THE JOURNAL OF EXPERIMENTAL ZOOLOGY 272:475-483 (1995)

Changes in the Distribution of Fibroblast Growth Factor in the Teleostean Testis During Spermatogenesis

AKIHIKO WATANAEiE AND KAZUO ONITAKF: Department of Biology, Faculty of Science, Yamagata University, 1-4-12 Kojirakawa-machi, Yamagata-city, Yamagata, Japan 990

ABSTRACT The distribution of fibroblast growth factor (FGF) in the testes of Salvelinus leucomaenis and Oryzias Zatipes was investigated by immunofluorescence staining. In S. leucomaenis, spermatogonia were strongly stained in the infantile testis. When human chorionic gonadotrophin (HCG) was injected into individual fish, the uptake of 5-bromo 2'-deoxyuridine (BrdU) occurred within a day At the same time, FGF became undetectable in spermatogonia, but appeared for the first time around the testicular somatic cells. A similar distribution was seen in the mature testes of S. leucomaenis and 0. latipes. The positively immunostained area corre- sponded to the predominant site of the Sertoli cells. This pattern of distribution of FGF suggests that FGF plays an important role in the initiation and progression of spermatogenesis in the teleostean testis. Q 1995 Wiley-Liss, Inc.

Spermatogenesis in vertebrates is controlled by interactions between germ cells and somatic cells in the testis, under the influence of certain hor- mones. Many studies have been reported of at- tempts to understand the mechanisms responsible for the control of spermatogenesis in various spe- cies. In mammals there is evidence that some growth factors, including activins, fibroblast growth factor 2, insulin-like growth factor 1, in- terleukin 1, transforming growth factors a and p, and seminiferous growth factor, might be involved in these interactions (Skinner, '93), but their roles in the testis are still unknown.

In the case of teleosts spermatogenesis can be initiated in the eel testis by a single injection of human chorionic gonadotrophin (HCG) (Miura et al., '91a). The treatment with HCG results in in- creased levels of ll-ketotestosterone (11-KT), the expression of mRNA for activin B, and morpho- logical changes in Leydig cells and Sertoli cells (Miura et al., '91a; Nagahama et al., '94).

Fibroblast growth factors (FGFs) are important for the control of proliferation and/or differentia- tion in various types of cell (Gospodarowicz, '90). They are characterized by their heparin-binding activity FGF has been reported in the testis (Ueno et al., '87) and it can affect Sertoli cells in vitro (Jaillard et al., '87; Smith, '89).

In this study, we investigated the distribution of FGF in the infantile and the mature testes of two teleosts by immunofluorescence staining. Te- 0 1995 WILEY-LISS, INC.

leostean testes can be divided into two groups on the basis of the distribution of spermatogonia in the seminiferous tubules (Grier, '81). In one group, spermatogonia are distributed over the entire length of the tubules unrestricted, and in the other, the spermatogonia are confined to the distal ter- mini of the tubules restricted. We used the testes of two species, namely, Saluelinus leucomaenis, which belongs to the first group mentioned above, and Oryzias latipes, which belongs to second.

MATERIALS AND METHODS Animals

Adult specimens of Salvelinus leucomaenis were provided by IWANA-Center (Oguni-machi, Yama- gata, Japan). The individuals used for the experi- ments were 19-21 cm in length in March and July, and 30-40 cm in length in November. They were kept at 10°C. Adult specimens of Medaka, Oryzias latipes, were purchased from Morikawa fish farm (Yamato-koriyama, Nara, Japan) and kept at 26°C in our laboratory before use in experiments. They were sexed by the morphological features of their maxillae (S. leucomaenis) and fins (0. latipes).

Received January 10, 1995; revision accepted April 17, 1995. Address reprint requests to A. Watanabe, Department of Biology,

Faculty of Science, Yamagata University, 1-4-12 Kojirakawa-machi, Yamagata-city, Yamagata, Japan 990.

476 A. WATANBE AND K. ONITAKE

Injection of HCG At least five individuals were used for each

experiment. Injection were given in March and April when no spermatocytes or spermatids were visible in the testes. Human chorionic go- nadotrophin (HCG; Teikoku Zoki, Tokyo, Japan) was injected of lOpg/g body weight into the muscle of each individual. One day after the in- jection, fish were pithed and their testes were removed for analysis.

Incorporation of Brd U Tests were cut into approximately 500-pm

cubes. They were incubated in Leibovits’ L15 me- dium contained 50 pg/rnlEi-bromo 2’-deoxyuridine (BrdU; Nakarai, Kyoto, Japan), 10% FBS at 20°C for 12 hr.

Immunofluorescence staining The methods used were the same as previ-

ously reported (Watanabe e t al., ’93). In brief, testes were dissected out and fixed in PLP fixa- tive that contained 2% paraformaldehyde at 4°C for 1 hr, immersed in 10% sucrose in PBS and

in 20% sucrose in PBS sequentially at 4°C for 1.5 hr each. They were embedded in 0. C. T. com- pound (Miles) and frozen in liquid nitrogen. Fro- zen sections of 8 pm in thickness were prepared. They were reacted with an human FGF2-spe- cific monoclonal antibody at room temperature for 30 min and then with FITC-labeled antibod- ies raised in goat against mouse IgG (MBL; di- luted 1: 30 in PBS that contained 3% BSAj at room temperature for 40 min.

For the detection of BrdU, frozen sections were washed in 20 mM PBS. They were treated with 80 mM HC1 at room temperature for 20 min. After washing in 20 mM PBS, they were treated with 1 mM cacodylate buffer. (0.89 mM sodium cacodylate 0.11 mM HC1, pH 7.0) at 75°C for 3 min and washed in 20 mM PBS. They were reacted with a BrdU-specific monoclonal antibody (Becton Dickinson, San Jose, CA) at room temperature for 30 min and then stained at room temperature for 60 min with the FITC- labeled goat antibodies against mouse IgG de- scribed above.

In control, a chick-specific monoclonal antibody

Fig. 1. Sections of the testis of S. leucomaenis. The testes were removed for analysis in March (a), July (b), November (c). Six-km paraffin sections were cut and stained with hematoxylin-eosin solution. g, spermatogonium; c, spermatocyte; t, spermatid; sp, sperm; sc, somatic cell. Bar, 100 pm.

DISTRIBUTION OF FGF IN THE TELEOSTEAN TESTIS 477

or PBS that did not include any antibodies was used for the first immunoreaction instead of the FGFB- specific or the BrdU-specific monoclonal antibody

Histology Testes were dissected from the bodies of S.

leucomaenis or 0. latipes and immediately fixed in Bouin’s fixative. They were then cut into 10- pm sections and stained in Delafield’s hematoxy- lin and eosin solution.

RESULTS Structure ofthe testis of^. leucomaenis In S. Leucomaenis, all germ cells in the testis

were exclusively spermatogonia in March (Fig. la). In July, many cysts were seen in the testes and not only spermatogonia but also spermatocytes and spermatids were seen within these cysts (Fig. lb). In November, every cyst was occupied by sperm and a layer of Sertoli cells was seen around

Fig. 2. Immunofluorescence staining of the infantile testis of S. leucomaenis with the monoclonal antibody against FGF protein. a: An 8-pm frozen section was stained. b: Control: the FGF-specific antibody was not included in the immunoreaction. c,d Bright field view of a and b. e: High-magnification view of a. Bars, 100 pm (a) and 25 pm (e).

478 A. WATANABE AND K. ONITAKE

the cyst (Fig. lc). In teleosts, the testes have two different types of structure, as discussed above: unrestricted and restricted (Grier, '81). Our results indicate that the testis of S. leucomaenis is of the former type.

Distribution of FGF in the infantile testis of S . leucomaenis

Before spermatogenesis had been initiated, sper- matogonia were strongly immunostained by the FGF-specific antibody (Fig. 2). The antigen was

seen not in the nucleus but in the cytoplasm and on the cell membrane (Fig. 2e). Very weak sig- nals were seen around the numerous cysts.

Effects on an injection of HCG on the testis of S . leucomaenis

It is known that an injection of HCG causes the initiation of spermatogenesis in the eel testis (Miura et al., '9lb). "0 examine the effects of HCG on spermatogenesis in S. leucomaenis, the syn- thesis of DNA in spermatogonia was examined by

Fig. 3. Labeling with BrdU of the testis of S. leucomaenis 1 day after the injection of HCG. a: An 8-pm frozen section of the testis that had been treated with BrdU for 12 hr and stained with the BrdU-specific antibody. c: High-magnifica- tion view of a. e: Control. The testis without prior injection

of HCG was treated with BrdU for 12 hr and stained with the BrdU-specific antibody. b, d, fi Bright-field view of a, c and e. Asterisks show the cysts that contain BrdU-labeled spermatogonia. Bars, 100 pm (a) and 25 pm (c).

DISTRIBUTION OF FGF IN THE TELEOSTEAN TESTIS 479

monitoring incorporation of BrdU. In the control testis, from fish that had not been treated with HCG, uptake of BrdU was rarely detected in sper- matogonia with the antibody against BrdU (Fig. 3a,c). One day after the injection of HCG, many immunofluorescent cells were detected in the tes- tis. We often observed that spermatogonia in ev- ery cyst were clearly labeled with BrdU. This result indicates that HCG can stimulate the syn- thesis of DNA in spermatogonia. Furthermore, the distribution of FGF was examined in the same testis. No immunofluorescence were seen in sper- matogonia 1 day after the injection (Fig. 4a,b). In these testes, signals were clearly detected around each cyst.

Distribution of FGF in the mature testis of S. leucomaenis

When spermatogenesis was proceeding, in July, the area around each cyst was strongly immunostained with the antibody against FGF (Fig. 5a,b). Testicular somatic cells, for the most part Sertoli cells, were distributed in this area.

No signals were detected in the developing germ cells.

When sperm occupied the entire testis, in No- vember, signals were detected around each cyst (Fig. 6a). This pattern was similar to that seen in July. Sertoli cells forming each cyst were strongly stained with the antibody against FGF (Fig. 6b). The number of Sertoli cells was apparently lower in November than in July (Figs. 5d, 6d).

Distribution of FGF in the mature testis o f 0 . latipes

To compare the distribution of FGF between the testis of S. leucomaenis and that of other species, frozen sections of the testis of 0. latipes were im- munostained with the antibody against FGF. Strong signals were detected around all cysts that contained germ cells at each stage (Fig. 7a,c,e). These areas corresponded to the site of Sertoli cells and their derivatives, the efferent duct cells. This observation indicates that FGF was distrib- uted in the testicular somatic cells or in the ex- tracellular space around them.

Fig. 4. Immunofluorescence staining of the testis of S. leucomaenis 1 day aker the in- jection of HCG. a: An 8-pm frozen section was stained with the monoclonal antibody against FGF. b: High-magnification view of a. c, d: Bright-field view of a and b. Bars, 100 Frn (a) and 25 pm (b).

480 A. WATANBE AND K. ONITAKE

Fig. 5. Immunofluorescence staining of the testis of S. leucomaenis in July with the monoclonal antibody against FGF. a: An 8-pm frozen section was stained. b: High- magnification view of a. c , d: Bright-field view of a and b. Bars, 100 pm (a) and 25 pm (b).

DISCUSSION Fibroblast growth factors are heparin-binding

factors that control the proliferation and/or the differentiation of many types of cell (Gospodarowicz, '90). In this study, we used a monoclonal antibody against human FGFB (Reilly et al., '89). Human FGFB protein is effective in other species (Dawid et al., '90; Niswander, '93). Thus it appears that the FGF of S. leucomaenis and 0. latipes has an epitope that is recognized by the antibody and can be detected by immunofluorescence staining.

In teleosts, the testes can be classified into two types. In one type, spermatogonia are found along the entire length of the seminiferous tubules (Grier, '81). Before spermatogenesis is initiated, spermatogonia and Sertoli cells form nearly solid cords in the tubules, When spermatogenesis is ini- tiated, Sertoli cells form the borders of cysts in which the proliferation and differentiation of sper- matogonia occur. In the other type, spermatogonia are localized in the distal termini of the tubules. In this type, as development of germ cells proceeds, the cysts that contain them are seen in the inner

area of the testis. S. leucomaenis has the former type of the testis (Fig. 1) and 0. latipes the latter.

In this study, we showed that FGF protein is localized in spermatogonia in the infantile testis of S. leucomaenis (Fig. 2a). It has been reported that FGF is also localized in rat spermatocytes (Lahr, '92). However, the effect of this protein on germ cells is still unknown. FGFB is known as a maintenance factor or a mitogen for some types of cell (Tilly et al., '92; Matsuda et al., '90; Gospodarowicz, '90; Watanabe and Ide, '93). In the testis, it may also act as a factor that controls the survival and/or the proliferation of germ cells.

It has been reported that pituitary gonadotro- pins cause the infantile testis of rainbow trout to mature (Robertson and Rinfrer, '57). It is also known that spermatogenesis can be initiated in the eel testis by treatment with HCG (Miura et al., '91a). In this report, it was shown that Leydig cells and Sertoli cells were activated 1 day after the injection of HCG, and the proliferation of sper- matogonia, meiosis and spermiogenesis occurred subsequently. In this study, it appeared that the

DISTRIBUTION OF FGF IN THE TELEOSTEAN TESTIS 48 1

Fig. 6. Immunofluorescence staining of the testis of S. leucomaenis in November with the monoclonal antibody against FGF protein. a: An 8-pm frozen section was stained. b: High-magnification view of a. c, d Phase-contrast image of a and b. sp, sperm; sc, somatic cells. Bars, 100 Krn (a) and 25 pm (b).

synthesis of DNA occurred in spermatogonia within 1 day after the treatment with HCG (Fig. 3a,c). This result suggests that HCG can trigger the initiation of spermatogenesis in S. leucomaenis.

It is thought that FGF might be a paracrine factor that acts as a signal for the initiation of spermatogenesis in the testis. Our results suggest that, upon the stimulation by HCG, FGF becomes localized on the borders of the cysts (Fig. 4a,b). This pattern is quite similar to that seen in the mature testis. These results suggest that, upon the stimulation by hormones, FGF might be se- creted from spermatogonia and stimulate the so- matic cells around them and that FGF protein may act as one of the factors that control the ini- tiation of spermatogenesis in vivo. In order to identify the role of FGF in germ cells, it is neces- sary to confirm where and when FGFs and their receptors are produced.

Activin is involved in the control of the initia- tion of spermatogenesis in the vertebrate testis. It is reported that activin stimulates the sper- matogonial proliferation in a Sertoli cell-germ cell co-culture system from the rat (Mather et al., '90). Furthermore, the expression of mRNA for activin

B is stimulated in eel testis prior to the initiation of spermatogonial proliferation under the control of some hormones such as gonadotropin and 11- ketotestosterone (Nagahama et al., '94). In this case, the activation of Leydig cells and Sertoli cells occurs 1 day after the initiation (Miura et al., '91aj. This result corresponds closely to the tim- ing of the changes in the distribution of FGF that were seen in this study

FGF might cooperate with activin to initiate spermatogenesis in the testis of S. leucomaenis.

In the mature testis, FGF protein was distrib- uted on the borders of the cysts, which were com- posed of some types of somatic cell (Figs. 5, 6). The same distribution was seen in 0. Zatipes (Fig. 7a,c). These results indicate that FGF affects so- matic cells during the process of spermatogenesis in the teleostean testis. In the rat, FGF is dis- tributed in Leydig cells and on the basement membrane of the seminiferous tubules of the de- veloping testis (Gonzalez et al., '90; Mullaney and Skinner, '92). It is likely that germ cells and each type of somatic cell in the teleostean testis are homologous to those in mammals (Grier, '81). Then, our results suggest the probability that FGF

482 A. WATANABE AND K. ONITAKE

Fig. 7. Immunofluorescence staining of the mature testis of 0. latipes with the monoclonal antibody against FGl? a: An 8-ym frozen section was stained. b: Control. The FGF- specific antibody was not included in the immunoreaction. c: High magnification in a. d Bright-field view of c. e: A 6-pm

paraffin section was stained with hematoxylin-eosin solution. Arrowheads indicate the distal terminis of seminiferous tu- bules. g , spennatogonium; c, spermatocyte; t, spermatid. Bars, 100 ym (a) and 25 km (c).

protein has the same effect on the somatic cells in the teleostean testis as it does in the develop- ing mammalian testis. In mammals, FGFZ is se- creted by Sertoli cells (Smith et al., '89) and stimulates the proliferation of immature Sertoli cells (Jaillard et al., '87) in vit.ro. Further experi- ments are necessary to identify the role of FGF protein in the testis.

The details of the role of FGF in spermatoge- nesis in the vertebrate testis are still unknown.

The teleostean testis is relatively simple and, in addition, the germ cells and somatic cells within it are homologous to those in mamma- lian testis (Grier, '81). Furthermore, it is known that the spermatogonia can develop sequen- tially into sperm in organ culture or cell cul- ture (Miura et al., '91b; Saiki and Onitake, '90). Therefore, the teleost is a useful material for investigations of control mechanisms in sper- matogenesis.

DISTRIBUTION OF FGF IN

ACKNOWLEDGMENTS The authors thank Dr. T. Reilly and Dr. H.

Walton of E.I. du Pont de Nemours and Company for the gift of the monoclonal antibody against hu- man FGF2 protein.

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