Histology, Ultrastructure and Gonadal Steroids of Two Male Phenotypes of the Protogynous Fish, Scarus Ferrugineus (Scaridae)
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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 plas- ma steroid levels are clearly detected. Initial-phase (IP) males are small primary males have drab color and large testes with high sperm produc- tion. 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 ultra- structural examination revealed that testes of primary and secondary males are almost identical in the terms of the organization of the sper- matogenic 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 bright- er 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 herm- aphrodite. 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 spermi- ation 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 termi- nal 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 radioimmunoas- say 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