THE JOURNAL OF EXPERIMENTALZOOLOGY 261:194-203 (1992)

Plasticity of Gonadal Development and Protandry in Fishes DOUGLAS Y.SHAPIRO Department of Marine Sciences, University of Puerto R ico, Mayaguez, Puerto Rico 00681 ABSTRACT Sexual differentiation in eutherian mammals follows a simple governing paradigm: development proceeds in a female direction unless a masculinizing mechanism intervenes. Sexual development in fishes is much more plastic than in mammals. It permits the intervention of environmental factors and follows several different types of sequences that produce successive hermaphrodites and alternative pathways for the development of the same final sex. In spite of this plasticity, the primacy of female development is suggested by the initial ovarian phase in the development of gonads of both sexeS in some gonochoristic fishes and by protogynous sex change. One barrier to the application of this principle to fishes generally is the existence of protandric hermaphrodites. Recent evidence suggests a reinterpretation of gonadal differentiation in a protandric anemonefish and a protandric sparid. In both cases, testicular development is both preceded and followed by ovarian development. These patterns are interpreted to mean that female development is primary and that male development is a temporary phase initiated by a masculinizing mechanism and terminated by its cessation.

In mammals, the development of the gonad involves a set of differentiating events early in embryonic life followed by a sequence of developmental steps. The differentiating events occur once and the subsequent developmental steps are regular and relatively invariant (Austin and Edwards, '81; van Tienhoven, '83). In fishes, in contrast, the development of the gonad is much more plastic (Atz, '64; Yamamoto, '69; Reinboth, '83). In this paper I compare the plasticity of gonad development in fishes with the regularity of development in mammals. Several patterns of fish gonadal development, particularly that seen in sexually "undifferentiated species and in protogynous hermaphroditism, suggest that an important paradigm of development in mammals, namely the primacy of female development, may apply t o some fishes. A major conceptual barrier to drawing this parallel is the existence of protandric hermaphroditism. I then present recent evidencethat suggests a new interpretation of protandric hermaphroditism, which removes this barrier.

DEVELOPMENT OF THE MAMMALIAN GONAD AND SEXUAL PHENOTYPE In mammals, the sex of the individual is determined normally by the chromosomal constitution established at the time of fertilization (Jost et al., '73; Bull, '83).XY individuals become male; XX individuals become females. Page et al. ('87) described a gene called ZFY (zinc-finger-Y) located in the 01992 WILEY-LISS, INC.

testis-determining region of the human Y-chromosome. Early findings indicated that ZFY was the mammalian sex-determining gene, but this interpretation has recently been questioned (Palmer et al., '89; Koopman et al., '89).The testis-detennining gene(s) on the Y chromosome directs the production of an unknown factor, possibly H-Y antigen (Wachtel et al., '75; Wachtel, '83; Simpson, '86; Simpson et al., '871, which induces testicular organization in the somatic portion of undifferentiated embryonic gonadal tissue. Ovarian organization is induced in the absence of the testis-determining factor, possibly under the direction of ovary-determining genes (Eicher and Washburn, '86). Embryologically,primordial germ cells originate in the inner cell mass of the blastocyst, outside of the presumptive gonad (Hann, '27; George and Wilson, '88; Byskov and HQyer,'88). Using amoeboid movements, the germ cells migrate through the entoderm of the gut into the mesoderm of the mesentery and finally settle in the coelomic epithelium of the genital ridge. Once the undifferentiated gonad has become a testis or ovary, the subsequent development of the sexual phenotype depends on the presence or absence of gonadal hormones. Female embryos and castrated embryos of both sexes develop female urogenital ducts (Jost, '53; Jost, '61). The fetal testis secretes two hormones, a non-steroid, Mullerianinhibiting factor (Josso et al., '771, which causes

DEVELOPMENTAL PLASTICITY AND PROTANDRY IN FISHES

regression of the Mullerian duct, and androgen, which induces development of the Wolfian duct and the male urogenital system (Josso, 731). Development thus proceeds in a female direction unless testicular secretions redirect it onto a male pathway. Similarly, in the absence of gonadal hormones just before or after birth, the mammalian pituitary of both sexes developsthe capacity for cyclical release of gonadotrophins,the female pattern, and the individual responds to circulating estrogens, as an adult, with female sexual behavior. If androgens are present during the critical perinatal period, the pituitary fails to develop a cyclical capacity and the individual responds to androgens, as an adult, with male sexual behavior (Goldman, '78; Baum, '87). In mammals, then, sexual development is preset to move in the female direction unless virilizing influences intervene. As Jost ('72) put it, " . . . the gonads as well as the body sex would become feminine if not diverted from doing so by a masculinizing mechanism." While this developmental paradigm is not universally accepted in all respects (Lepori, '80, pp. 169-1841, it is widely employed as a fundamental principle of mammalian sexual development (Money and Schwartz, '78; Byskov, '81; Josso, '81; Winter et al., '81; Wartenberg, '83; Baum, '87; Byskov and Hdyer, '88; George and Wilson, '88; Williams-Ashman, '88) and, in a reverse manner, also applies to birds and some amphibians, where the heterogametic sex is female (van Tienhoven, '83, pp. 49-69; Adkins-Regan, '81; Adkins-Regan et al., '82).I will call this paradigm the primacy of female development.

PLASTICITY OF GONADAL DEVELOPMENT IN FISHES Gonadal development in fishes may follow several sequences and is more readily influenced by exogenous events than in mammals. First, the direction of germ cell and gonadal somatic differentiation can be affected by administration of steroid hormones during critical phases of development (Hunter and Donaldson, '83).Exogenous estrogens given to fry in food or placed directly into the water medium cause genetic males to develop ovaries and become functional adult females. For example, when 0.5-5 pg/L estradiol-17P was given to masu salmon at ages 5-22 days after hatching, 100%of individuals developed as females (Nakamura, '81a). Similarly, exogenous androgens given to genetic females direct the developing gonad to become a testis, e.g., in Tilapia mossambica (Nakamura, '81b). Clearly, steroid hormones are capable of redirecting development so that the primordial germ cells

195

themselves differentiate in a direction opposite to that of the genetic sex of the individual (in fishes, unlike mammals, it is the gender of mature germ cells in the gonad rather than the gender of the duct systems that forms the basis for classifying the individual as male or female). This process of steroidal sex reversal (Yamamoto,'69) is commonly employed in aquaculture to produce monosexual populations (Hunter and Donaldson, '83).Although exogenous steroids can influence gonadal sex in marsupials, gonadal differentiation in eutherian mammals is impervious to such influences (van Tienhoven, '83; Chan and 0, '81; Byskov and Hdyer, '88). Temperature can affect gonadal differentiation in some fishes (D'Ancona, '591, as it can in many reptiles (Bull, '831. In the Atlantic silversides, Menidiu menidia, eggs andlarvaereared at 11-19°C produced significantly higher proportionsof females than eggs and larvae reared at warmer temperatures (ConoverandKynard, '81). Temperature and steroid hormones may act on gonadal differentiation in fishes by inducing or inhibiting the production of H-Y antigen early in development, as suggested in chickens and sea turtles (Zaborski, '82; Wellins, '87). In any case, temperature has no such influence on gonadal differentiation in eutherian mammals (van Tienhoven, '83). Substantial numbers of fishes are sequential hermaphrodites (Reinboth, '70; Atz, '64; Chan and 0, '81; Shapiro, '87a). In protogynous species, individuals develop ovaries and reproduce first as females. Later in adult life the ovaries degenerate and are replaced by functional testes, turning the previous female into a reproductive male. In protandric fishes, development follows the opposite course. Individuals reproduce initially as males and later change sex and function as females. Adult sex change involves the degeneration of functional gonadal tissue of the first sex and the growth and maturation of tissue of the opposite sex. It is not known whether most proliferating tissue of the second sex develops from gonial cells that had differentiated at the same time as differentiating gonia of the first sex and subsequently remained dormant within the gonad or whether germinal tissue of the second sex differentiated at the time of sex change from a standing stock of undifferentiated stem cells (Reinboth, '82, '83). In some species, varying numbers of immature germ cells of the second sex are imbedded in the matrix of the gonad of the first sex or cluster in a contiguous part of the gonad (Reinboth, '62;Smith, '65; Sadovy and Shapiro, '871, but in other species no germ cells of the second sex are recognizable in the first-

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functioning gonad (Shapiro,'81).In spite of our ignorance on this point, it is clear that major processes of gonadal differentiation are delayed in sex-changing fishes until adulthood. In effect, the gonad differentiates twice. These fishes contrast starkly with mammals, in which differentiating events occur only once and sequential hermaphroditism is unknown. Other fishes are simultaneous hermaphrodites (Reinboth, '70; Atz, '64). Gonadal tissues of both sexes differentiatetogether, although not necessarily synchronously (Sadovy and Shapiro, ,871, as the individual matures. As an adult the individual contains functional germinal tissue of both genders. In these cases, as in gonochoristic fishes, where individuals remain only one sex throughout life, differentiating events occur only once. Within some protogynous and protandric species, individuals may follow alternative developmental sequences to reach the same final sex. Many wrasses, for example, contain two alternative pathways for becoming males. Primary males differentiate testes asjuveniles and function reproductively as males in adulthood. Secondary males differentiate and function initially as females and later change sex and become males. Consequently, these species are diandric (Reinboth,'70; Warner and Robertson, '78). Some protandric sparids are digynic (Malo-Michele, '77)' with primary females differentiating ovaries that remain functional throughout adult life and secondary females that have changed sex from an initial male phase. The timing of adult sex change, i.e., of the onset of the second phase of gonadal differentiation, and the pursuit of a primary or secondary developmental pathway by an individual in diandric and digynic species are not necessarily genetically predetermined. For example, in many sex-changing species, individuals begin to change sex not because they have reached any particular age, size, or stage of development, but because of the occurrence of particular behavioral or demographic changes within their social system (Shapiro, '84; Chan and 0, '81). Because these social events can occur virtually at any time in the life of the individual, there is great variability in the age and size at which the second phase of gonadal differentiation begins (Shapiro, '87b). In the diandric bluehead wrasse, the proportion of individuals developing according to the primary male, as opposed to the secondary male sequence, varies widely: from virtually none on small patch reefs to as many as 50%of the local population on larger patch reefs (Warner and Hoffman,'80). An important segment of sex allocation theory has been

devoted to predicting the proportion of the population following each of these two developmental sequences (Charnov,'82; Warner and Hoffman, '80; Shapiro, '89). Variability in the proportion of primary males could result from three processes. Genetically programmed primary and secondary male juveniles could assort among ree& of different size at the time they settle onto the reef from the pelagic, larval phase or could suffer differential post-settlement mortality. Alternatively, environmental cues associated with reef or local population size could induce one or the other of these two sequences of gonadal development in juveniles at the time of settlement (Shapiro, '89). Behavioral initiation of sex change and environmental induction of alternative developmental sequences, should it occur, both enhance the degree of plasticity of gonadal development in fishes. How do we make physiological sense of such plasticity within fishes? In mammals, a simple rule governs gonadal ontogeny, namely, the primacy of female development. There is descriptive evidence t o suggest such a scheme in fishes. In some gonochoristic species, e.g., masu salmon (Nakamura, %la,,841, lampreys (Gorbman,'83; Hardisty, '65a,b), carp (Davies and Takashima, 'SO), zebrafish (Takahashi, ,771, and Sumatra barb (Takahashi and Shimizu, ,831,the gonads of both sexes pass through a juvenile ovarian phase prior to differentiating definitively as an immature ovary or testis (Dildine, '36; Bertin, '58).This sequence of gonadal development has been called "undifferentiated (Ymamoto, '69) t o distinguish it from the sequence in other gonochores in which the primitive gonad develops directly either into ovary or testis. The "undifferentiated pattern is seen also in protogynous species, where all individuals (exceptpossibly primary males, which differentiate testes as juveniles) differentiate ovaries prior to differentiating testes, and where ovarian remnants in the testes strikingly resemble histological remnants in juvenile testes which underwent a prior ovarian phase (Takahashi and Takano, '72; Takahashi, '77; Takahashi and Shimizu, '83). The pattern is also seen in cases of pre-maturational sex change (Alekseev, '82, '83; Huang et al., '74). The similarity among these developmental sequences has led t o the suggestion that adult female-to-male sex change is the delayed appearance of the final stage of gonochore sexual development (Bullough,'47; Atz, '64; Shapiro, '87a). Since protogynous sex change is induced by a specific set of external behavioral events in the social

DEVELOPMENTAL PLASTICITY AND PROTANDRY IN FISHES

system (Shapiro, '881, the redifferentiation of ovary into testis can be said to occur in response to a masculinizing mechanism. Thus, the mammalian rule of primacy of female development might find a parallel among secondary males of protogynous fishes and among "undifferentiated" gonochores. One of the main barriers to applying the mammalian scheme to fishes is protandry. If most individuals of these species differentiate functional testes prior to differentiating functional ovaries, the primacy of female development is clearly in doubt. Modern definitions of successive hermaphroditism are based on reproductive function (Policansky, '82; Sadovy and Shapiro, '87; Chan and Yeung, '89). If an individual functions as one sex and then switches and functions as the opposite, the individual is said t o have changed sex. This approach to hermaphroditism focuses attention on gonadal changes during adulthood. Recent evidence, summarized below, from sparids and pomacentrids, families in which protandry is common, suggests that when protandry is viewed from a broader ontogenetic perspective there is reason to believe that female o r ovarian development is the primary developmental scheme. Male development is inserted into a female developmental sequence as a temporary phase, initiated and terminated by a male inductor mechanism. To the extent to which this interpretation is correct, protandry, like protogyny and gonochorism in some fishes, can be subsumed under the mammalian paradigm of the primacy of female development.

A DEVELOPMENTAL VIEW OF PROTANDRY Anemonefishes Amphiprion anemonefisheslive in colonies within clusters of sea anemones (Dunn, '81). Generally, only the largest two individuals in each colony are reproductively active: the largest is a functional female and the second largest is a functional male (Fricke and Fricke, '77; Fricke, '79; Moyer and Bell, '76; Moyer and Nakazono, '78; Ross, '78a,b; Wood, '81, '86), although occasionally more than one breeding pair is active (Moyer,'80). The gonads of all nonbreeding individuals contain both ovarian and testicular tissue, in varying amounts. These small individuals have been classified variously as juvenile, subadult, or inactive males (Moyerand Nakazono, '78; Fricke and Fricke, '77; Fricke, '79; Wood, '86). Ontogeny is generally assumed to involve initial development toward a functional male state. If the functional female disappears from a colony,

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TABLE 1. T h e proportion of the gonad of Amphiprion melanopus devoted to ovarian tissue (% OV), as estimated by the relative diameters ofovarian and testicular tissue in gonadal cross-sections' Size rank 1 2 3 4

5 6 7 8 9 10-23

Colony 1 (N = 23fish) Sex %OV

F F M M J J J J J J

98 100 33 25 93 87 77 70 50

Colony 2

Colony 3

(N = 9fish)

(N = 5 fish) Sex %OV

Sex

%OV

F M J

100 35 50 80 60 60

J J J J J J

F M J J J

100 20 90 58 2

2 2 2

2

'Females (F), males (MI, and juveniles (J)are listed from top to bottom in order of decreasing size within each of three colonies. Data from Shapiro and Fautin (in preparation). 'Individuals with rudimentary gonads only a few cells in thickness. These gonads were too small for a meaningful estimate of % OV.

the functional male changes sex protandrically and becomes a reproducing female. Recently, Shapiro and Fautin (in preparation) examined the gonads of all individual A . melanopus from three colonies containing five, nine, and 23 individuals, respectively. Most gonads contained discrete ovarian and testicular sections that each ran the length of the gonad. Microscopic cross-sections were prepared for anterior, central, and posterior locations of each lobe of the gonad of each colony member. The proportions of each cross-section that were devoted t o ovarian and testicular tissues were estimated by measuring the diameters of both types of tissue with an ocular micrometer. Very small individuals contained tiny, rudimentary gonads several cell diameters in thickness. By the time individuals were 36-48 mm in total length, their gonads were large enough to evaluate and contained 50-60% ovarian tissue. As the size rank of individuals within the colony increased, the proportion of the gonad devoted to ovarian tissue also increased (Table 1).Excluding the gonads of functional males, the ovarian portion rose progressively from approximately 50% to 100% with increasing size rank. The only gonads consistently out of this sequence were those of the active males, usually the second-ranking individual in the colony (colony 1,unusually large with 23 members, had two mature males and two mature females). Shapiro and Fautin (in preparation) interpret these data to mean that rudimentary gonads, containing roughly equal amounts of tissue of both

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D.Y. SHAPIRO

sexes, develop progressively in a female direction as individuals grow and move up the size hierarchy within their colony. When the individual attains the second-ranking position, the gonad is redirected from developing into an ovary to developing as a testis. The initiating event is probably behavioral because gonadal redirection only occurs following the disappearance of the largest member of the colony, an event causing all colony members to move up in the size hierarchy by one position (Fricke and Fricke, '77; Fricke, '79; Moyer and Bell, '76; Moyer and Nakazono, '78; Ross, '78a). The gonad then matures as a testis and the second-ranking individual is a reproductively active male. Male status continues until the female disappears, when the second-ranking individual suddenly becomes the largest colony member. At this point, the gonad reverts t o the female developmental sequence from which it began and matures into a functional ovary. According t o this interpretation, the primary developmental scheme is mainly ovarian. Testicular differentiation predominates only if the individual is diverted from the underlying female sequence by a behaviorally induced virilizing mechanism. When this mechanism ceases to operate, the gonad once more takes up the underlying female sequence and completes its maturation as an ovary. Of course, this interpretation leaves open the questions of how and why both ovarian and testicular tissues appear together, albeit in differing quantities, during gonadal development. If female development is primary in Amphiprion, then the gonads ofjuveniles should be able to develop directly into ovaries without passing through a functional testicular stage. When 40juvenile A. bicinctus were placed together as 20 pairs for up to 5 months, gonads of 17 of the 20 dominant fish contained mainly growing oocytes, while the gonads of the 20 subordinate fish were either indifferent (N = 8 ) or were developing in a male direction (N = 12) (von Brandt, '79). A similar result was also found in A. frenutus (Moyer and Nakazono, '78). When five juvenile A . bicinctus were each held alone, the gonads of three contained mainly growing ovarian tissue. These data suggest that juveniles can develop into females without first developing testes (von Brandt, '79). Long-term observations of another anemonefish species in the field yielded equivalent findings (Wood, '81, '86). The similarity of these results among four species suggest that the primacy of female development may be common to all protandric anemonefishes. The application of this principle to protandric fishes

is strengthened by the pattern of development in the sparid, Spurus uurutu.

Sparids For 45 years, gonadal development in S. uurutu has been used as a simple standard for comparison with more complicated developmentalsequences in other protandric sparids (D'Ancona, '45, '50; Bertin, '58; Atz, '64; Reinboth, '70, Fig. 1;Lissia Frau et al., '76). Juveniles contain a bisexual gonad, with a ventral testicular portion separated from a dorsal ovarian portion by a band of connective tissue. As the individual matures, the testicular portion develops extensively and the fish spawns as a male. Later in life, the testicular portion diminishes in size and finally is reduced to a small, non-functional remnant, while the ovarian portion expands and suborns virtually the entire gonad. The individual functions as a female thereafter. Recently, sexual development has been reexamined in this species in what is probably the most detailed and controlled study yet done on protandric sparids (Zohar et al., '78, '84). Juveniles of age 2-3 months were captured from the eastern Mediterranean basin and maintained under uniform conditions in large outdoor ponds filled with seawater adjacent to the Gulf of Aqaba, Red Sea. Water temperature was maintained close to 21°C. Under these conditions, the fish grew and reproduced annually. Every month for 2 years ten individuals were removed and their gonads examined histologically. Until the age of 4 months, gonads were rudimentary (Fig. 1).From 4 to 8 months, a connective tissue barrier grew between ventral and dorsal parts of the gonad. The dorsal ovarian part developed a central cavity filled with lamellae containing nests of gonial cells. This region progressively outgrew the ventral testicular part until it was approximately 2.5 times the size of the future testis. Germ cells matured in neither part of the gonad. During this period of time, then, the gonad developed primarily and progressively in an ovarian direction (Zohar et al., '78, '84). Between the ages of 8 and 12 months, gonadal development reversed itself (Fig. 1).Gonial cells in the dorsal ovarian section degenerated, the lamellar structure disappeared, and the entire dorsal region shrank in proportion to the progressively growing ventral testicular region, which supported active spermatogenesis (Zohar et al., '84). By the 12th month, the testicular region was 4-5 times the size of the ovarian region and all individuals of this age spawned as males. Immediately aRer spawning,gonadal development

DEVELOPMENTAL PLASTICITY AND PROTANDRY IN FISHES Months of life 1

6

12

18

24

MS

30

36

FS

-

80% I MS

MS 80%

20% I

20 %

-

Rudimentary gonad

Y

.

I

PO"

Ovarian growth predominates

TT

Testicular growth predominates

Fig. 1. Sequences ofgonadal differentiation ofsparus aumtu followed by 80% and 20%of the population (based on Zohar et al., '84). Bar coloration represents the period of rudimentary development (open bar) and periods in which ovarian (OV) and testicular ('IT)tissue growth predominates (black bar and crosshatched bar, respectively). MS = spawning as a male; FS = spawning as a female.

in all individuals returned to its initial, female direction (Fig. 1).The male part of the gonad emptied of spermatogenic cells except for spermatogonia in the testicular lobules. In the female part, oogonia multiplied rapidly and primary oocytes entered a phase of rapid previtellogenic growth. By the age of 16 months, the ovarian part was approximately four times the size of the testicular region in all individuals. For 80% of the population, the gonad continued its development as an ovary and the individual spawned during the 24th month as a female. These individuals remained females thereafter (Fig. 1). For the remaining 20% of individuals, gonadal development was again temporarily diverted from a female into a male pathway. Between the ages of 18 and 24 months, the testicular region became large and filled with active spermatogenic tissue, while the ovarian region degenerated. After these individuals spawned as males, the gonad again developed in the ovarian direction, with 80%of these individuals thereafter functioning as females and 20% repeating the process (Zohar et al., '78, '84). Thus, initially the gonads of all individuals developed primarily in a female direction. In spite of the fact that all individuals spawned as males at the age of 12 months, predominantly ovarian development both preceded and followed a 4-month period

199

of testicular growth. Essentially the same sequence was repeated for that 20% of the population that did not complete a functional male-to-female sex change during the second year.

DISCUSSION Gonadal development in S. auratu paralleled to a striking degree the sequence of development in Amphiprion. In both cases, individuals developed initially in a predominantly ovarian direction and testicular development was preceded and followed by ovarian growth. It is difficult to avoid interpreting these results to indicate primacy of female development in these protandric species. Male development in these representatives of both families occupies discrete time periods that are probably switched on and off by a masculinizing mechanism. At a descriptive level at least, as opposed to a physiological level, where little is known about gonadal differentiation in protandric species (Yeung and Chan, '85, '87a; Reinboth et al., '861, the mammalian paradigm applies. The literature contains hints that other sparids may follow a similar pattern to S. auratu. Ovarian growth exceeded testicular growth during juvenile stages of the protandric yellowfin bream, although the figures suggesting this pattern are not fully consistent (Pollock,'85). After spawning as a male, testicular tissue diminished as ovarian tissue grew so that the ovarian portion of the gonad exceeded the testicular portion in size. Some individuals then completed a male-to-female sex change, while others continued with the ovarian area exceeding the testicular area until just before the breeding season when the testes again grew and the individuals spawned again as males. Unfortunately, the conclusions from this study are not perfectly clear (Pollock, '85). Predominantly ovarian development was said to precede and follow male function in Inegocia m e r dervoorti (Okada, '66) but these conclusions are confused by a subsequent publication (Okada, '68). Juvenile Boops salpa were described as developing ovarian cavities to a high degree within bisexual gonads before individuals differentiated into males, females, or protandric hermaphrodites (MaloMichele, '77). Other protandric sparids may, however, follow sequences of gonadal differentiation that are not consistent with primacy of female development (Yeung and Chan, '87b; Okada, '65a,b; Kinoshita, '39; Reinboth, '62, '70). There are two difficulties with most studies in the literature yielding these counterexamples. Some involve small sample sizes,

200

D.Y. SHAPIRO

e.g., 21 specimens in Sparus longispinis where the verbal description of testicular development preceding ovarian development in the bisexual gonad is unusually clear (Kinoshita, ’36). In others, specimens are collected monthly but include individuals of all ages and sizes so that it is not easy to separate annual patterns within an age or size class from age-related, developmental patterns (Lissia Frau et al., ’76; Malo-Michele, ’77). Patterns may also be confused by collections relying on commercial catches that sample multiple sites. The captivity study of S.auratu, which suggested primacy of female development (Zohar et al., ’78, ’84), while susceptible to the criticism that its study animals may not typify natural populations, has the virtue of following a fixed sample of individuals of the same age through 2 years of growth and development under uniform environmental conditions. One feels confident that the resulting sequences of gonadal changes are truly attributable to developmental processes and not to other confounding factors. If primacy of female development correctly describes the physiology of gonadal differentiation in these fishes, then we might expect similar processes to influence development in fishes and mammals. Recent studies of the endocrinology of sex differentiation in fishes suggest that, in spite of the ease with which exogenous steroid hormones influence the direction of differentiation (Yamamoto, ,691, there is no consistent evidence that androgens and estrogens play an initiating role in natural processes of differentiation (Nakamura and Nagahama, ’85, ’89; Reinboth, ’87). Fishes may be similar to mammals in this regard. We also might expect the second phase of sex differentiation in successive hermaphrodites to involve similar physiological processes to initial differentiation both in gonochoristic fishes and in mammals. The recent implication of H-Y antigen in natural sex reversal is consistent with this expectation (Duchac and Buhler, ’83; Pechan et al., ’86; Reinboth et al., ’87). In conclusion, the plasticity of gonadal differentiation in fishes may obscure the operation of a simple developmental paradigm, at least for many species, that is similar to the central paradigm governing sex differentiation in mammals. If the paradigm holds true at the level of physiology, as well as at the descriptive level discussed here, then what will remain to be explained in fishes is the relative ease with which differentiating processes can be influenced by environmental factors and the timing with which those factors intervene to produce the several types of successive hermaphrodites. Even

if the paradigm applies to many species of fish, it remains to be seen if and how it will apply to primary males in diandric fishes, to simultaneous hermaphrodites, and to “differentiated” species of gonochores in which gonads develop directly from the undifferentiated state into testes or ovaries.

ACKNOWLEDGMENTS Work on this paper was supported by NIH grant S06RR08103 and NSF grant RII-8606320. I thank S. Wachtel, D. Fautin, R. Cochran, M. Shpigel, A. Marconato, and Y. Zohar for reviewing the manuscript and Y. Zohar for helpful discussions on the development of gonads in sparids. LITERATURE CITED Adkins-Regan, E. (1981)Hormone specificity, androgen metabolism, and social behavior. Am. Zool., 21:257-271. Adkins-Regan, E., P. Pickett, and D. Koutnik (1982) Sexual differentiation in quail: conversion of androgen to estrogen mediates testosterone-induced demasculinization of copulation but not other male characteristics. Horm. Behav., 16:259-278. Alekseev, F.E. (1982) Hermaphroditism in sparid fishes (Perciformes, Sparidae) I. Protogyny in porgies, Pagrus pagrus, P. ehrenbergi, and P. auriga, from West Africa. J. Ichthyol., 22:85-94. Alekseev,F.E. (1983)Hermaphroditism in porgies (Perciformes, Sparidae). 11.Sexual structure of the populations, mechanism of its formation and evolution in scups, Pagrus pagrus, P. orphus, P. ehrenbergi, and P. auriga. J. Ichthyol., 23:61-73. Atz, J.W. (1964) Intersexuality in fishes. In: Intersexuality in Vertebrates, Including Man. C.N. Armstrong and A.J. Marshall, eds. Academic Press, London, pp. 142-232. Austin, C.R., and R.G. Edwards (1981) Mechanisms of Sex Differentiation in Animals and Man. Academic Press, London, 603 pp. Baum, M.J. (1987) Hormonal control of sex differences in the brain and behavior of mammals. In: Psychobiology of Reproductive Behavior, An Evolutionary Perspective. D. Crews, ed. Prentice-Hall, Inc., Englewood Cliffs, NJ, pp. 231-257. Bertin, L. (1958) Sexualite et fecondation. Traite Zool., 13(A): 1584-1652. Bull, J.J. (1983) Evolution of Sex Determining Mechanisms. The BenjaminiCummings Publishing Company,Menlo Park, CA, 316 pp. Bullough, W.S. (1947)Hermaphroditism in the lower vertebrates. Nature, I60:9-11. Byskov, A.G. (1981) Gonadal sex and germ cell differentiation. In: Mechanisms of Sex Differentiation in Animals and Man. C.R. Austin and R.G. Edwards, eds. Academic Press, London, pp. 145-164. Byskov, A.G., and P.E. HQyer (1988) Embryology of mammalian gonads and ducts. In: The Physiology of Reproduction. E. Knobil and J. Neill, eds. Raven Press, Ltd., New York, pp. 265-302. Chan, S.T.H., and W.S. 0 (1981) Environmental and nongenetic mechanisms in sex determination. In: Mechanisms of Sex Differentiation in Animals and Man. C.R. Austin and R.G. Edwards, eds. Academic Press, London, pp. 55-112. Chan, S.T.H., and W.S.B. Yeung (1989) Sex steroids in intersexual fishes. Fish Physiol. Biochem., 7:229-235.

DEVELOPMENTAL PLASTICITY AND PROTANDRY IN FISHES Charnov, E.L. (1982) The Theory of Sex Allocation. Princeton University Press, Princeton, 355 pp. Conover, D.O., and B.E. Kynard (1981)Environmental sex determination: interaction of temperature and genotype in fish. Science, 213:577-579. D'Ancona, U. (1945) Sexual differentiation of the gonad and the sexualization of the germ cells in teleosts. Nature, 156:603-605. D'Ancona, U. (1950) Determination et differenciation du sexe chez les poissons. Arch. Anat. Microsc. Morphol. Exp., 39:274-294. D'Ancona, U. (1959) Distribution of the sexes and environmental influence in the European eel. Arch. Anat. Microsc. Morphol. Exp., 48bis:61-70. Davies, P.R., and F. Takashima (1980) Sex differentiation in commoncarp,Cyprinuscalpio. J.TbkyoUniv.Fish., 66:191-199. Dildine, G.C. (1936) Studies in Teleostean reproduction. I. Embryonic hermaphroditism in Lebistes reticulatus. J. Morphol., 60:261-277. Duchac, B.J., and E.M. Biihler (1983) Expression of H-Y antigen in the sex-changefish Corkjulis.Experientia, 39:767-769. Dunn, D.F. (1981)The clownfish sea anemones: Stichodactylidae (Coelenterata: Actiniaria) and other sea anemones symbiotic with pomacentrid fishes. Trans. Am. Phil. SOC.,71:l-115. Eicher, E.M., and L.L. Washburn (1986) Genetic control of primary sex determination in mice. Annu. Rev. Genet., 20:327-360. Fricke, H.W. (1979) Mating system, resource defence and sex change in the anemonefish Amphiprion akallopisos. Z. Tierpsychol., 50: 313-326. Fricke, H., and S. Fricke (1977) Monogamy and sex change by aggressive dominance in coral reeffish. Nature, 266:830-832. George, F.W., and J.D. Wilson (1988) Sex determination and differentiation. In: The Physiology of Reproduction. E. Knobil and J. Neill, eds. Raven Press, Ltd, New York, pp. 3-26. Goldman, B.D. (1978) Developmental influences of hormones on neuroendocrine mechanisms of sexual behaviour: comparisons with other sexually dimorphic behaviours. In: Biological Determinants of Sexual Behaviour. J.B. Hutchison, ed. John Wiley & Sons, Chichester, pp. 128-152. Gorbman, A. (1983) Reproduction in cyclostome fishes and its regulation. In: Fish Physiology. W.S. Hoar, D.J. Randall, and E.M. Donaldson, eds. Academic Press, New York, pp. 1-29. Hann, H.W. (1927)The history of the germ cells of Cottus bairdzz Girard. J . Morphol. Physiol., 43:427-497. Hardisty, M.W. (1965a)Sexual di&rentiation and gonadogenesis in lampreys. Part I: the ammocoete gonads of the brood lamprey, Lampetraplaneri. J . Zool., 146:305-345. Hardisty, M.W. (1965b)Sexual differentiation and gonadogenesis in lampreys. Part 11: the ammocoete gonads of the landlocked sea lamprey, Petromyzon marinus. J. Zool., 146:346-387. Huang, C.C., C.F. Lo, and K.H. Chang (1974) Sex reversal in one sparid fish, Chrysophrys major (Perciformes, Sparidae). Bull. Inst. Zool. Acad. Sin. (Tapei), 1355-60. Hunter, G.A., and E.M. Donaldson (1983) Hormonal sex control and its application to fish culture. In: Fish Physiology, Vol. IX, Reproduction, Part B, Behavior and Fertility Control. W.S. Hoar, D.J. Randall, and E.M. Donaldson, eds. Academic Press, New York, pp. 223-303. Josso, N. (1981) Differentiation of the genital tract stimulators and inhibitors. In: Mechanisms of Sex Differentiation in Animals and Man. C.R. Austin and R.G. Edwards, eds. Academic Press, London, pp. 165-204. Josso, N., J.-Y. Picard, and D. Tran (1977) The antimullerian hormone. Recent Prog. Horm. Res.,33:117-167.

201

Jost, A. (1953) Problems of fetal endocrinology: the gonadal and hypophyseal hormones. Recent Prog. Horm. Res., 8:379-418. Jost, A. (1961) The role of fetal hormones in prenatal development. Harvey Lect., 55:201-226. Jost, A. (1972) A new look at the mechanisms controlling sex differentiation in mammals. Hopkins Med. J., 130:38-53. Jost, A., B. Vigier, J. Prepin, and J.P. Perchellet (1973) Studies on sex differentiation in mammals. Recent Prog. Horm. Res., 29:l-41. Kinoshita, Y. (1936) On the conversion of sex in Sparus longispinis (Temminck and Schlegel) (Teleostei).J. Sci. Hiroshima Univ., Ser. B. Div. 1(4):69-79. Kinoshita, Y. (1939) Studies on the sexuality of genus Sparus (Teleostei).J. Sci. Hiroshima Univ., Ser. B., Div. 1(7):25-37. Koopman, K., J. Gubbay, J. Collignon, and R. Lovell-Badge (1989) Zfy gene expression patterns are not compatible with a primary role in mouse sex determination. Nature, 342: 940-942. Lepori, N.G. (1980) Sex Differentiation, Hermaphroditism and Intersexuality in Vertebrates Including Man. Piccin Medical Books, Padua, 345 pp. Lissia Frau, A.M., M. Pala, and S. Casu (1976) Observations and considerations on protandrous hermaphroditism in some species of sparid fishes (Teleostei,Percifonnes). Studi Sassar., 54:147-167. Malo-Michele, M. (1977) Contribution a l'etude histologique de la gonade, en particulier de l'ovaire, chez la Saupe, Boops salpa (L.)(Teleosteen, Sparidae). Donnees nouvelles sur son type d'hermaphrodisme. Invest. Pesq.,41 :165-183. Money, J., and M. Schwartz (1978) Biosocial determinants of gender identity, differentiation and development. In: Biological Determinants of Sexual Behaviour. J.B. Hutchison, ed. John Wiley & Sons, Chichester, pp. 766-784. Moyer, J.T. (1980) Influence of temperate waters on the behavior of the tropical anemonefish Amphiprion clurkii at Miyakejima, Japan. Bull. Mar. Sci., 30:261-272. Moyer, J.T., and L.J. Bell (1976) Reproductive behavior of the anemonefish Amphiprion clarkii at Miyake-jima, Japan. Jpn. J . Ichthyol., 23:23-32. Moyer, J.T., and A. Nakazono (1978) Protandrous hermaphroditism in six species of the anemonefish genus Amphiprion in Japan. Jpn. J . Ichthyol., 25:lOl-106. Nakamura, M. (1981a) Feminization of masu salmon Oncorhyncus masou by administration of estradiol-17p. Bull. Jpn. SOC.Sci. Fish., 47:1529. Nakamura, M. (1981b)Effects of 11-ketotestosterone on gonadal sex differentiation in Tilapia mossambica. Bull. Jpn. SOC.Sci. Fish., 47:1323-1327. Nakamura, M. (1984) Effects of estradiol-17P on gonadal sex differentiation in two species of salmonids, the masu salmon, Oncorhyncus masou, and the chum salmon, 0. keta. Aquaculture, 43:83-90. Nakamura, M., and Y. Nagahama (1985)Steroid producing cells during ovarian differentiation of the tilapia, Sarotherodon niloticus. Dev. Growth Differ., 27:701-708. Nakamura, M., and Y. Nagahama (1989) Differentiation and development of Leydig cells, and changes of testosterone levels during testicular differentiation in tilapia Oreochromis niloticus. Fish Physiol. Biochem., 7:211-219. Okada, Y.K. (1965a) Bisexuality in sparid fishes. I. Origin of bisexual gonads in Mylio macrocephalus. Proc. Jpn. Acad., 41:294-299. Okada, Y.K. (1965b) Bisexuality in sparid fishes. 11. Sex segregation in Mylio mucrocephnlus. Proc. Jpn. Acad.,41:300-304.

202

D.Y. SHAPIRO

Okada, Y.K. (1966)Sex reversal inlnegocia m.eerderuoortiwith special reference to repetition of hermaphroditic state. Proc. Jpn. Acad., 42:497-502. Okada, Y.K. (1968)A further note on the sex reversal inznegock meerderuoorti. Proc. Jpn. Acad., 44:374-378. Page, D.C., R. Mosher, E.M. Simpson, E.M.C. Fisher, G. Mardon, J. Pollack, B. McGillivray, A. de la Chapelle, and L.G. Brown (1987) The sex-determining region of the human Y chromosome encodes a finger protein. Cell, 51:1091-1104. Palmer, M.S., A.H. Sinclair, F? Berta, N.A. Ellis, P.N. Goodfellow, N.E. Abbas, and M. Fellous (1989) Genetic evidence that ZFY is not the testis-determining factor. Nature, 342:937-939. Pechan, P., D.Y. Shapiro, and M. Tracey (1986) Increased H-Y antigen levels associated with behaviorally-induced, femaleto-male sex reversal in a coral reef fish. Differentiation, 31:106-110. Policansky, D. (1982) Sex change in plants and animals. Annu. Rev. Ecol. Syst., 13:471-495. Pollock, B.R. (1985) The reproductive cycle of yellowfin bream, Acanthopagrus australis (Gunther),with particular reference to protandrous sex inversion. J. Fish Biol., 26:301-311. Reinboth, R. (1962) Morphologische und funktionelle Zweigeschlechtigkeit bei marinen Teleostieren (Serranidae,Sparidae, Centracanthidae, Labridae). Zool. Jahrb. Abt. Allg. Zool. Physiol. Tiere, 69:405-620. Reinboth, R. (1970) Intersexuality in fishes. Mem. SOC.Endocrinol., 18:5 15-542. Reinboth, R. (1982) The problem of sexual bipotentiality as exemplified by teleosts. Reprod. Nutr. Dev., 22:397-403. Reinboth, R. (1983) The peculiarities of gonad transformation in teleosts. Differentiation, 23(SuppZ.):S82-S86. Reinboth, R. (1987) Natural sex inversion. In: Proc. 3rd Int. Symp. Reprod. Physiol. Fish. D.R. Idler, L.W. Crim, and J.M. Walsh, eds. Marine Sciences Research Laboratory, Memorial University, St. John’s, Newfoundland, pp. 124-127. Reinboth, R., B. Becker, and M. Latz (1986) Zn uitro studies on steroid metabolism by gonadal tissues from ambisexual teleosts. 11. Conversion of 14C androstenedione by the heterologous gonadal tissues of the protandric sea bream Pagellus acarne (Risso).Gen. Comp. Endocrinol., 62:335-340. Reinboth, R., A. Mayerova, C. Ebensperger, and U. Wolf (1987) The occurrence of serological H-Y antigen (Sxs antigen) in the diandric protogynous wrasse, Coris julis (L.)(Labridae, Teleostei). Differentiation, 34: 13- 17. Ross, R.M. (1978a) Reproductive behavior of the anemonefish Amphiprion melanopus on Guam. Copeia, 1978:103-107. Ross, R.M. (1978b) Territorial behavior and ecology of the anemonefishAmphiprion melumpus on Guam. Z. Tierpsychol., 46:7 1-83. Sadovy, Y., and D.Y. Shapiro (1987) Criteria for the diagnosis of hermaphroditism in fishes. Copeia, 1987:136-156. Shapiro, D.Y. (1981) Size, maturation, and the social control of sex reversal in the coral reef fish Anthias squamipinnis (Peters).J . Zool., 193:105-128. Shapiro, D.Y. (1984) Sex reversal and sociodemographic processes in coral reef fishes. In: Fish Reproduction: Strategies and Tactics. G.W. Potts and R.J. Wooton, eds. Academic Press, London, pp. 103-118. Shapiro, D.Y. (1987a) Differentiation and evolution of sex change in fishes. BioScience, 37.490-497. Shapiro, D.Y. (198713) Reproduction in groupers. In: Tropical Snappers and Groupers: Biology and Fisheries Management. J.J. Polovina and S.Ralston, eds. Westview Press, Boulder, Colorado, pp. 295-327. Shapiro, D.Y. (1988) Behavioural influences on gene structure

and other new ideas concerning sex change in fishes. Environ. Biol. Fish., 23:283-297. Shapiro, D.Y. (1989) Sex change as an alternative life-history style. In: Alternative Life-History Styles of Animals. M.N. Bruton, ed. Kluwer Academic Publishers, Dordrecht, pp. 177-195. Simpson, E. (1986) The H-Y antigen and sex reversal. Cell, 44:813-814. Simpson, E., P. Chandler, E. Goulmy, C.M. Disteche, M.A. Ferguson-Smith, and D.C. Page (1987) Separation of the genetic loci for the H-Y antigen and for testis determination on human Y chromosomes. Nature, 326:876-878. Smith, C.L. (1965) The patterns of sexuality and the classification of serranid fishes. Am. Mus. Novit., 2207:l-19. Takahashi, H. (1977) Juvenile hermaphroditism in the zebrafish, Brachydanio rerio. Bull. Fac. Fish. Hokkaido Univ., 28:57-65. Takahashi, H., and M. Shimizu (1983) Juvenile intersexuality in a cyprinid fish, the Sumatra barb, Barbus tetruzona tetrazona. Bull. Fac. Fish. Hokkaido Univ., 34:69-78. Takahashi, H.,and K. Takano (1972) Morphogenesis of accessory reproductive organs in male goldfish, Camssius auratus. Bull. Fac. Fish. Hokkaido Univ., 23.53-64. van Tienhoven, A. (1983)Reproductive Physiologyof Vertebrates. Cornell University Press, Ithaca, 491 pp. von Brandt, I. (1979) Soziale Kontrolle des Wachstums und Geschlechtsdifferenzierung bei Jungen Anemonenfischen (Amphiprion bicinctus). University of Munich, 81 pp. Wachtel, S.S. (1983) H-Y Antigen and the Biology ofSex Determination. Grune and Stratton, New York, 302 pp. Wachtel, S.S., S. Ohno, G.C. Koo, and E.A. Boyse (1975) Possible role for H-Y antigen in the primary determination of sex. Nature, 257:235-236. Warner, R.R., and S.G. Hoffman (1980) Local population size as a determinant of mating system and sexual composition in two tropical marine fishes (Thalussoma spp.). Evolution, 34:508-518. Warner, R.R., and D.R. Robertson (1978) Sexual patterns in the labroid fishes of the western Caribbean: 1. The wrasses (Labridae).Smithson. Contrib. Zool., 254:l-27. Wartenberg, H. (1983) Structure aspects of gonadal differentiation in mammals and birds. Differentiation, 23(Suppl.): S64-S71. Wellins, D.J. (1987) Use of an H-Y antigen assay for sex determination in sea turtles. Copeia, 1987:46-52. Williams-Ashman, H.G. (1988) Perspectives in the male sexual physiology of eutherian mammals. In: The Physiology of Reproduction. E. Knobil and J. Neill, eds. Raven Press, Ltd., New York, pp. 727-751. Winter, J.S.D., C. Faiman, and F. Reyes (1981) Sexual endocrinology of fetal and perinatal life. In: Mechanisms of Sex Differentiation in Animals and Man. C.R. Austin and R.G. Edwards, eds. Academic Press, London, pp. 603. Wood, E.M. (1981) Sex change and other social strategies in anemonefish. Prog. Underwater Sci., 6:61-64. Wood, E.M. (1986) Behaviour and social organization in anemonefish. Prog. Underwater Sci., 11:53-60. Yamamoto T. (1969) Sex differentiation. In: Fish Physiology. W.S. Hoar and D.J. Randall, eds. Academic Press, New York, pp. 117-175. Yeung, W.S.B., and S.T.H. Chan (1985) The in uitro metabolism of radioactive progesterone and testosterone by the gonads of the protandrous Rhabdosargus sarba at various sexual phases. Gen. Comp. Endocrinol., 59:171-183.

DEVELOPMENTAL PLASTICITY AND PROTANDRY IN FISHES Yeung, W.S.B., and S.T.H. Chan (1987a) A radioimmunoassay study of the plasma levels of sex steroids in the protandrous, sex-reversingfish Rhabdosargus sarba (Sparidae). Gen. Comp. Endocrinol., 66:353-363. Yeung, W.S.B., and S.T.H. Chan (198713) The gonadal anatomy and sexual pattern of the protandrous sex-reversing fish, Rhabdosargus sarba (Teleostei: Sparidae). J. Zool., 212: 521-532. Zaborski, P. (1982) Expression of H-Y antigen in non-mammalian vertebrates and its relation to sex differentiation. In: Reproductive Physiology of Fish. C.J.J. Richter and H.J.T.

203

Goos, eds. Centre for Agricultural Publishing and Documentation (Pudoc), Wageningen, pp. 64-68. Zohar, Y., M. Abraham, and H. Gordin (1978)The gonadal cycle of the captivity-reared hermaphroditic teleost Sparus aurata (L.)during the first two years of life. Ann. Biol. Anim. Biochim. Biophys., 18:877-882. Zohar, Y., R. Billard, and C. Weil(1984) La reproduction de la daurade (Sparus aurata) et du bar (Dicentrarchus tabrax): connaissance du cycle sexuel et contr6le de la gametogenese et de la ponte. In: “L’Aquaculture du Bar et des Sparid&. G. BarnabeandR. Billard, eds. INRA Publications,Paris, pp. 3-24.