Histology, Ultrastructure and Gonadal Steroids of Two Male Phenotypes of the Protogynous Fish, Scarus Ferrugineus (Scaridae)

Histology,JKAU: Mar. Ultrastructure Sci., Vol. 17, and pp: Gonadal33-58 (2006 Steroids A.D. of / 1427Two Male...A.H.) 33

Histology, Ultrastructure and Gonadal Steroids of Two Male Phenotypes of the Protogynous Fish, Scarus ferrugineus (Scaridae)

*Al-Saydah H. Abdel-Aziz, Fayzah A. Bawazeer and Mashael A. Otaibi *Oceanography Department, Faculty of Science, Alexandria University, Egypt, and Zoology Department, Girls College, Jeddah, Saudi Arabia

Abstract. Two male phenotypes of the protogynous scarid, Scarus ferrugineus differ in external appearance, gonad morphology and plasma steroid levels are clearly detected. Initial-phase (IP) males are small primary males have drab color and large testes with high sperm production. Terminal-phase (TP) males are larger primary and secondary (derived from females by sex reversal) males have bright and gaudly color and much smaller testes than do IP males. Histological and ultrastructural examination revealed that testes of primary and secondary males are almost identical in the terms of the organization of the spermatogenic cysts, association of sertoli cells and developing germ cells but differ in clustering and development of Leydig cells. Differences in plasma steroid levels between the two male phenotypes are measured by radioimmunoassay. Results indicate a relationship of Leydig cell development and high levels of plasma 11-KT in the terminal phase males, to be perhaps, due to secondary sexual characters such as brighter coloration and reproductive behaviour rather than to male gameto- genesis. While testosterone is considered an important androgen responsible for spermatogenesis for the two male phenotypes. Keywords: Scaridae, diandric, protogynous, sex-reversal, primary males, secondary males, sex steroids.

Introduction In many animal populations there exist two or more types of males differing in reproductive behaviour. Among fishes, such "alternative male reproductive

33 34 Al-Saydah H. Abdel-Aziz et al. behaviours'' are often associated with different gonadal and external body morphology. This phenomenon occurs especially among the sex changing coral reef wrasses (Family Labridae: Reinboth, 1973; Robertson and Warner, 1978; Warner, 1984 and Hourigan et al., 1991) and the related parrotfishes (Family Scaridae: Randall and Randall, 1963; Robertson and Warner, 1978; Warner, 1984; Streelman et al., 2002, and Abdel-Aziz et al., 2006). Despite the wide- spread occurrence of different male phenotypes in at least 13 families of teleosts (Hourigan et al., 1991), most studies have been limited to behavioural and histological observations. Gonadal steroids have important functions in the reproduction and behavior of vertebrates. These functions are often deduced from the temporal correlation of steroidogenesis with reproductive events. Complications arise when several events, such as reproductive behavior and germ cell maturation, are closely associated in time. Studies of differences in steroidogenesis between male phenotypes may help distinguish the production of steroids required for general processes of male reproduction from that specifically related to alternative male behaviours. The rusty parrotfish, Scarus ferrugineus (Scaridae) is a protogynous hermaphrodite. As in other scarids the pattern of sexual ontogeny in this species is complicated by the existence of two types of males: following sex change most males undergo a dramatic color change, displaying distinctly colorful bright "terminal phase" (TP) coloration. However, a small portion of males retain the female-like initial phase (IP) coloration after sex change (Cardwell and Liley, 1991). The present study, aimed to investigate the histology and ultrastructure of the testis of primary and secondary males of S. ferrugineus, with special emphasis on steroid-producing Leydig cells. The results are then compared with plasma steroid levels of the two male phenotypes. The androgen measured, testosterone (T) and 11-ketotestosterone have been implicated in spermatogenesis or spermiation in several fishes. Estradial-17 β (E2) is the major naturally occurring estrogen in many fishes (Fosteir et al., 1983, Hourigan et al., 1991 ; Kawaga et al., 1981 and Nakamura et al., 1989).

Materials and Methods Scarus ferrigineus fish were collected monthly from the eastern coast of the Red Sea, Saudi Arabia. The standard length (SL), total body weight and body color were recorded. About 344 fishes were examined. They are characterized by two sharply discontinous color patterns. 244 fishes were in initial phase (IP) Histology, Ultrastructure and Gonadal Steroids of Two Male... 35 which has reddish brown to brown body color shading to yellow on caudal peduncle and caudal fin (Fig. 1a). 130 ones were in terminal phase (TP) whose color was bluish green with bluish caudal fin (Fig. 1b).

( a )

( b )

Fig. 1. Rusty parrotfish, Scarus ferrugineus: (a) Initial phase. (b) Terminal phase. 36 Al-Saydah H. Abdel-Aziz et al.

The gonads of all fish were removed and weighed and the gonadosomatic index (GSI) was calculated (GSI = Gonad weight / Body weight × 100). The ovaries of all females were discarded after they had been sexed macroscopical- ly. The testes of all males, and the gonads of fishes whose sex could not be determined by naked eye, were fixed in 10% neutral buffered formaline for a minimum of 24 h, before progressive dehydration in ethanol, clearing in xylene and embedding in paraffin wax. Transverse sections (TS) were cut at 3 µm from the mid region of the gonad and stained using haematoxylin and eosin (H & E) and periodic acid Schiff's (PAS). Then males were classified according to the pattern of coloration observed at time of sampling into IP males, or TP males and according to the histological character of their gonads to primary or secondary males, reversed from females based on Reinboth criteria (1973). All testes were examined for the presence of oocytes, atretic follicles and a central cavity in conjunction with an alternative path for the transport of sperm (suggesting that the central cavity was non-functional and hence an ovarian remnant). Any of these findings would be considered evidence that testis had developed from a preceding ovarian stage (Hourigan et al., 1991). For electron microrscope observations, a subsample from each gonad was immersed in 3.7% glutaraldehyde buffered in pH 7.3 with 0.13 M cacodylate buffer for 3 hr at –4ºC. Samples were postfixed in 1% osmium tetroxide buffered with 0.1 M cacodylate buffer for 2 hr at room temperature. After dehydration in agraded aceton series, the subsamples were embedded in epoxy resin and sectioned. One micrometer sections were stained with 1% toluidene blue in 0.1 M phosphate buffer. Ultrathin sections were stained with uranyl acetate solution and lead citrate (Reynolds, 1963) and viewed under the trans- mission electron microscopy (TEM. 100 × ).

Determination of Steroid Levels Blood samples were collected from the caudal artery of alive fish. Plasma was separated by centrifugation, frozen at –80ºC and stored until use at –20ºC. Plasma samples were collected for initial phase (IP) primary males and terminal phase (TP) primary and secondary males.

Assays Estradiol-17B (E2), testosterone (T) and 11-ketotestosterone (11-KT) levels in plasma were measured following diethyl ether extraction by radioimmunoassay as described by Chang et al. (1995). Mean extraction efficiencies for E2, T and 11-KT were 88.1%, 93.61, and 92.5%, respectively. Histology, Ultrastructure and Gonadal Steroids of Two Male... 37

Antigen used were [2,4,6,7-3H] – estradiol – 17 β (99 Ci/mmol), [1,2,6,7-3H] – testosterone (89.1 Ci/mmol), and [1,2,-3H] – 11-Keto tertosterone (57.4 Ci/ mmol) were purchased from NEN Research Broducts (Boston, MA). Cross-reactions of the antisera against E2, T and 11-KT were evaluated by Kagawa et al. (1981), Kime and Manning (1982), and Fostier et al. (1983), respectively. All samples were measured in duplicates. The sensitivities of the assay for E2, T and 11-KT were 10, 12.5 and 12.5 pg per assay, respectively. Mean plasma steroid concentrations for the two male phenotypes were compared using student's t-test at p < 0.05 and p < 0.01 significant level. One way analysis of variance (ANOVA) was used to compare between plasma steroid concentration in the IP and TP primary males and TP secondary males according to statistical analysis system (SPSS +) with an IBM computer.

Results From Table 1, it is clear that initial phase (IP) fish comprise primary males (6.07%), females (84.58%) and intersex (9.35%) and terminal phase (TP) fish include primary males (11.54%), secondary males (85.38%) and intersex(3.08%).

Table 1. Distribution of the color phases in relation to sex and size of rusty parrotfish "Scar- us ferrugineus".

Color phases Initial phase(IP) Terminal phase (TP) N = 214 % = 62.21 N = 130 % = 37.79 Primary Primary Secondary Females Intersex Intersex Parameters males males males Number (N) 13 181 20 15 4 111 Percentage (%) 6.07 % 84.58 % 9.35 % 11.54 % 3.08 % 85.38 %

Standard length 10 - 20 16 - 32 19 - 24 21 - 31 23 - 26 20 - 47 range (cm)

Mean length & standard 15.62 ± 2.40 21.90 ± 2.90 21.30 ± 1.27 26.20 ± 2.71 24.50 ± 1.12 34.19 ± 3.80 deviation (cm)

Initial Phase Males (IP Males) IP males were generally smaller than TP males (mean SL = 15.62 ± 2.40 cm) but with much larger gonads. Mean GSI (1.759) of IP males were significantly higher (P < 0.01) than from that of TP males throughout the year (Table 2). 38 Al-Saydah H. Abdel-Aziz et al.

Table 2. Mean monthly variations in the gonado-somatic index (GSI) of the two male phenotypes of rusty parrotfish "Scarus ferrugineus". Color phases & sex Initial phase (IP) Terminal phase (TP)

Months Primary males Primary males Secondary males September 0.550 ± 0.000** 0.190 ± 0.110 0.130 ± 0.080 October 2.010 ± 0.550** 0.630 ± 0.090 0.100 ± 0.040 November 2.630 ± 0.300** 0.290 ± 0.210 December 0.650 ± 0.270** 0.270 ± 0.000 0.081 ± 0.040 January 1.480 ± 0.000** 0.640 ± 0.160 0.138 ± 0.070 February 1.940 ± 0.000** 0.660 ± 0.360 0.130 ± 0.070 March 2.990 ± 0.690** 0.896 ± 0.460 0.265 ± 0.260 April 0.990 ± 0.011 0.231 ± 0.330 May 2.595 ± 1.050** 0.128 ± 0.040 June 0.980 ± 0.000** 0.054 ± 0.020 Mean ± SD 1.758 ± 0.846** 0.611 ± 0.273 0.155 ± 0.076

P < 0.01**: highly significant

Histological examination of the gonads of IP males revealed that all individu- als were primary males, i.e. fishes that had initially differentiated as males, the gonads had a single centrally located tubular vas deferens draining each of the two lobes of the large testis and no remnant of ovarian tissue or of an ovarian lumen was observed (Fig. 3). The testis of IP primary male is surrounded by tunica albuginea of connective tissue that emits septa to the interior of the organ, which delimit seminiferous lobules (Fig. 2). The germ cells within the seminiferous lobules (Sl) are arranged in cysts or clusters enclosed by thin interdigitating processes of a few Sertoli's cells. All of the germ cells within each cyst appeared to be at the same stage of development. Germ cells within neighbouring cysts, however, were at different stages of development from early spermatocytes to late spermatids (Fig. 2 and 3). The seminiferous lobules of ripe testis are destended with spermatozoa (Fig. 4) whereas appear nearly empty in spent testis (Fig. 5). The interstitial tissue between the seminiferous lobules contains blood vessels, fibroblasts and few singly scattered interstitial or Leydig cells (Fig. 3). The ultrastructure of the spermatogenic cells of IP males are shown in Fig. (6-12). Histology, Ultrastructure and Gonadal Steroids of Two Male... 39

Fig. 2. T.S of testis of initial phase (IP) primary male showing spermatogenesis (stage III). sd, central sperm duct; tunica albugina (blue arrow); s, sperm cluster [H & E; bar = 200 µm].

Fig. 3. T.S of IP primary male testis showing spermatogenesis ongoing (stage III). Note, seminiferous lobules with spermatocytes (sc) and spermatids (st) clusters and sperms (s) being present in lumen of seminiferous lobules. Note also, scarcity of Leydig cells (L) in interstitial tissue [H & E; bar = 40 µm]. 40 Al-Saydah H. Abdel-Aziz et al.

Fig. 4. T.S of ripe testis (stage IV) of IP primary male showing active spermatogenesis. Expanded seminiferous lobules distended with spermatozoa (s) and reduction in marginal cysts of spermatogenic cells [H & E; bar = 40 µm].

Fig. 5. T.S of spent testis (stage V) of IP primary male. Note, empty lobule (EL); thickned interstitial tissue (It) and may be vacuolated Leydig cells (L) [H & E; bar = 40 µm]. Histology, Ultrastructure and Gonadal Steroids of Two Male... 41

Fig. 6. Electron micrograph of T.S in testis of IP primary male showing spermatogonium with large nucleus with prominent nucleolus (Nu); and cytoplasm with electron dense masses free (nauge) (arrow) or attached with mitochondrial groups (M); endoplasmic reticulum (ER) and free ribosomes.

Fig. 7. Electron micrograph of T.S in testis of IP primary male showing cyst of primary spermatocytes (Ps) surrounded by Sertoli's cells (Sc). 42 Al-Saydah H. Abdel-Aziz et al.

Fig. 8. High magnification from Fig. 7 showing Pachytene stage of primary spermatocyte (Ps) with large nucleus, moderately compacted chromatin and characteristic synaptonemal complex (red arrow). Note, SER; m, mitochondria and free ribosomes in primary spermatocyte.

Fig. 9. Electron micrograph of T.S in testis of IP primary male showing secondary spermatocytes (SS) with compact mottled nuclei and scarce cytoplasm and late spermatids (LS) have eccentric nuclei with very dense chromatin and cytoplasmic oraganelles (mitochondria (M) and centeriole (C))at one pole. Histology, Ultrastructure and Gonadal Steroids of Two Male... 43

Fig. 10. Electron micrograph of T.S in testis of IP primary male showing spermatids with compact electron dense nucleus; marginal hyper trophoidmitochondria (M) with lucent matrix and scarce tubular cristae; ribosomes; v, vesicles and glycogen particles (g) in cytoplasm. Note also, cross sections of sperm flagellum (blue arrow).

Fig. 11. Electron micrograph of T.S in testis of IP primary male showing sperm with round compact head, short mid piece with mitochondrial (M) ring and flagellum (arrow). 44 Al-Saydah H. Abdel-Aziz et al.

Fig. 12. Electron micrograph of T.S in testis of IP primary male showing part of Leydig cell have large nucleus contain chromatin blocks (arrow). Note, pleomorphic mitochondria (M) with tubulovesicular cristae; vesicle of SER (red arrow); lysosomes (1y); and lipid droplets (1).

1 – Spermatogonia They are found at the periphery of the seminiferous lobules and characterized by their low electron density (Fig. 6). Spermatogonium has a central rounded nucleus with loose chromatin and distinct one or two nucleoli with fibrilar and granular chromatin. The cytoplasm contains granular materials that are either free (nauge) or associated with mitochondria (cement). Mitochondria are unevenly distributed throughout the cytoplasm while endoplasmic reticulum is concentrically organized around the nucleus.

2 – Spermatocytes Primary spermatocytes are the largest germ cells which has a dense cytoplasm, a nucleus with slightly clumped mottled nuclear chromatin and characteristic synaptonemal complexes (Fig. 7 and 8). The latter consist each of two parallel electron-dense strips as lateral arms and a medial electron lucent strip (Fig. 8). Secondary spermatocyte (Fig. 9) has a scant cytoplasm, a central nucleus with chromatin forming electron dense masses that may be scattered or clus- tered at one pole. The nucleolus seems to fade away and is never seen again in the subsequent stages. Histology, Ultrastructure and Gonadal Steroids of Two Male... 45

3 – Spermatids Round spermatid (initial stage) has a scant cytoplasm, with electron-lucent vesicles, few hypertrophoid mitochondria and a dense round nucleus. In inter- mediate spermatid stage, the compacted electron dense nucleus moves to an eccentric position while the cell organelles (mitochondria, centeriols and Golgi vesicles) migrate and concentrate at the opposite pole of the cell (Fig. 9 and 10). In advanced stage the nucleus is very dense and the flagellum is in process of differentiation.

4 – Spermatozoa The sperm is appeared consisting of 3 regions, a round head, a short mid piece and a flagellum (Fig. 11). The head has no acrosome and consists mostly of a round nucleus, that has very dense chromatim material, and the nucleur envelope and the plasma membrane are applied tightly to the interior part of the nucleus. The mid piece is short and contains a mitochondrial ring of 2 to 4 unequal-sized spherical mitochondria. The flagellum is surrounded by flagellar plasma membrane and has the classic nine double outer and two single central microtubular construction (Fig. 10 and 11). Sertoli's cells (SC) are distributed on the inner wall of the lobules emits cytoplasmic processes that delimit the cysts and are connected by desmosomes (Fig. 7). Each SC contained a large oval nucleus that appeared finely granulated with few scattered heterochromatin blocks. The cytoplasm of these cells was greatly reduced, and contained a few mitochondria, cisternae of endoplasmic reticulum and free ribosomes. Leydig cells of initial-phase primary males were few in number, and were distributed singly in the interstitial regions between the lobules. They exhibit a vesiculoues nucleus and a cytoplasm with moderate vesicles of a smooth endoplasmic reticulum (SER) and mitochondria with tubuluovesicular cristae. Few scattered oil droplets, free ribosomes and lysosomes can also be detected (Fig. 12).

Terminal-Phase Males (TP Males) TP males were divided into two classes on the basis of gonadal histology, i.e., primary TP males and secondary TP males. Primary TP males ranged in size from 21 to 31 cm SL with a mean length of 26.20 ± 2.71 cm (Table 1), with small testis (mean GSI = 0.611 ± 0.273) but otherwise histologically similar to those of primary IP males having a single centrally located vas deferens and a central testicular sperm duct and no ovarian lumen remnant (Fig. 13-15). 46 Al-Saydah H. Abdel-Aziz et al.

Fig. 13. T.S of testis of terminal phase (TP) primary male showing spacious central sperm duct (sd) supported by thick connective tissue septae (green arrows). SL, seminiferous lobules, [H & E; bar = 200 µm].

Fig. 14. T.S in the testis of terminal phase (TP) primary male (stage III) showing active spermatogenesis. Note, Leydig cell groups (green arrow) in the interstitial tissue between the seminiferous lobules; primary spermatocytes (Ps); spermatozoa (s) and spermatids (st) [H & E; bar = 40 µm]. Histology, Ultrastructure and Gonadal Steroids of Two Male... 47

Fig. 15. Magnification of Fig. 14 showing Leydig cell (L) group with distinct nucleus and granular eosinophilic cytoplasm in the interstitial tissue between seminiferous lobules. PS, primary spermatocytes st, spermatids.

Fig. 16. Electron micrograph of T.S in testis in TP primary male showing Part of Leydig cell. Note mitochondria (M) with vesiculotubular cristae, abundant vesiculated SER (green arrow); free ribosomes (R) and Golgi apparatus (G). N, nucleus with dilated nuclear membrane (red arrow). 48 Al-Saydah H. Abdel-Aziz et al.

Secondary males (sex changed) ranged in size from 20 to 47 cm SL (mean SL = 34.19 ± 3.80 cm) and had the color of TP fish. The gonad morphology of these secondary TP males differed greatly from that of primary males. Their was very small or minute (Mean GSI = 0.155 ± 0.076) (Table 2) and lacked a central vas deferens. The testis of secondary male has a lamellar structure and efferent sperm ducts are situated longitudinally along the testicular wall and are filled with large numbers of mature sperms (Fig. 17 and 19). A remnant of the membrane bound cavity or lumen is present; however, there is no evidence that sperms were released into this cavity. There appeared to be little differences in the organization of lobules with spermatogenic cysts surrounded by Sertoli's cells and ultrastructure of the spermatogenic cells among primary IP males (Fig. 2-5 & 6-11) and TP primary (Fig. 13-15) and secondary males (Fig. 17-20 & 21-23), despite the de novo occurrence of testicular tissue in the latter. Testes from all TP males, regardless of the month of collection showed active spermatogenesis and spermiation (Fig. 13-15 & 17-20).

Fig. 17. T.S in the testis of TP secondary testis (stage III). Note, marginal sperm ducts (sd) with spermatozoa; remnants of membrane bound ovarian cavity (OC) and active spermatogenesis in male lamellae (ML) [H & E; bar = 200 µm].

Leydig cells showed the greatest differences between IP and TP males. Leydig cells in both primary (Fig 14 and 15) and secondary (Fig. 18 and 20) TP males occurred in clusters distributed in the interstitial region among lobules. They appeared to be more numerous, larger, and contained more cytoplasm Histology, Ultrastructure and Gonadal Steroids of Two Male... 49

Fig. 18. Magnified from Fig. 17 showing Leydig cell groups (green arrows) characterized by PAS positive cytoplasm and large nuclei with prominent nucleoli. Ps, primary spermatocytes; st, spermatids [PAS; bar = 40 µm].

Fig. 19. T.S of ripe testis of TP secondary male. Note, spaceous marginal sperm duct (sd) full of spermatozoa and active spermatogenesis in male lamellae. Oc, ovarian cavity; [H & E; bar = 200 µm]. 50 Al-Saydah H. Abdel-Aziz et al.

Fig. 20. Magnified from Fig. 19. Note, numerous seminiferous lobules, full of spermatozoa (s), other lobules contain spermatocytes; spaceous interstitial tissue contain inactive Leydig cells (green arrows) [H & E; bar = 40 µm].

Fig. 21. Electron micrograph of T.S in testis of TP secondary male showing spermatogonium. Note, nucleus(N), dense electron masses free and attached with mitochondrial (M) group in cytoplasm. Histology, Ultrastructure and Gonadal Steroids of Two Male... 51

Fig. 22. Electron micrograph of T.S in testis of TP secondary male showing spermatocyte containing large nucleus with chromatin blocks; surrounded by Sertoli's cell with long nucleus and scant cytoplasm with vesiculated ER (blue arrow) and free ribosomes, secondary spermatocytes (ss).

Fig. 23. Electron micrograph of T.S in testis of TP secondary male showing sperm with spherical electron dense head (H), and a short mid piece (mp) with mitochondtial ring (M) and cytoplasmic canal (arrow) and flagellum with axoneme (ax) [bar = 0.5 µm]. 52 Al-Saydah H. Abdel-Aziz et al. than those in the gonads of IP males (Fig. 3). Unlike IP males (Fig. 12) the Leydig cells of TP males (Fig. 16 & 24) contained more mitochondria with vesiculotubular cristae; extensive smooth endoplasmic reticulum (SER) distributed throughout the cytoplasm, a character of active steroidogenesis. Also secondary TP male Leydig cells had extensive vesiculated SER, numerous mitochondria with tubuluvesicular cristae and numerous glycogen particles than a terminal phase primary males (compare Fig. 16 & 24).

Fig. 24. Electron micrograph of T.S in testis of TP secondary male showing part of Leydig cell with prominent hypertrophoid SER vesicles (V); numerous glycogen particles (g) and large mitochondria (M) with tubulovesicular cristae. N; nucleus [bar = 1 µm]. Plasma Steroids Level Plasma levels of the steroids (estradiol-17β, testosterone and 11-ketotestosterone) of two male phenotypes of S. ferrugineus are shown in Table 3 and Fig. (25). It is obvious that, mean plasma levels of 11-KT were significantly higher (p < 0.01) in TP males (both primary and secondary) than in IP primary males concomitant with better development of steroid producing Leydig cells. Also plasma 11-KT levels were the highest (2.868 ± 0.624 ng/ml) in TP secondary males (reversed from females). On the other hand, IP primary males and TP males (both primary and secondary) had similar moderate levels of testosterone (Table 2). 11-KT and T have nearly the same value in the IP primary males (0.946 ± 0.041 ng/ml & 0.842 ± 0.152 ng/ml respectively. Estradiol-17 β were relatively low in the two male phenotypes. Histology, Ultrastructure and Gonadal Steroids of Two Male... 53

Table 3. Mean steroid concentrations, in the plasma (ng / ml plasma) of the two male phenotypes of rusty parrotfish "Scarus ferrugineus" . Sex steroids Estradiol – 17 b 11-Ketotestosterone Testosterone Sex E2 11-Kt T Initial phase (IP) Primary males 0.290 ± 0.148 0.946 ± 0.041 0.842 ± 0.052 Terminal phase (TP) Secondary males 0.128 ± 0.084 2.868 ± 0.624 0.673 ± 0.184 Primary males 0.184 ± 0.168 1.940 ± 0.124 0.702 ± 0.164

Discussion Recent studies revealed that the rusty parrotfish Scarus ferrugineus is a diandric protogynous hermaphrodite. They comprise primary males (develop testis as juveniles and remain males throughout life) and secondary male develop ovaries as juveniles and later in adult life the functional ovary redifferentiates into a functional testis (Streelman et al., 2002; and Abdel-Aziz et al., 2006). In the present study the morphological examination of S. ferrugineus revealed that its dichromatic as most scared fish (Reinboth, 1962; Cardwell and Liley, 1991; Streelman et al., 2002; and Abdel-Aziz et al., 2006), since the largest adult male (TP) male have bright and goodly color differ from drap color (IP) of the smaller adult which usually males and females. Primary and secondary testes of S. ferrugineus male can be distinguished histologically and were similar to those described for diandric protogynous wrasses (Reinboth, 1962; Cardwell and Liley, 1991; Streelman et al., 2002; and Abdel-Aziz et al., 2006) and scarids (Ross, 1984; Sadovy and Shapiro, 1987; Abdel-Aziz and Ramadan, 1988; and Hourigan et al., 1991). The present study showed that the testes of primary and secondary males were almost identical, in terms of the organization of spermatogenic cysts, association of Sertoli's cells and developing germ cells but differ in clustering and development of Leydig cells. This indicates that the testicular organization of Sertoli's cells and spermatogenic cysts in the testes of the two male phenotypes do not depend on initial gonad development, but may result instead from cell- cell interaction and holocrine secretions within the gonad (Hourigan et al., 1991). The ultrastructure of spermatogenesis and spermatozoon of S. ferrugineus don't show any peculiarities and are generally similar to those described in other teleosts (Brusle, 1981; Billard, 1983; Lopes et al., 2004; and Shahin, 2006). 54 Al-Saydah H. Abdel-Aziz et al.

Also the Leydig cells in the testes of the two male phenotypes were similar to those observed in other fishes (Nagahama, 1983; Nakamura et al., 1989; and Hourigan et al., 1991) and were the major cell type with characteristics of steroid synthesis. These characteristics include numerous large mitochondria with tubulovesicular cristae and abundant smooth endoplasmic reticulum (Christensen and Gillim, 1969). Differences between IP males and TP males of S. ferrugineus in the present study were most distinct in terms of these cells. Both primary and secondary TP males had clusters of Leydig cells, each cell containing more mitochondria and well developed SER than the solitary dispersed IP Leydig cells, suggesting a better developed steroidogenic capacity in TP males. Also, in terminal phase male, Leydig cells of secondary testes were larger and contain extensive vesiculated (SER) and larger mitochondria than those of primary testes. On the other hand, Sertoli's cells had very little cytoplasm, containing a few mitochandria and vesicles of smooth endoplasmic reticulum. Thus, Sertoli's cells may be involved in limited steroidogenesis. They did not appear to differ among the two male phenotypes. As mentioned in the protogynous wrasses, Thalassoma dupperey (Nakamura et al ., 1989; and Hourigan et al., 1991). Studies of steroidogensis in marine hermaphroditic fishes have been limited to some protogynous labrids (e.g. Idler et al., 1976; Nakamura et al., 1989; and Hourigan, et al., 199), scarids (Liley et al., 1987 and Cardwell and Liley, 1991) and protandrous sparids (Idler et al., 1976 and Yeung and Chan, 1987). Idler et al. (1976) suggested that the functional androgens of gonochoristic and hermaphroditic fishes may be 11-KT and 11 β, OH- testosterone, respectively. Hourigan et al., (1991) mentioned that different male phenotypes of the protogynous Thalassoma duperrey differ greatly in testicular androgen synthesis and in plasma steroid levels and that they may be related to reproductive roles rather than to sex change. In the present study, IP males and TP primary and secondary males of S. ferrugineus exhibited distinct differences in gonad size, similar to those recorded for diandric wrasses (Warner and Robertson, 1978; Abdel-Aziz and Rama- dan, 1988; Hourigan et al., 1991; and Nakamura et al., 1989). Unexpectedly how-ever, smaller gonads of TP males contained a higher concentration and better development of steroid producing Leydig cells than did the larger testes of IP males which was associated with high plasma levels of 11-KT in TP males and especially in secondary males. 11-KT has been implicated in spermatogenesis in numerous gonochoristic fishes (Fostier et al., 1983; Liley and Stacey, 1983; and Sakai et al., 1989). However, if high levels of production are related primarily to spermatogenesis, then 11-KT levels would be expected to be highest in IP males whose large gonads produced copious amounts of sperm. Histology, Ultrastructure and Gonadal Steroids of Two Male... 55

Instead, plasma levels of 11-KT in IP of S. ferrugineus males were very low compared to those of TP primary and secondary males. This suggests that high levels of 11-KT in the plasma of TP males may be independent of spermatogenesis and related instead to some other function specific to terminal phase males. Generally, in the diandric protogynous fish, IP primary males spawn in groups and TP primary and secondary males spawn singly with females, (Warner and Robertson, 1978 and Warner, 1984). Also, Warner and Robertson (1978) mentioned that under condition of high-sperm competition, individual IP males with greater sperm production relative to TP males in a spawning group should experience greater reproductive success. On the other hand, TP males have bright color and much small gonads, and generally spawn singly with a female, and sperm competition among males is probably low but courtship behavior occur (Hourigan et al., 1991 and Andrew et al., 1996). Thus, secondary sexual characteristic of TP males, such as brighter coloration and spawning behavior may be a consequence of high levels of this androgen. It is important to mention that, the near absence of large primary IP males and small primary TP males indicate that primary TP males developed from IP males through change of coloration as proposed by Ross (1984) and Hourigan et al., (1991); in Thalassoma duperrey and Abdel-Aziz and Ramadan, (1988) in Coris julis. On the other hand, IP primary males and TP males (both primary and secondary) had nearly similar moderate levels of testosterone. Indicating that testosterone may be considered an important androgen (i.e. for spermatogenesis ) in the two males.

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