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Biology of Reproduction 61, 944-947 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Recombinant Human Insulin-Like Growth Factor I Stimulates All Stages of 11-Ketotestosterone-Induced Spermatogenesis in the Japanese Eel, Anguilla japonica, In Vitro1

Manal R. Nader2,a, Takeshi Miuraa,b, Nobutaka Andoa, Chiemi Miuraa, and Kohei Yamauchia

a Department of Biology, Faculty of Fisheries, Hokkaido University, Hakodate 041-8611, Japan b PRESTO, Japan Science and Technology Corporation, Kawaguchi City, Saitama Pref. 332-0012, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we examined the in vitro effects of insulin-like growth factor I (IGF-I) in the presence or absence of 11-ketotestosterone (11-KT: the spermatogenesis-inducing hormone) on the proliferation of Japanese eel (Anguilla japonica) testicular germ cells. Initially, a short-term culture (15 days) of testicular tissue with only type A and early type B spermatogonia (preproliferated spermatogonia) was carried out in Leibovitz-15 growth medium supplemented with different concentrations of recombinant human IGF (rhIGF)-I or -II in the presence or absence of 10 ng/ml of 11-KT. Late type B spermatogonia (proliferated spermatogonia) were observed in treatments of 100 ng/ml of both rhIGF-I and -II in combination with 11-KT, indicating the onset and progression of spermatogenesis. In all tested rhIGF-I concentrations (except 0.1 ng/ml) supplemented with 11-KT, late type B spermatogonia were detected in at least one individual. Then, we proceeded with an in vitro 45-day culture of testicular tissue with 100 ng/ml of rhIGF-I in the presence or absence of 10 ng/ml of 11-KT to test the long-term effects of rhIGF-I on the spermatogenetic cycle. The presence of all types of germ cells, including spermatozoa, in the testis cultured with the admixture of the two hormones indicated that the germ cells underwent complete spermatogenesis whereas no germ cell proliferation was observed when the rhIGF-I was applied alone. These results suggest that IGF-I in the presence of 11-KT plays an essential role in the onset, progress, and regulation of spermatogenesis in the testis of the Japanese eel.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis is a complex process that can be divided into three major phases: spermatogonial proliferation, meiosis, and spermiogenesis [1, 2]. Several factors appear to be involved in the control of the spermatogenetic process [3, 4], and recently, attention has focused on growth factors as potential mediators of autocrine/paracrine interactions between testicular cells [5, 6]. Increasing evidence points to the insulin-like growth factors (IGFs), more specifically IGF-I, as important regulators of testicular function [2, 3]. Both IGF-I and -II are mitogenic peptide hormones that play an important role in the regulation of growth, differentiation, regeneration, and metabolism [7]. IGF-I is mainly produced in the liver [8–10] and has been known for some time to mediate most of the growth-promoting actions of growth hormone (GH) [11–13].

Spermatogenesis in most mammalian species is a continuous process involving the presence of several successive generations of germ cells [1]. In contrast, the germ cells within each testicular cyst in migratory teleosts (salmonids, eels, etc.) develop synchronously [14, 15]. Previously, we reported that, under culture conditions, type A and early type B spermatogonia (nonproliferated spermatogonia) are the only germ cells present in the testes of Japanese eel, Anguilla japonica [4]. It is believed that this phenomenon is due to insufficient gonadotropin in the pituitary [16]. It has been proven that in most cases a single injection of hCG can induce complete spermatogenesis in the Japanese eel [17, 18] and that the hormonal induction is achieved via stimulation of Leydig cells to produce 11-ketotestosterone (11-KT), which is a major androgen in male eels [19]. In other work, we demonstrated that in vitro, 10 ng/ml of 11-KT in the presence of 1 µg/ml of bovine insulin (bIns) is optimal for the induction of all stages of spermatogenesis in Japanese eel after a 36-day culture period [15]. To our knowledge, in vitro induction of the full cycle of spermatogenesis, from germ stem cells to sperm production, has only been achieved in eels [4]. The establishment of a viable organ culture methodology, coupled with the presence of only type A and early type B spermatogonia, make the testes of the Japanese eel an excellent system for studying the control mechanisms of spermatogenesis.

This work was undertaken to analyze the short- and long-term in vitro effects of different concentrations of IGFs in combination with 11-KT on the proliferation of germ cells in the testis of the Japanese eel, Anguilla japonica.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Cultured male Japanese eels (1 yr old; 180–200 g in body weight) were purchased from a commercial supplier and kept in recirculating fresh water at 23°C.

Culture Technique

In vitro organ culture techniques were performed as described in Miura et al. [17–19]. In brief, eels were anesthetized, killed, and then sterilized in 70% alcohol. Fresh testes were removed and cut into small pieces (1 x 1 x 0.5 mm). For each tested concentration, two fragments from each eel were placed on elder pith covered with a nitrocellulose membrane (autoclaved and dried at 80°C), and each elder pith was floated separately in culture medium. The basal culture medium consisted of Leibovitz-15 medium supplemented with 0.1 mM L-glutamic acid, 0.1 mM L-aspartic acid, 1.7 mM L-proline, 0.5% BSA, and 10 mM HEPES (pH 7.4).

Short-term culture. Testes extracted from nine eels were used for the recombinant human IGF (rhIGF)-I experiment, while testes extracted from five eels were used for each of the rhIGF-II and bIns experiments. Culture was carried out for 15 days with various rhIGF-I (Mediagnost, Tuebingen, Germany) or II (Genzyme, Cambridge, MA) concentrations (0.01, 0.1, 1, 10, 100 ng/ml) or bIns (0.01, 0.1, 1, 10, 100, and 1000 ng/ml; Sigma, St. Louis, MO) in the presence or absence of 11-KT in order to establish the optimum effective concentration of each substance on germ cell proliferation. 11-KT was administered at a constant dose of 10 ng/ml, this being the effective dose for the induction of spermatogenesis in this species [16, 19]. Medium was changed and supplemented with the respective peptides and hormones on the 7th day of culture.

Long-term culture. After establishing the effective concentration of rhIGF-I (100 ng/ml) on germ cell proliferation over a 15-day culture period, we proceeded to carry out a 45-day organ culture experiment of Japanese eel testes extracted from five eels to test whether rhIGF-I in the presence or absence of 10 ng/ml of 11-KT induces all stages of spermatogenesis. Culture of testis fragments with neither substance was also undertaken for the length of the experimental period. Medium was changed and supplemented with peptide and hormone every 7th day of the experimental period.

Microscopy Techniques

Fragments were sampled for light and electron microscopy. For each treatment, the two cultured testis fragments of each eel were fixed in 1% paraformaldehyde, 1% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 and transferred to 10% sucrose in the same buffer for a minimum of 20 min. The fragments were then postfixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 h and embedded in epoxy resin according to standard procedures.

One-micrometer sections were stained with toluidine blue for light microscopic examination. Ultrathin sections were stained with uranyl acetate and lead citrate for electron microscopic examination.

Five random toluidine-blue-stained 1-µm-thick sections from the two fixed fragments of each treatment in each animal were examined. The presence of late type B spermatogonia meant that germ cells had already begun mitotic divisions. In the short-term experiments, all type A and cysts of late type B spermatogonia in each section were counted, ratios were calculated, and data were presented in percentage average of late type B/total germ cell count. The analysis of the data was carried out by the Scheirer, Ray, and Hare extension of the Kruskal-Wallis test (a two-way ANOVA design for ranked data) followed by the post hoc Bonferroni adjustment, and a P < 0.05/number of comparisons was considered significant. In the long-term experiment, cysts of the following germ cell types were estimated, and the percentage average was calculated as germ cell type/total germ cell count: 1) type A and early type B spermatogonia, 2) late type B spermatogonia, 3) spermatocytes, 4) spermatids, and 5) spermatozoa.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Short-Term Culture Experiments

Before culture, Japanese eel testes possess only type A and early type B spermatogonia (Fig. 1a). In all three experiments, the combination of the two hormones was found to be highly significant (P < 0.01). Even though proliferation of spermatogonia was observed in all concentrations of rhIGF-I coupled with 11-KT except in 0.1 ng/ml, the post hoc test showed that only 100 ng/ml of rhIGF-I coupled with 11-KT was significant (P < 0.0001), thus classifying it as the effective concentration. Cysts containing late type B spermatogonia (23.1 ± 6.7%) can be clearly observed in six out of nine animals in the experimental group cultured with 100 ng/ml of IGF-I in combination with 11-KT (Fig. 1c). One eel treated with 100 ng/ml rhIGF-I in the absence of 11-KT displayed late type B spermatogonia, but this group did not statistically differ from control, while fragments from eight eels maintained their original structure (Fig. 1b). In the IGF-II experiment, only a concentration of 100 ng/ml coupled with 11-KT was successful in inducing spermatogenesis in three out of five eels and was found to be highly significant in comparison to the zero dose (P < 0.0001), with late type B spermatogonia constituting 26.4 ± 11.4% of total germ cells. In the experimental group combining 1000 ng/ml of bIns with 11-KT, spermatogenesis was induced in four out of five animals, and even though late type B spermatogonia were observed in one eel in the group cultured with 100 ng/ml of bIns in combination with 11-KT, only 1000 ng/ml of bIns in the presence of 11-KT proved to be significant (P < 0.0001), with late type B spermatogonia amounting to 28.1 ± 12.42% of total germ cell count. In all cultures supplemented with rhIGF-II or bIns but not with 11-KT, the structure of the testis was not altered.



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  		FIG. 1. Light micrograph of a toluidine blue-stained 1-µm section of Japanese eel testis a) before culture, b) cultured with rhIGF-I (100 ng/ml) for 15 days, and c) cultured with 11-KT (10 ng/ml) and rhIGF-I (100 ng/ml) for 15 days. GA: Type A and early type B spermatogonia; L-GB: late type B spermatogonia (bars = 10 µm)

Long-Term Culture Experiment

In cultures supplemented with 11-KT and rhIGF-I, all stages of germ cells were present in four out of the five experimental animals (Fig. 2a) in the following percentages: type A spermatogonia, 19 ± 7.8%; late type B spermatogonia, 9 ± 4.2%; spermatocytes, 20 ± 5.8%; spermatids, 17 ± 5.6%; and spermatozoa, 13 ± 6.1%. The spermatozoon nucleus was crescent-shaped, becoming more round posteriorly with one mitochondrion located at the anterior end and a flagellum with a 9+0 axonemal structure located at the caudal end of the base of the nucleus (Fig. 2b). No distinctive midpiece was observed between the head and the tail of the observed spermatozoa (Fig. 2b). After 45 days of culture, no effect on germ cells was observed in cultures carried out only in the presence of either rhIGF-I or 11-KT, or in the absence of the two hormones.



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  		FIG. 2. Testis cultured with 11-KT (10 ng/ml) and rhIGF-I (100 ng/ml) for 45 days. a) Light micrograph of a toluidine blue-stained 1-µm section of Japanese eel testis. C, spermatocytes; T, spermatids; Z, spermatozoa. Bar = 10 µm). b) Electron micrograph of spermatozoon (bar = 10 µm) with the insert indicating the 9+0 axonemal structure (bar = 0.1 µm)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
After 45 days of culture, the appearance of all stages of spermatogenesis only in the experimental group cultured with 100 ng/ml of rhIGF-I in combination with 10 ng/ml of 11-KT demonstrated that the admixture of the two substances is essential for the induction of the spermatogenetic cycle from spermatogonial proliferation to spermiogenesis.

While the effect of IGF-I on spermatogenesis has been widely documented [5, 6], many aspects of its action remain unclear. On the other hand, the actions of 11-KT are known to be mediated through Sertoli cells [16, 18]. To date, and to our knowledge, no report has been published describing the actions of IGF-I combined with a steroid hormone on the full spermatogenetic process in fish testis. This is probably due to the lack of adequate culture techniques allowing the long-term culture of testicular tissue. For the first time, we demonstrated that rhIGF-I coupled with the fish spermatogenesis-inducing hormone 11-KT gives rise to full spermatogenesis and spermiogenesis from the proliferation of type A spermatogonia (primitive spermatogonia) to the production of spermatozoa in the Japanese eel, Anguilla japonica.

In all tested rhIGF-I concentrations except 0.1 ng/ml, supplemented with a constant concentration of 10 ng/ml of 11-KT, at least one eel out of 9 displayed late type B spermatogonia (proliferating spermatogonia) after 15 days of culture. On the other hand, only 100 ng/ml of rhIGF-II combined with 11-KT was able to induce spermatogenesis after 15 days, while very high concentrations of 1 µg/ml of bIns with 11-KT were necessary. This suggests that the eel testis, like the trout testis, displays affinity for rhIGF-I > rhIGF-II >> Ins, and the Ins effect is believed to be due to the nonspecific binding of this peptide to rhIGF-I receptor sites [1].

The shape of the spermatozoa obtained in this in vitro experiment displayed the general features of those in previously published reports on in vivo eel spermatozoa [20,21]. In the New Zealand freshwater eel [20], as in the Japanese eel [21], the mitochondrion is also located at the anterior end of the crescent-shaped nucleus, with a 9+0 axonemal structure attached to the caudal end. In future research, it will be necessary to assess the viability of these in vitro spermatozoa to gain a better understanding of their development in comparison with their in vivo counterparts.

In our work, only 100-ng/ml levels of rhIGFs yielded statistically positive results, whereas lower concentrations had no clear effects in the case of rhIGF-I and no effect in the case of rhIGF-II. This might be due to the fact that we used rhIGFs, which are 80% homologous to teleost IGFs [9]. In a study carried out on coho salmon, Moriyama et al. [10] found that the IGF-I plasma levels vary between 45.2 ± 5.4 and 117.4 ± 19.1 ng/ml, placing our effective concentrations within the range found in the plasma of another teleost species and confirming that the positive effects of IGFs observed in our experiment are not pharmacological.

It is widely accepted that 11-KT is the spermatogenesis-inducing hormone in the Japanese eel and that its actions are mediated through Sertoli cells [19]. On the other hand, the production sites of IGF-I in the testis appear to be numerous while an involvement of Sertoli cells has been demonstrated [6]. Given that Sertoli cells mediate the actions of 11-KT and that IGF-I is produced by these somatic cells, it can be speculated that 11-KT may have a positive effect on the production of IGF-I by Sertoli cells and, hence, stimulate the spermatogenetic process. Our results do not support this hypothesis as no statistically significant results were obtained with treatments of only 10 ng/ml of 11-KT or with 11-KT and rhIGF-I or rhIGF-II concentrations equal to or lower than 10 ng/ml. We can, therefore, conclude that IGF-I production is not mediated through the presence of 11-KT and that another factor is responsible for IGF-I secretion in the testis. Consequently, it appears that IGF-I plays an essential supporting role in allowing the germ cells to proceed through spermatogenesis. Recently, Gomez et al. [22] have localized GH receptors in the testis of rainbow trout. The pathway linking IGF-I and GH, in which IGF-I seems to mediate many of the actions of GH, is well established [11, 12]. GH treatments have been shown to modify testicular rhIGF-I mRNA content in rainbow trout, and data supporting the occurrence of GH receptors in Sertoli cells have been obtained [22]. Since Sertoli cells are one of the suggested sites of IGF-I production [6], a direct stimulation of IGF-I output by GH from Sertoli cells appears to be very plausible.

Furthermore, the liver has been reported to be the major source of IGF-I. In the testis of mammals, an endocrine role for IGF-I has been proposed [23], with the inference that the peptide can be made available to the testis from the liver or some other organ through the blood stream. In a separate experiment using polymerase chain reaction, we have detected an IGF-I mRNA product in the testis and liver of Japanese eels after 6 days of being injected with hCG (data not shown). Therefore, in eel, the source of IGF-I to the germ cells could be endocrinological (through the liver), paracrine (somatic cells to germ cells), autocrine (germ cells to germ cells), or all three combined as has been suggested in mammals [23].

In conclusion, we have clearly demonstrated that rhIGF-I in combination with 11-KT can induce all stages of spermatogenesis from type A spermatogonia to spermatozoa, whereas when applied alone, it has no statistically significant effect on germ cell proliferation. This suggests that, in agreement with other studies, 11-KT is necessary for the induction of spermatogenesis while IGF-I is necessary for the continuation of the process. Our results offer the first proof of the necessity of the presence of both IGF-I and 11-KT for the stimulation of all stages of spermatogenesis in fish testis.


   ACKNOWLEDGMENTS
 
We would like to thank Dr. Noda Takashi from the Laboratory of Marine Zoology, Hokkaido University, and Mr. Amer Mohamed Abdel-Baky, Department of Biology, Hokkaido University, for their technical help and advice in the statistical analyses.


   FOOTNOTES
 
1 This study was supported in part by grants-in-aid from the Ministry of Agriculture, Forestry, and Fisheries of Japan (BDP 98-IV-2-1), a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and "Research for the Future Program," The Japan Society for the Promotion of Science. Back

2 Correspondence and current address: Department of Biology, Faculty of Fisheries, Hokkaido University, 3-1-1, Hakodate 041-8611, Japan. FAX: 81 138 40 5546; mnsqr{at}pop.fish.hokudai.ac.jp Back

Accepted: May 12, 1999.

Received: August 24, 1998.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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