this study was to describe the immunolocalization of FGFR-1, -2, -3, and -4 in rat testes at each stage of Leydig cell development, and in association with these findings we report the localization of the FGFRs in the seminiferous epithelium at these stages.

1138

FGFRS IN FETAL, IMMATURE, AND ADULT RAT TESTES MATERIALS AND METHODS Animals Sprague-Dawley rats were obtained from Central Animal Services, Monash University. All procedures and animal care were administered in accordance with the requirements and approval of the Standing Committee of Ethics in Animal Experimentation, Monash University. Four intact male rats at 80 days (adult) and four at 20 days (immature) postpartum were anesthetized with ether and were perfusion fixed with Bouin's fixative; the testes were dissected from the animals and immersion fixed for a further 18 h. Three pregnant female rats at 19 days postcoitum were anesthetized with ether, and fetal rats were removed from the uterus and quickly decapitated. Testes were dissected from a total of 15 embryos and immersion fixed in Bouin's fixative for 2-3 h. All testes were processed into paraffin, and 3-jim serial sections were cut. Immunohistochemistry Immunohistochemistry was performed at least three times on each animal from the three groups examined. Sections were dewaxed, rehydrated, and treated with 3% H 20 2 in PBS for 15 min and rinsed with PBS. Sections were digested with 0.025% trypsin (Sigma Chemical Co., St. Louis, MO) for 30 min at room temperature and washed with PBS. Sections were then blocked with CAS Block solution (Zymed, San Francisco, CA) for 1 h. Primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were affinity-purified rabbit polyclonal antibodies raised to peptides mapping at the carboxy-terminus of FGFR- 1 (residues 808-822 of the human filg gene product), FGFR-2 (residues 805-821 of the human bek gene product), FGFR-3 (to residues 792-806 of human FGFR-3), and FGFR-4 (residues 789-802 of human FGFR-4); they were used at 0.5 jig/ml and incubated on tissue sections overnight at 4C. The FGFR antibodies used were characterized by Santa Cruz Biotechnology as specific for each receptor and did not cross-react with other FGFRs. Control sections were incubated with rabbit serum (Sigma) or with antibodies that had been preabsorbed with 0.5 ig/ml or 2 jig/ml of immunizing peptides (Santa Cruz Biotechnology) instead of the primary antibody. Sections were washed with PBS and incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) for 1 h. After rinsing with PBS, sections were incubated with avidin-biotin-horseradish peroxidase complex (Vectastain Elite ABC kit; Vector Laboratories) for 1 h and then rinsed with PBS. Sections were reacted to color with a brown substrate (3'3-diaminobenzidine HCI [DAB]; Zymed) or a purple substrate (VIP; Vector Laboratories). Sections with a brown reaction product were counterstained with Mayer's hematoxylin. RESULTS Adult Testis Differential immunostaining for each of the FGFRs, i.e., FGFR-1, -2, -3, and -4, was observed in the adult rat testis. FGFR-1 (Figure 1). Immunostaining for FGFR-1 was localized to the epithelium and interstitium (A-F). In germ cells, cytoplasmic immunoreactivity for FGFR-I was localized to pachytene spermatocytes from stage VI to XII (C and D), and pericellular immunostaining was observed in round spermatids of stage I-VIII tubules (A-C). Immunostaining was also localized to elongating spermatids of

1139

stage IX-XIV (D and E) and I-VIII (A-C) tubules. No immunoreactivity was observed in Sertoli cells (A, B, and D). The basal compartment of tubules was not immunoreactive, suggesting that spermatogonia, which have very little cytoplasm, did not contain FGFR-1 (B). In the interstitium, immunostaining for FGFR- 1 was present in Leydig cells and peritubular cells (A and F). FGFR-2 (Figure 1). FGFR-2 immunoreactivity was detected in the interstitium and seminiferous epithelium (HJ and L-O). Cytoplasmic FGFR-2 immunoreactivity was observed in leptotene and zygotene spermatocytes (L and M), with more prominent staining in all pachytene spermatocytes (H-J and N). FGFR-2 immunostaining was also present in early stages of spermatid development and was observed in step 1-12 spermatids of stage I-XII tubules (H-J and L). FGFR-2 immunoreactivity was not detected in Sertoli cells of the testis epithelium at any stage of spermatogenesis (I, J, and L). Similar to findings for FGFR-1, the basal compartment of tubules was not immunoreactive for FGFR-2, suggesting that spermatogonia did not contain FGFR-2. In the interstitium, immunostaining for FGFR-2 was present in Leydig cells (O). FGFR-3 (Figure 2). Immunoreactivity for FGFR-3 was most prominently localized to germ cells, and no immunostaining was present in Sertoli cells at any stage of spermatogenesis (A-D). Spermatogonia did not appear to be immunoreactive for FGFR-3. In contrast to FGFR-1 and -2, immunostaining for FGFR-3 was not present in spermatids (A-D). Cytoplasmic immunostaining was observed in preleptotene spermatocytes (stage VII-VIII; A and D) and was more prominent in leptotene (stage IX-XI; C), zygotene (stage XII-XIII; D), and pachytene spermatocytes of stage XIV and I-VI tubules (A, B, and D). However, immunostaining for FGFR-3 was not seen in pachytene spermatocytes from stage VII to XII (A-D). In the interstitium, no immunoreactivity for FGFR-3 was present in Leydig cells

(E). FGFR-4 (Figure 2). In contrast to observations for FGFR1, FGFR-2, and FGFR-3, cytoplasmic immunostaining for FGFR-4 was present in Sertoli cells at all stages of spermatogenesis (G-K). Germ cell staining was observed in late spermatids, similar to staining for FGFR-1; FGFR-4 immunoreactivity was present in stage I-VIII tubules, where elongating spermatid cytoplasm was immunoreactive as well as Sertoli cells (G and H). As with FGFR-1, -2, and -3, immunostaining for FGFR-4 was also seen in pachytene spermatocytes (G-J), but not leptotene or zygotene spermatocytes (J and K). In contrast to findings for FGFR-1, -2, and -3, the basal compartment of tubules was immunostained for FGFR-4, suggesting that spermatogonia were immunoreactive for FGFR-4 (G and H). This basal staining was also seen in stage VII-VIII tubules, indicating that FGFR-4 was also localized to preleptotene spermatocytes (I). Intense immunostaining for FGFR-4 was present in the interstitium of adult testes, in particular in Leydig and peritubular cells (L and M). Controls. No specific immunostaining was observed when rabbit serum was used instead of primary antibody. Preabsorption of FGFR antibodies with the appropriate FGFR peptide resulted in decreased immunostaining (0.5 jg/ml of peptide), or abolished FGFR immunostaining (2 jig/ml of peptide; Fig. 1G for FGFR-1, Fig. 1K for FGFR2, Fig. 2F for FGFR-3, and Fig. 2N for FGFR-4). In summary, the localization of each of the FGFRs in the epithelial and interstitial compartments of the adult rat testis was cell specific. This pattern is summarized in a

1140

CANCILLA AND RISBRIDGER

FIG. 1. Photomicrographs of adult rat testis (80 days postpartum) immunostained for FGFR-1 and FGFR-2, detected with DAB. A) Stage Il-IV tubule, B) stage V tubule, C) stage VII tubule, D) stage X tubule, and E) stage XIV tubule immunostained for FGFR-1. Immunostaining is seen in pachytene spermatocytes (PA) of stage VII (C) and X (D) tubules but not in stage Ill-IV (A), V (B), or XIV (E)tubules. All spermatids were immunoreactive for FGFR1, with cytoplasmic staining of elongating spermatids (ES) (A, B, D, E) and pericellular staining of round spermatids (RS) (A-C). No immunoreactivity is seen in Sertoli cells (S) (A, B, D) or spermatogonia (SG) (B). F) In the interstitium, immunostaining for FGFR-1 is observed in Leydig cells (L) and peritubular cells (PT). G) Preabsorption control for FGFR-1. H) Stage II tubule, I) stage V tubule, ) stage VIII tubule, L) stage IX-X tubule, M) stage XIII tubule, and N) stage XIV tubule immunostained for FGFR-2. FGFR-2 immunostaining is seen in all pachytene (PA) spermatocytes (H-J, L, N) and in leptotene (LE) (L) and zygotene (Z) (M) spermatocytes. Round spermatids (RS) (H-J) and step 10 elongating spermatids (ES) (L) are also immunostained for FGFR-2. O) Micrograph of interstitium demonstrating FGFR-2 immunoreactivity in Leydig cells (L). K) Preabsorption control for FGFR-2. Bar = 10 .m (A-J, L-O) and 25 iLm (K).

diagram indicating stages of spermatogenesis and cell types immunoreactive for each receptor (Fig. 3). Sertoli cells were immunoreactive for FGFR-4. FGFR-1, -2, -3, and -4 were present in germ cells, but specific receptors were localized to specific stages of germ cell development. Spermatogonia appeared to have immunostaining only for FGFR-4. In the interstitium, FGFR-1, -2, and -4 were present in mature Leydig cells of the adult testis. FGFR-1 and -4 were also present in peritubular cells.

Immature (Pubertal) Testis (Figure 4) Immunostaining for each of the FGFRs was seen in the immature rat testis in the epithelium and interstitium. FGFR-1 (Figure 4). Immunoreactivity for FGFR-1 was localized to spermatogonia and spermatocytes, but no immunostaining was detected in Sertoli cells (A and B). Immunostaining was also observed for FGFR-1 in the inter-

FGFRS IN FETAL, IMMATURE, AND ADULT RAT TESTES

1141

FIG. 2. Photomicrographs of adult rat testis (80 days postpartum) immunostained for FGFR-3 and FGFR-4, detected with DAB (A-L, N) or VIP (M). A) Cross section of a stage XIV tubule, demonstrating FGFR-3 immunostaining in pachytene (PA) spermatocytes; weak immunostaining is seen in preleptotene (PL) but not pachytene spermatocytes (PA) of adjacent stage VII tubule. FGFR-3 immunostaining is observed in pachytene spermatocytes (PA) of stage 11-111 tubule (B) but not in pachytene spermatocytes (PA) at stage IX (B, C), where staining isseen in leptotene (LE) spermatocytes. D) Immunostaining for FGFR-3 is present in preleptotene (PL) and pachytene spermatocytes (PA) of stage VIII tubule but not in pachytene spermatocytes (PA) of stage XIIXIII tubule, where intense immunoreactivity is localized in zygotene spermatocytes (Z). D, diplotene spermatocytes. E) High-power micrograph of interstitial Leydig cells (L), which are not immunoreactive for FGFR-3. F) Preabsorption control for FGFR-3. CG)Stage I-III tubule, H) stage V-VI tubule, I) stage VII tubule, ) stage XI tubule, and K) stage XIII tubule immunostained for FGFR-4. FGFR-4 immunoreactivity is seen in Sertoli cells (S) (I, K), pachytene spermatocytes (PA) (G-I), and elongating spermatids (ES) of step I-VII tubules (G-I). FGFR-4 immunoreactivity was not seen in leptotene spermatocytes (LE) (J), diplotene spermatocytes (D) (K), round spermatids, or step 9-14 elongating spermatids (ES) (I, K). Spermatogonia (SG) were also immunoreactive (GC, H). L) High-power micrograph of interstitium, showing immunostaining in Leydig cells (L). M) FGFR-4 immunostaining detected with purple substrate, demonstrating staining in Leydig cells (L) and peritubular cells (PT). N) No immunoreactivity is seen in the preabsorption control for FGFR-4. Bar = 100 i.m (N), 25 pm (A, B, D, F,M), and 10 pLm (C, E, G-L).

stitium, where immature adultlike Leydig cells and peritubular cells were immunoreactive (A and B). FGFR-2 (Figure 4). Immunoreactivity for FGFR-2 was detected in developing pachytene spermatocytes but not in Sertoli cells (D and E). Intense immunostaining for FGFR2 was detected in immature adultlike Leydig cells and in peritubular cells (E). FGFR-3 (Figure 4). Immunostaining for FGFR-3 was found in developing leptotene, zygotene, and pachytene

spermatocytes (G-I); FGFR-3 immunostaining was more intense than FGFR-1 or -2 immunostaining. In the interstitium, immunostaining for FGFR-3 was detected in immature adultlike Leydig cells and peritubular cells (J); this was similar to findings for FGFR-1 and -2. FGFR-4 (Figure 4). Immunoreactivity for FGFR-4, like FGFR-1, -2, and -3, was present in pachytene spermatocytes but was not detected in Sertoli cells in immature testes (L and M). FGFR-4 immunoreactivity was detected in

1142

CANCILLA AND RISBRIDGER FGFR peptide abolished FGFR immunostaining (2 g/ml of peptide; C, F, K, and N for, respectively, FGFR-1, FGFR2, FGFR-3, and FGFR-4). In summary, in the immature, pubertal testis, all four FGFRs were present in spermatocytes, immature adultlike Leydig cells, and peritubular cells; no immunoreactivity was detected in Sertoli cells. In contrast to what was seen in the adult testis, FGFR-1 was present in spermatogonia. Fetal Testis (Figure 5) In the Day 19 fetal rat testis, when the fetal generation of Leydig cells was present, no immunoreactivity for FGFR-2 or -4 was detected in either the interstitium or epithelium. FGFR- 1 (Figure 5). Although weak immunoreactivity for FGFR-1 was detected in fetal Sertoli cells (A and B), specific immunostaining for FGFR-1 was most prominent in gonocytes, in a pericellular and cytoplasmic pattern (B). FGFR-1 immunostaining was also localized to the fetal Leydig cells and mesenchyme surrounding the developing tubules (A and B). FGFR-3 (Figure 5). In contrast to FGFR-1 immunoreactivity, FGFR-3 was discretely localized to the gonocytes in fetal testes (D and E). No immunoreactivity was localized to the interstitial cells. Controls (Figure 5). No immunostaining was observed when rabbit serum was used instead of primary antibody. FGFR immunostaining was abolished when FGFR-1 and -3 antibodies were preabsorbed with the corresponding FGFR peptide (2 g/ml of peptide; C and F for, respectively, FGFR-1 and FGFR-3). In summary, only one receptor, FGFR-1, was detected by immunohistochemistry in the interstitium, and it was localized to fetal Leydig cells and surrounding mesenchyme. Differential localization for FGFR-1 and -3 was present in the epithelium, with FGFR-1 immunoreactivity in Sertoli cells and gonocytes and FGFR-3 immunoreactivity in gonocytes. DISCUSSION

FIG. 3. Diagram of rat testis indicating stage of spermatogenic cycle (stage I-XIV). Cell types indicated are spermatogonia (IN and B), sper0 matocytes (PL, LE, Z, P, and D), secondary spermatocyte (m2 m), and spermatids (steps 1-19). Boxes indicate other spermatogonia (SG), Sertoli cells (S), Leydig cells (L), and peritubular cells (PT). Shaded cells and boxes indicate immunostaining for FGFR-1, FGFR-2, FGFR-3, and FGFR-4.

the interstitium of immature rat testes, where intense immunostaining was observed in immature adultlike Leydig cells and peritubular cells (M). Controls (Figure 4). No immunostaining was observed when rabbit serum replaced primary antibody. Preabsorption of antibodies for FGFR-1 to -4 with the corresponding

We report in this paper that in the fetal, immature, and adult testis there is specific FGFR localization within the interstitial and seminiferous tubule compartments, and that this changes during development of the testis and with the stages of spermatogenesis. The results show cytoplasmic and pericellular immunostaining for FGFR-1 through -4. Cytoplasmic immunostaining of transmembrane receptors has been reported previously; in those studies, several antibodies were used to localize splice variants of FGFR-1 and FGFR-3 in different cellular compartments, including cytoplasmic and nuclear compartments of a number of cell types and tissues [2225]. Many splice variants of FGFRs have been identified, including a splice variant of FGFR-1 that lacks a transmembrane domain [25] and splice variants of FGFR-1 and -4 that lack a hydrophobic signal sequence [26, 27]; these splice variants are not transported to the cell surface, and localize to the cell cytoplasm. In the rat testis, pericellular localization of transmembrane FGFRs may be masked if the cytoplasmic variant of that receptor is also expressed in the same cell. Since we have observed pericellular and cytoplasmic localization of FGFRs, it is probable that several structural and functional receptor variants are expressed in the rat testis at different times of development and spermatogenesis.

FGFRS IN FETAL, IMMATURE, AND ADULT RAT TESTES

1143

FIG. 4. Photomicrographs of immature rat testis (20 days postpartum) immunostained for FGFR-1 to -4, detected with DAB. A) FGFR-1 immunoreactivity is seen in type B spermatogonia (SG), and preleptotene (PL) and pachytene spermatocytes (PA), but not Sertoli cells (S). B) FGFR-1 immunostaining localized in immature adultlike Leydig cells (L) and peritubular cells (PT). C) Preabsorption control for FGFR-1. D) FGFR-2 immunostaining is seen in pachytene spermatocytes (PA) and E) in peritubular (PT) and immature adultlike Leydig cells (L). F) Preabsorption control for FGFR-2. G-I) Prominent immunoreactivity for FGFR-3 is localized in leptotene (LE), zygotene (Z), and pachytene spermatocytes (PA). ) FGFR-3 immunostaining is seen in peritubular (PT) and immature adultlike Leydig cells (L). K) Preabsorption control for FGFR-3. L) Pachytene spermatocytes (PA) and M) immature adultlike Leydig cells (L) and peritubular cells (PT) demonstrate immunoreactivity for FGFR-4. N) Preabsorption control for FGFR-4. Bar = 10 jIm.

This study showed a change in expression of FGFRs in the fetal Leydig cell and during development of the adult Leydig cell population. Previous studies determined that FGFR-1 is expressed in adult Leydig cells [7, 8]. Our results confirm this but further demonstrate that the fetal Leydig cells also express FGFR-1. A significant difference between these two generations of Leydig cells was the emergence of immunoreactivity for FGFR-2, -3, and -4 in immature adultlike Leydig cells; these are not detected in fetal Leydig cells. Although most studies on Leydig cells have focused on FGF-1 and FGF-2, the presence of these receptors implies that a wider range of FGF ligands can act on immature adultlike Leydig cells.

Previous data from our laboratory have demonstrated that there is a developmental response by Leydig cells to FGF-1 and FGF-2, so that in vitro, the effects of FGF-1 and FGF-2 on steroidogenesis are dependent on the specific stage of Leydig cell development and differentiation; for example, adult Leydig cells can discriminate between the closely related ligands FGF-1 and FGF-2 [21]. Our data show that although all four FGFRs localize to the immature adultlike Leydig cells, adult Leydig cells do not express FGFR-3, suggesting a functional requirement for FGFR-3, in addition to FGFR-1, -2, and -4, in the immature adultlike Leydig cell population. Although FGF-1 can bind all four receptors with varying affinity [3], selective binding of oth-

1144

CANCILLA AND RISBRIDGER

FIG. 5. Photomicrographs of fetal rat testis (19 days postcoitum) immunostained for FGFR-1 and FGFR-3 detected with DAB. A) FGFR-1 immunoreactivity is seen in fetal Sertoli cells (S), gonocytes (G), and interstitial cells (I).B)At higher power, immunostaining for FGFR-1 is seen in fetal Leydig cells (L)and there is prominent staining in gonocytes (G). C) Preabsorption control for FGFR-1. D, E)Intense FGFR-3 immunoreactivity localized in fetal gonocytes (G). F) Preabsorption control for FGFR-3. Bar = 50 lpm (A, C, D) and 10 pm (B,E,F).

er FGFs occurs through FGFR isoforms. Isoforms of FGFRs have unique ligand-binding properties; "IIIc" variants have a large range of FGF ligands, whereas "IIIb" variants are more discriminating [3]. Further studies to determine which FGFR splice variants are present may identify other members of the FGF family that are important for Leydig cell development or function. An additional mechanism for the regulation of FGF activity is the interaction between HSPGs and FGFRs. These interactions are essential for ligand/receptor binding and signaling and may further affect the specificity of the FGFRs. We have previously reported that HSPGs mediate Leydig cell response to exogenously added FGF-2 [19]. In addition, Leydig cell steroidogenesis requires functional HSPGs [20], suggesting an autocrine or paracrine interaction between FGFs and FGFRs produced by Leydig cells. It is interesting to note that the peritubular cells in the immature testis showed immunoreactivity for FGFR-1, -2, -3, and -4, and that this was similar to that of the immature adultlike Leydig cell in the 20-day testis. The peritubular cell population is believed to contain precursors of the adult generation of Leydig cells, and it may be possible that FGFs are involved in the processes of Leydig cell development and differentiation in the immature testis. In contrast, in the adult testis, the differentiated peritubular cells express only FGFR-1 and -4. FGFR-1 has been detected in immature and adult rat testis in Leydig cells, peritubular cells, spermatocytes, spermatids, and Sertoli cells [7, 8]. In the present study, FGFR1 immunoreactivity was detected in all these except Sertoli cells; the failure to detect FGFR-1 in Sertoli cells of immature and adult rat testis may be due to expression of FGFR-1 splice variants in Sertoli cells. A splice variant of FGFR-1 with a different carboxy-terminal region, or truncated intracellular domain, would still be detected by antibodies raised to the extracellular domain [71, whereas the antibody used in the current study was raised against the intracellular carboxy-terminal region and therefore would

not detect these variants. In immature testes, the mitogenic and differentiating functions of FGFs on the Sertoli cell [28, 29] may be due to FGFR-1 variants expressed by these cells or due to interactions with FGFRs on adjacent cells. A significant difference between fetal and adult Sertoli cells was the detection of FGFR-4 in adult testis at all stages of spermatogenesis. Together, these results suggest that in the adult testis, FGF ligands acting through FGFR-4 on Sertoli cells may be required throughout spermatogenesis. Intense immunolocalization of FGFR-3 to spermatocytes of adult rat testes suggests a specific function of FGFs in spermatogenesis; high expression of FGFR-3 homologue in the amphibian testis suggests an important role for this receptor in testis function across species [30]. Localization of all four FGFRs in spermatocytes indicates that FGFs are essential in these cells; a number of FGFs may interact with these receptors to produce specific effects in these cells. The stage-specific localization of FGFRs during maturation of spermatids also suggests a functional role for FGFs and FGFRs in spermatogenesis. A switch in FGFR localization appears to occur in spermatogonia with development. In the immature rat testis, spermatogonia were immunoreactive for FGFR-1, whereas in the adult rat testis it appeared that spermatogonia were immunoreactive for FGFR-4. The difference in localization suggests that these cells have different ligand requirements or that the same ligand may have a different function at these different developmental stages. In the fetal testis, several FGF ligands may act on gonocytes by signaling through FGFR-I and -3; the exact specificity of the receptor will depend on the receptor variant that is present. For example, FGFR-3(IIIb) has a restricted ligand-binding capacity that is limited to FGF-1 and FGF-9, whereas FGFR3(IIIc) can bind more ligands, including FGF-2, which is implicated in gonocyte proliferation in vitro [14]. FGFR-2 was not immunolocalized in the fetal rat testis; this is consistent with a previous report showing that FGFR-2(IIIb) mRNA was not expressed in the fetal mouse testis [31].

FGFRS IN FETAL, IMMATURE, AND ADULT RAT TESTES

It has been suggested that FGF ligands from the seminiferous tubules can regulate tubular and interstitial cell functions. FGFs bind to cell surface and extracellular matrix HSPGs, which act as an extracellular store of growth factor [1]. It is unlikely that FGFs produced in the epithelium are able to freely diffuse between the epithelium and into the interstitium. Our data, showing discrete and specific localization of FGFRs to testicular cells, suggest that the interactions between FGFRs and ligands are highly regulated at each stage of spermatogenesis and during development. Several FGFs are expressed in the adult testis [32]: FGF-1, FGF-2, FGF-3, FGF-6, and FGF-8. Some FGFs have very specific receptor-binding properties, such as FGF-3, which activates only FGFR-l(IIIb) and FGFR2(IIIb) [3]. Identification of the receptor splice variants will provide further insights into the roles of FGFs in spermatogenesis, in Leydig cell function, and during testicular development. ACKNOWLEDGMENT The authors wish to thank Sarah Meecham for assistance with identification of spermatogonia and stages of spermatogenesis.

REFERENCES 1. Baird A, Bohlen P. Fibroblast growth factors. In: Sporn MB, Roberts AB (eds.), Peptide Growth Factors and Their Receptors. Handbook of Experimental Pharmacology. Berlin: Springer-Verlag; 1990: 369-418. 2. Goldfarb M. Functions of fibroblast growth factors in vertebrate development. Cytokine Growth Factor Rev 1996; 7:311-325. 3. Ornitz DM, Xu J, Colvin JS, McEwen DG, MacArthur CA, Coulier E Gao G, Goldfarb M. Receptor specificity of the fibroblast growth factor family. J Biol Chem 1996; 271:15292-15297. 4. Yayon A, Klagsbrun M, Esko JD, Leder P, Ornitz DM. Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 1991; 64:841-848. 5. Spivak-Kroizman T, Lemmon MA, Dikic I, Ladbury JE, Pinchasi D, Huang J, Jaye MM, Crumley G, Schlessinger J, Lax K. Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation and cell proliferation. Cell 1994; 79: 1015-1024. 6. Kan M, Wang E Xu J, Crabb JW, Hou J, McKeehan WL. An essential heparin-binding domain in the fibroblast growth factor receptor kinase. Science 1993; 259:1918-1921. 7. Han IS, Sylvester SR, Kim KH, Schelling ME, Venkateswaran S, Blanckaert VD, McGuinness MP, Griswold MD. Basic fibroblast growth factor is a testicular germ cell product which may regulate Sertoli cell function. Mol Endocrinol 1993: 7:889-897. 8. Le Magueresse-Battistoni B, Wolff J, Morera A-M, Benahmed M. Fibroblast growth factor receptor type I expression during rat testicular development and its regulation in cultured Sertoli cells. Endocrinology 1994; 135:2402-2411. 9. Ueno N, Baird A, Esch F, Ling N, Guillemin R. Isolation and partial characterization of basic fibroblast growth factor from bovine testis. Mol Cell Endocrinol 1987; 49:189-194. 10. Mayerhofer A, Russell LD, Grothe C, Rudolf M, Gratzl M. Presence and localization of a 30-kDa basic fibroblast growth factor-like protein in rodent testes. Endocrinology 1991; 129:921-924. 11. Koike S, Noumura T Cell- and stage-specific expression of basic FGF in the developing rat gonads. Growth Regul 1994; 4:77-81. 12. Hughes SE, Hall PA. Immunolocalization of fibroblast growth factor receptor 1 and its ligands in human tissues. Lab Invest 1993; 69:173182. 13. Mullaney BP, Skinner MK. Basic fibroblast growth factor (bFGF) gene expression and protein production during pubertal development of the seminiferous tubule: follicle-stimulating hormone-induced Sertoli cell bFGF expression. Endocrinology 1992; 131:2928-2934.

1145

14. Van Dissel-Emiliani FM, De Boer-Brouwer M, De Rooij DG. Effect of fibroblast growth factor-2 on Sertoli cells and gonocytes in coculture during the perinatal period. Endocrinology 1996; 137: 647-654. 15. Murono EP, Washburn AL, Goforth DP, Wu N. Evidence for basic fibroblast growth factor receptors in cultured immature Leydig cells. Mol Cell Endocrinol 1992; 88:39-45. 16. Murono EP, Washburn AL, Goforth DP, Wu N. Effects of acidic fibroblast growth factor on 5-ene-313-hydroxysteroid dehydrogenaseisomerase and 5a-reductase activities and [12 51]human chorionic gonadotrophin binding in cultured immature Leydig cells. J Steroid Biochem Mol Biol 1993; 45:477-483. 17. Murono EP, Washburn AL, Goforth DP, Wu N. Evidence that both receptor- and heparan sulfate proteoglycan-bound basic fibroblast growth factor are internalized by cultured immature Leydig cells. Mol Cell Endocrinol 1993; 98:81-90. 18. Fauser BC, Baird A, Hsueh AJ. Fibroblast growth factor inhibits luteinizing hormone-stimulated androgen production by cultured rat testicular cells. Endocrinology 1988; 123:2935-2941. 19. Laslett AL, McFarlane JR, Hearn MT, Risbridger GP. Requirement for heparan sulfate proteoglycans to mediate basic fibroblast growth factor (FGF-2)-induced stimulation of Leydig cell steroidogenesis. J Steroid Biochem Mol Biol 1995; 54:245-250. 20. McFarlane JR, Laslett A, de Kretser DM, Risbridger GP Evidence that heparin binding autocrine factors modulate testosterone production by the adult rat Leydig cell. Mol Cell Endocrinol 1996; 118:5763. 21. Laslett AL, McFarlane JR, Risbridger GP. Developmental response by Leydig cells to acidic and basic fibroblast growth factor. J Steroid Biochem Mol Biol 1997; 60:171-179. 22. Maher PA. Identification and characterization of a novel, intracellular isoform of fibroblast growth factor receptor-i (FGFR-1). J Cell Physiol 1995; 169:380-390. 23. Stachowiak MK, Maher PA, Joy A, Mordechai E, Stachowiak EK. Nuclear localization of functional FGF receptor 1 in human astrocytes suggests a novel mechanism for growth factor action. Mol Brain Res 1996; 38:161-165. 24. Kilkenny DM, Hill DJ. Perinuclear localization of an intracellular binding protein related to the fibroblast growth factor (FGF) receptor 1 is temporally associated with the nuclear trafficking of FGF-2 in proliferating epiphyseal growth plate chondrocytes. Endocrinology 1996; 137:5078-5089. 25. Johnston CL, Cox HC, Gomm JJ, Coombes RC. Fibroblast growth factor receptors (FGFRs) localize in different cellular compartments. A splice variant of FGFR-3 localizes to the nucleus. J Biol Chem 1995; 270:30643-30650. 26. Hou J, Kan M, McKeehan K, McBride G, Adams P, McKeehan WL. Fibroblast growth factor receptors from liver vary in three structural domains. Science 1991; 251:665-668. 27. Horlick RA, Stack SL, Cooke GM. Cloning, expression and tissue distribution of the gene encoding rat fibroblast growth factor receptor subtype 4. Gene 1992; 120:291-295. 28. Smith EP, Hall SH, Monaco L, French FS, Wilson EM, Conti M. A rat Sertoli cell factor similar to basic fibroblast growth factor increases c-fos messenger ribonucleic acid in cultured Sertoli cells. Mol Endocrinol 1989; 6:954-961. 29. Jaillard C, Chatelain PG, Saez JM. In vitro regulation of pig Sertoli cell growth and function: effects of fibroblast growth factor and somatomedin-C. Biol Reprod 1987; 37:665-674. 30. Shi D-L, Fromentoux V, Launay C, Umbhauer M, Boucaut J-C. Isolation and developmental expression of the amphibian homolog of the fibroblast growth factor receptor 3. J Cell Sci 1994; 107:417-425. 31. Finch PW, Cunha GR, Rubin JS, Wong J, Ron D. Pattern of keratinocyte growth factor and keratinocyte growth factor receptor expression during mouse fetal development suggests a role in mediating morphogenetic mesenchymal-epithelial interactions. Dev Dynam 1995; 203:223-240. 32. Yamasaki M, Miyake A, Tagashira S, Itoh N. Structure and expression of the rat mRNA encoding a novel member of the fibroblast growth factor family. J Biol Chem 1996; 271:15918-15921.