A rapid activation of immature testis of Japanese eel (Anguilla japonica) by a single injection of human chorionic gonadotropin

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1 J. Biosci., Vol. 20, Number 5, December 1995, pp Printed in India. A rapid activation of immature testis of Japanese eel (Anguilla japonica) by a single injection of human chorionic gonadotropin 1. Introduction D Ν SAKSENA*, Τ MIURA, J JIANG and Υ NAGAHAMA Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444, Japan *School of Studies in Zoology, Jiwaji University, Gwalior , India MS received 19 April 1995; revised 18 August 1995 Abstract. The testis of Japanese eel (Anguilla japonica) consists of type A and early type B spermatogonia together with inactive Leydig and Sertoli cells. A single injection of human chorionic gonadotropin induced marked changes in the morphology of the testis and in the scrum androgen levels within a period of 72 h. Morphological changes include spermatogonial proliferation, activation of Leydig and Sertoli cells, organization of seminiferous lobules and formation of lobular lumen in the testis. Leydig cells were enlarged, exhibiting characteristics of steroid-producing cells. Sertoli cells become elongated, show signs of high cellular activity and remain in close contact with spermatogonia. The lobular organization was achieved much earlier than the progression of spermatogenesis to late type Β spermatogonia. Even 6 h after hcg injection, a significant increase in plasma levels of 11-ketotestosterone was observed, followed by a further time dependent increase. Plasma testosterone levels were also increased after injection, but the increase was much less than that of 11-ketotestosterone. Keywords Testicular reorganization; spermatogenesis; Sertoli-Leydig cell function; Anguilla japonica. Generally in the vertebrates various components of testis form a well defined cellular organization. The association of cells and sequence of their appearance in seminiferous tubules are highly organized. However, in fishes, each spermatogenic cycle is followed by a resting stage which in turn is led by a testicular reorganization once again. The lobular lumen is formed, for the release of spermatozoa, during the process of late spermatogenesis by the rearrangement of germ cell cysts and somatic cells especially Sertoli cells. Spermatogenesis in male eels remains suspended and sexual maturity is never attained in freshwater or under conditions of culture However, induction of spermatogenesis and maturation has been achieved by the administration of exogenous gonadotropin in varying doses for different durations (Boetius and Boetius 1967; Yamamoto and Yamauchi 1974; Sugimoto and Takahashi 1979; Khan et al 1987; Colombo et al 1987). Recently, we have been successful in inducing spermatogenesis up to spermiation in Japanese eel within three weeks after injecting a single dose of human chorionic gonadotropin (hcg) (Miura et al 1991a,b). Thus, eel testis, provides an excellent model for studying testicular structure and function. In the *Corresponding author. 675

2 676 D Ν Saksena et al present paper, therefore, an attempt has been made to study the organization of testis, structure of germ and somatic cells and serum titers of androgens during induction of early spermatogenesis in Japanese eels under the influence of hcg. 2. Materials and methods Cultivated male eels (weighing g) were purchased from a commercial cel supplier in April, 1991 and were kept in circulating freshwater tanks having a capacity of 500 litres maintained at 20 C. hcg (Sigma) was dissolved in saline solution (150mM NaCl), and was administered intraperitoneally to 25 eels at a dose of 5 IU/g body weight. An even number of eels received the same amount of saline solution and served as controls. Five eels each from the above two groups were sacrificed after anesthetising them in 0.1 % ethyl aminobenzoate solution; 6, 12, 18, 24 and 72 h after hcg or saline injection The eels were not fed during the experiment. The blood samples were collected from the caudal vasculature by a syringe. The serum was separated by centrifugation at 4 C and was stored at -80 C until used for assay. The testis pieces were removed carefully after dissecting eels and were fixed in Karnovsky's solution at 4 C overnight. The tissue pieces were then postfixed in 1% osmium tetraoxide in cacodylate buffer After processing, they were embedded in spurr or epon (Polyscience Inc., Warrington, USA) following a standard procedure. One μm thick sections stained with 0 1% toluidine blue were prepared for light microscopical examination. Ultra thin sections (70-90 nm) were prepared by a diamond knife and Sorval MT 5000 ultramicrotome, stained with uranyl acetate and lead citrate, and were examined under a JEOL 100-cx transmission electron microscope. The serum levels of androgens (testosterone and 11-ketotestosterone) were determined by specific radioimmunoassay following the method of Ueda et al (1985). All the results were expressed as means ± Standard error and the changes in the serum androgen levels were statistically analysed by two way ANOVA and differences between mean values within each group were measured by paired 't' test. 3. Results 3.1 Anatomy The testis of cultivated eels, when observed under light microscopy, consist of germinal tissue and interspersed connective tissue. The germinal tissue is disposed into chord-like testicular lobules containing spermatogonia and a few somatic Sertoli cells. The interlobular connective tissue also contains interstitial cells and blood capillaries. The spermatogonia are rounded in shape having round nuclei with prominent nucleoli. The cell boundaries, nuclear boundaries, and darkly stained granules representing mitochondria are distinctly visible. The Sertoli cells are found among spermatogonia with irregular shape but with Dell defined nuclei (figure 1A). The histoarchitecture of testes after 6, 12 and 18 h of hcg treatment remains unchanged. A few mitotic figures can be seen after 24 h of hormonal injection. A

3 Activation of immature testis of Japanese eel by hcg 677 Figure 1. Semithin sections of the testis of Japanese eel (toluidine blue preparations). (A) Before hcg treatment. Note connective tissue (CT), type A spermatogonia (GA) and Sertoli cells (arrow heads) ( 650). (B) 72 h after hcg treatment. Note the formation of seminiferous tubules with lumen (L.). Arrow heads point to the mitotic figures in early type Β spermatogonia and LGB late type Β spermatogonia ( 650).

4 678 D Ν Saksena et al dramatic change, however, takes place after 72 h when the spermatogonia type A and both early and late spermatogonia type Β along with Sertoli cells organize themselves in such a manner that a quite distinct lobular structure with a lumen is formed. The interlobular connective tissue is reduced considerably, and mitotic division of spermatogonia early type Β are observed frequently (figure 1B). Ultrastructurally, in the testis of cultivated eels, two types of spermatogonia (type A and early type B) are recognized (figure 2A-B). Type A spermatogonia are isolated and scattered in the testicular lobule, while early type Β spermatogonia lie in the cysts. Both type A spermatogonia and cysts are surrounded by Sertoli cells. Type A spermatogonia have a sheet of cytoplasm around big rounded homogeneously dense nuclei. Single sometimes double nucleoli with denser granules are also observed. The mitochondria are rounded in shape with parallel cristae and are located mostly at places where cytoplasmic accumulation is greater in the cell. Electron dense mitochondrial cement in intermitochondrial space of adjoining mitochondria is also seen. The endoplasmic reticulum is disposed throughout the cell. Early type Β spermatogonia remaining in the cysts are mostly similar to type A spermatogonia in shape and other details but are smaller in size. Sertoli cells are found surrounding the spermatogonia, whether inside or outside the cysts, in clusters deep inside the testicular lobules. They possess irregular nuclei and contain round lipid globules (figure 2B). Rounded but sometimes elongated Leydig cells are disposed singly or in groups at the periphery of the testicular cysts separated by basal lamina along with the fibroblast cells and other connective tissue. Mitochondria in Leydig cells are small, irregular and disorganized. The endoplasmic reticulum also remained with no sign of secretory activity (figure 2C). No change of the testicular structure was evident alter 6 h of hcg treatment as compared to that of the initial control group but Sertoli cells become elongated and are active with rounded mitochondria and irregular nuclei after 12 h (figure 3A). Leydig cells also show organization of mitochondrial elements with endoplasmic reticulum still developing (figure 3B). After 24 h of hormone injection, the testicular organization remains the same but the testicular cysts with early type Β spermatogonia are increased. Sertoli cells are elongated with an irregular nucleus containing more electron dense material towards its periphery (figure 4A). Mitochondria with tubular cristae are observed.at places Sertoli cells receive invaginations of spermatogonial cytoplasm indicating very close physiological association between them (figure 4B). Leydig cells are further activated with a greater number of enlarged mitochondria and smooth endoplasmic reticulum (figure 4C). Seventy two h after treatment, as a result of mitotic divisions, late type Β spermatogonia are produced. These cells have dense nuclei with heterogenous distribution of electron-dense granules (figure 5A). The cellular bridges between these cells are also observed (figure 5B). Sertoli cells are much elongated (figure 5C) with a comparatively more active Golgi apparatus (figure 5D). The lipid globules are usually present in Sertoli cells Leydig cells at this stage are large and found in groups (figure 5E) and assume characteristics of steroid synthesizing cells. The well organized mitocondria with tubular cristae, smooth endoplasmic reticulum and Golgi elements in the form of cisternae and vesicles are observed. The free ribosomes are distributed throughout the cytoplasm (figure 5F). The oocytic cysts with meiotic figures and synaptonemal complex were also observed in some specimens.

5 Activation of immature testis of Japanese eel by hcg 679 Figure 2. Testis of Japanese eel before hcg treatment. (A) Type A spermatogonia (GA) surrounded by Sertoli cells (SR). An asterisk marks nucleoli and white arrow head the mitochondrial cement ( 9,100). (B) Sertoli cells with irregular nuclei (N) and lipid globule (Li) ( 13,000). (C) Leydig cells with small disorganized mitochondria (M) with no signs of secretory activity ( 12,000).

6 680 D Ν Saksena el al Figure 3. Electron micrograph of the testis of Japanese eel after 12 h of hcg treatment showing (A) Sertoli cells and (B) Leydig cells started becoming active with organization of mitochondria (M) and other cellular components ( 12,000). Figure 4. Testis of Japanese eel after 24 h of hcg treatment (A) Elongated Sertoli cells with greater number of mitochondira (M) and endoplasmic reticulum (ER) ( 12,000). (Β) Association between spermatogonia (GS) and the Sertoli cells (SR) ( 15,000). (C) Leydig cells with better cellular organization. Note well developed mitochondria (M) ( 12,000).

7 Figure 4. Activation of immature testis of Japanese eel by hcg 681

8 682 D Ν Saksena et al Figure 5A and Β. Testis of Japanese eel after 72 h of hcg treatment (A) Mitotic figure in early type Β spermatogonia ( 6,200). (Β) Late type B spermatogonia ( 11,000).

9 Activation of immature testis of Japanese eel by hcg 683 Figure 5 C-F. Testis of Japanese eel alter 72 h of hcg treatment. (C) Cellular bridges between type late Β spermatogonia ( 6,500). (D) Sertoli cells with active Golgi element (GE) ( 14,000). (E) A group of Leydig cells ( 7,000). (F) An active Leydig cell with steroid-producing characteristics of mitochondria (M) and Golgi elements (GE) ( 14,000)

10 684 D N Saksena et al 3.2 Organization of lobules and lumen formation In the immature eel testis, the testicular lobules contain spermatogonia and Sertoli cells. Early type Β spermatogonia takes part in the cyst formation. The Sertoli cells are found surrounding the germ cells and the cysts, and are also located in clusters filling up the spaces in chord like lobules. With increased spermatogonial division, number and size of testicular cysts are increased and groups of Sertoli cells become localized in the center. Further, increase in the size of cysts takes place due to division of spermatogonia. As a result, central Sertoli cells are pushed apart leaving a gap in between, in the central part of the lobule (figure 6A). This gap is further widened and takes the shape of tubular lumen (figure 6B,C). The testicular lobules thus formed consist of cysts containing spermatogonia surrounded by Sertoli cells with a lumen in the centre and Leydig cells lying just outside the cyst. 3.3 Changes in the plasma level of androgens after hcg injection The plasma level of testosterone and 11-ketotestostcrone during the course of experiment have been shown in figure 7. Prior to the hcg treatment, the levels of testosterone and 11-ketotestosteronc were 0 32 ± 0 21 and 0 27 ± 0 05 ng/ml, respectively. After hcg administration, the levels of both the androgens increased significantly (f<0 01), reaching a maximum after 18 h (testosterone, 1 1 ±0 086 ng/ml and 11-ketotestosterone 5 7 ± 0 4 ng/ml) and slightly decreased after 24 h. 4. Discussion The testicular structure in teleosts has been found to be quite variable in comparison to higher vertebrates. The testis in most teleosts consists of compact paired structures lying in the abdominal cavity and composed of a mass of elongated, branched tubular structures with thin fibrous walls which lack a permanent lining, seminiferous epithelium and because of this reason, they are generally referred to as lobules, crystals or canals (Lofts 1969). On the basis of distribution of spermatogonia and spermatogenetic pattern, two kinds of testicular structures namely, tubular and lobular types have been identified (Hoar 1969; Billiard et al 1982; Nagahama 1983, 1986; Redding and Patino 1993). The former type corresponds to the restricted spermatogonial testis and is characteristic of fishes belonging to the order Atheriniformes while the later to the unrestricted spermatogonial testis which is similar to the mammalian testis and is typical of most teleosts (Grier 1981). A block in the pituitary gonadotropic function seems to be responsible for suspended maturity in male eels in rivers and under the culture conditions. Successful attempts have been made to induce sexual maturity in European and Japanese eels by means of hormonal injections. Colombo et al (1987) and Khan et al (1987) induced spermatogenesis and production of spermatozoa in European eels by administering a single dose of hcg after 1 month and 3 months, respectively. Sugimoto and Takahashi (1979) have shown that interstitial (Leydig) cells are activated in the testis of Japanese eel during hcg induced maturation Recently, Miura et al (1991a) have induced sexual maturation in Japanese male eels within 18 days by administering a single dose of hcg. In the present study, activation of Sertoli and Leydig cells

11 Activation of immature testis of Japanese eel by hcg 685 Figure 6. Testis of Japanese eel showing phases of lobular organization. (A) Localization of Sertoli cells in the centre ( 7,600). (B) Creation of a gap between Sertoli cells ( 8,000). (C) Widening of gap and lobular lumen formation (L) ( 8,000). after 12 h, mitotic spermatogonial division after 24 h and lobular organization after 72 h were achieved by injecting a single dose of hcg. Thus, the pituitary- gonadotropic function was restored by exogenous administration of hcg resulting in the induction of spermatogenesis.

12 686 D Ν Saksena et al Figure 7. Effects of a single dose of hcg on plasma androgen levels. Testosterone ( ) and 11-ketotestosterone ( ) after hcg treatment. Testosterone (O) and 11-ketotestosterone ( ) after saline treatment. The vertical bars represent the mean ±SEM. The structure and disposition of spermatogonia were very similar to the description of earlier workers (Gresik et al 1975; Sugimoto and Takahashi 1979; Grier 1975; Billard 1984; Colombo et al 1987; Miura et al 1991a). In A. japonica, isolated type A spermatogonia are always surrounded by Sertoli cells, while early type Β spermatogonia were restricted to testicular cysts and continue to divide until spermatozoa formation. Each cyst is enclosed in a covering of Sertoli cells and the cells extend in between and remain in close contact with spermatogonia. Clusters of Sertoli cells also occur inside the testicular lobule. The Leydig cells occurring singly or in groups always lie outside the cyst in the interstitium separated by basement membrane. The nucleolar subdivisions namely pars fibrosa and pars granulosa observed in spermatogonia of guppy, Poecilia reticulata (Billard 1984), were not identified in Japanese eel. The cytoplasmic bridges between dividing

13 Activation of immature testis of Japanese eel by hcg 687 spermatogonia first observed by Fawcett et al (1959) and later by Clerot (1971), Grier (1975) and Billard (1984), were also recognized between spermatogonia late type Β in the present study. The suggestion that these cytoplasmic bridges are responsible for synchronous division and development of spermatogenetic stages during spermatogenesis and help in maintaining a link between growing sister cells seems to be justified in the present study also. The Sertoli cells in the teleost testis perform several functions including support and structural organization of the cysts, lobules and tubules help in the formation of spermatozeugma (in some species) in transfer and eventual conversion of metabolites and hormones towards the germ cells or central cavity phagocytosis of germ cells and in isolation of cyst compartments beyond the spermatocytie stage (Billard 1986). These cells have also been implicated with the steroid production in certain species (Weib 1969; Bara 1969: Van den Hurk et al 1978; Abraham et al 1980). The Sertoli cells remain in very close and direct association with germ cells which they support physically and nurture by modifying the chemical environment (Redding and Patino 1993). The Sertoli cells become active within 12 h of hcg treatment as evidenced by increasing cell and nuclear size, organization of mitochondria and Golgi bodies and distribution of free ribosomes. The peak activity is achieved after 72 h. Normally, the lobular lumen is formed very late during the spermatogenesis in teleosts but in Japanese eel testis under the influence of hcg the Sertoli cells and spermatogonial cysts organized themselves to induce lobular organization and formation of lumen much earlier i.e., within a period of 72 h when the spermatogenesis has progressed only up to late type Β spermatogonia The Sertoli cells in the hcg treated eels had very close association with spermatogonia and not only provide mechanical support but also help in transfering the metabolites and other substances to the latter. The Leydig cells are typically interspersed in the connective tissue surrounding germ cell-sertoli cell unit and their primary function is to produce steroids needed for gametogenesis and expression of secondary sexual characters (Redding and Patino 1993). These are characteristically steroid-producing cells in the eel testis which become elongated alter hcg treatment in eels (Sugimoto and Takahashi 1979; Colombo et al 1987; Miura et al 1991b). The Leydig cells in the present study also become activated within 12 h after hcg treatment and their cellular components like mitochondria. Golgi bodies, endoplasmic reticulum and free ribosomes exhibit features of active secretory cells within 72 h. The lipoluscin bodies as reported by Follinius and Porte (1980) in Lebistes reticulatus Gresik et al (1973) in Oryzias latipes and Colombo and Burighel (1974) in Gobius jozo were not observed in Anguilla japonica On the contrary, the Sertoli cells always possessed lipid globules in them. It is assumed that in teleosts, gonadotropin does not act directly but works in conjunction with the somatic cells to produce steroids mediating induction of spermatogenesis and spermiation. 11- Ketotestosterone has been identified as a major androgen for differentiation and sexual maturation in several teleosts (Idter et al 1961; Billard et al 1982; Fostier et al 1983; Nagahama ; Billard 1986) but in hcg stimulated European eel, the pattern of steroid metabolism indicated greater production of androstenetrione and not 11-ketotestosterone (Eekstein et al 1982). Colombo et al

14 688 D Ν Saksena et al (1987) have shown that plasma testosterone levels were significantly increased after hcg treatment in comparison to untreated male European eels but a decrease in androgen level occurred when the testis had already organized itself into lobules. Miura et al (1991a,b) have reported an increase in the levels of both testosterone and 11-ketotestosterone in response to hcg in plasma and in the culture medium in which testis pieces were incubated. Ishi (1991) also suggested that gonadotropin stimulates testicular interstitial cells in fish to secrete androgens. In the present study, a continuous increase in plasma level of testosterone and 11-ketotestosterone was observed after hcg treatment, confirming the observation of Colombo et al (1987) and Miura et al (1991a). The changes in the plasma level of androgens have been found to be associated with the activation of Sertoli cells and Leydig cells, induction of spermatogonial mitosis and lobular organization in male Japanese eels. In conclusion, hcg first stimulated Leydig cells to produce of androgens; testosterone and mainly 11-ketotestosterone which in turn induce mitotic spermatogonial proliferation and organization of testicular lobules with a wide lumen within a short period of 72 h. The Sertoli cells remain in close contact with the spermatogonia, and maintain supply of metabolites and other substances to the later. They also take part in the lobular organization and formation of its lumen. This further strengthened our earlier contention regarding the stimulatory effect of hcg on the induction of spermatogenesis and sexual maturation in the males of Japanese eel (Miura et al 1991 a,b). It may, however, be added that besides acting through the androgens (mainly testosterone and 11-ketotestosterone), the hcg may also influence spermatogenesis directly. Acknowledgements One of us (DNS) wishes to thank the Indian National Science Academy and the Japan Society for Scientific Promotion for the award of a Visiting Fellowship and to Late Prof. J Bahadur and Prof R Mathur for their keen interest and encouragement. Thanks are also due to Ms S Fukada and Ms Κ Noda for their help during the tenure of this work. References Abraham M, Rahamim E. Tibika H. Golenser Ε and Kieselstein Μ 1980 Blood testes barrier in Aphanius dispar (Teleostei); Cell Tissue Res Bara G 1969 Histochemical demonstration of 3ß-, 3α-, 11ß- and 7ß- hydroxysteroid dehydrogenases in the testes of Fundulus heteroclitus; Gen. Comp. Endocrinol Billard R 1984 Ultrastructural changes in the spermatogonia and spermatocytes of Poecilla reticulate during spermatogenesis; Cell Tissue Res Billard R 1986 Spermatogenesis and spernatology of some teleost fish species; Reprod. Nutr. Dev Billard R, Fostier A. Weil C and Breton Β 1982 Endocrine control of spermatogenesis in teleost fish; Can. Fish ;Aquat. Sci Boetius I and Boetius J 1967 Studies in the European eel. Anguilla anguilla (L): Experimental induction of the male sexual cycle, its relation to temperature and other factors; Medd. Dan. Fisk-Havunders Colombo G. Grandi G and Rossi R 1984 Gonad differentiation and body growth in Anguilla anguilla L; J. Fish Biol Colombo G, Grandi G. Romes A, Giovannini G, Pelizzol D. Catozzi L and Piffanelli A 1987 Testis cytological structure, plasma sex steroids and gonadal cytosol free steroid receptor of heterologous gonadotropin (hcg)-stimulated silver eel, Anguilla anguilla L; Gen. Comp. Endocrinol

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