The novel mutant scl of the medaka fish, Oryzias latipes, shows no secondary sex characters

A new mutant that has neither male nor female secondary sex characters was found in the medaka, Oryzias latipes. Both XX and XY mature mutants had gonads with many spermatozoa, but spawning did not occur when the mutants were paired with normal males or normal females. F1 progeny were successfully obtained by artificial insemination using unfertilized eggs from wild-type females and spermatozoa of the XY mutant. The mutant phenotype did not occur in the F1 progeny from this cross. Incrossing among the F1 progeny produced 17 mutant offspring out of 68 progeny (25%), demonstrating that the mutant phenotype is caused by a single recessive mutation. This mutant was named scl (sex character-less). Because papillary processes, a male secondary sex character, were induced in the XY mutants by androgen administration, it seems that the androgen receptor is functioning normally. We found a loss-of-function type mutation in the P450c17 gene of the mutant; this gene encodes a steroidogenic enzyme required for the production of estrogen and androgen. The scl phenotype was completely linked to the mutant genotype of P450c17, strongly suggesting that mutation at the P450c17 locus is responsible for the scl mutant phenotype.     

Social control of sex change in the Red Sea razorfish Xyrichtys pentadactylus (Teleostei,Labridae)

Synopsis The Red Sea razorfish, Xyrichtys pentadactylus, a territorial haremic labrid with dominance hierarchies within the harems. Theory predicts that primary males (fish developing initially as males) should be rare or nonexistent in haremic territorial species because the larger secondary males (males which have undergone sex and/or color change) limit access to females. Histological examination of gonads of 95 specimens showed that all males are derived from females by sex change (i.e. they are secondary males). During five months of field studies 100% of more than 200 observed matings were pair spawnings — the usual mating practice for monandric (having one type of male) species. Sex change in females was induced by male removal in nature. Isolation of four groups of females in aquaria showed that the largest female in the social group changes sex in the absence of a male, demonstrating that sex change is socially-controlled in this species.     

H-Y Antigen Expression in Temperature Sex-Reversed Turtles (Emys orbicularis)

H-Y antigen has been used as a marker for the heterogametic sex and is assumed to be an organizing factor for the heterogametic gonad. In the turtle Emys orbicularis , H-Y antigen is restricted to the female cells, indicating a female heterogamety (ZZ/ZW) sex-determining mechanism. Moreover, the sexual differentiation of the gonads is temperature sensitive, and complete sex reversal can be obtained at will. In this framework the relationships between H-Y antigen, temperature, and gonadal phenotype were studied. Mouse H-Y antiserum was absorbed with blood and gonadal cells of control wild male and female adults, and with blood and gonadal cells from three lots of young turtles from eggs incubated at 25–26°C (100% phenotypic males), at 30–30.5°C (100% phenotypic females), or at 28.5–29°C (majority of females with some males and intersexes). The residual activity of H-Y antiserum was then estimated using an immunobacterial rosette technique. In adults, both blood cells and gonadal cells were typed as H-Y negative in males and as H-Y positive in females. In each of the three lots of young, blood cells were H-Y negative in some individuals and H-Y positive in others. The proposed interpretation is that the H-Y negative individuals were genotypic males (ZZ) and the H-Y positive were genotypic females (ZW). The gonads of these animals were then pooled in different sets according to their sexual phenotype and to the presumed genotypic sex (i.e., blood H-Y phenotype). Testicular cells were typed as H-Y negative in genotypic males as well as in the presumed sex-reversed genotypic females; likewise, ovarian cells were typed as H-Y positive in genotypic females as well as in the presumed sex-reversed genotypic males. These results provide additional evidence that H-Y antigen expression is closely associated with ovarian structure in vertebrates displaying a ZZ/ZW sex-determining mechanism.     

Differential Responses of Brain,Gonad and Muscle Steroid Levels to Changes in Social Status and Sex in a Sequential and Bidirectional Hermaphroditic Fish

Sex steroids can both modulate and be modulated by behavior, and their actions are mediated by complex interactions among multiple hormone sources and targets. While gonadal steroids delivered via circulation can affect behavior, changes in local brain steroid synthesis also can modulate behavior. The relative steroid load across different tissues and the association of these levels with rates of behavior have not been well studied. The bluebanded goby (Lythrypnus dalli) is a sex changing fish in which social status determines sexual phenotype. We examined changes in steroid levels in brain, gonad and body muscle at either 24 hours or 6 days after social induction of protogynous sex change, and from individuals in stable social groups not undergoing sex change. For each tissue, we measured levels of estradiol (E₂), testosterone (T) and 11-ketotestosterone (KT). Females had more T than males in the gonads, and more E₂ in all tissues but there was no sex difference in KT. For both sexes, E₂ was higher in the gonad than in other tissues while androgens were higher in the brain. During sex change, brain T levels dropped while brain KT increased, and brain E₂ levels did not change. We found a positive relationship between androgens and aggression in the most dominant females but only when the male was removed from the social group. The results demonstrate that steroid levels are responsive to changes in the social environment, and that their concentrations vary in different tissues. Also, we suggest that rapid changes in brain androgen levels might be important in inducing behavioral and/or morphological changes associated with protogynous sex change.     

Temperature-dependent gonadal differentiation in the turtle Emys orbicularis: concordance between sexual phenotype and serological H-Y antigen expression at threshold temperature

As in many other turtles, the sexual differentiation of gonads in embryos of Emys orbicularis is temperature-sensitive, 100% phenotypic males being obtained below 27.5 degrees C and 100% phenotypic females above 29.5 degrees C. The expression of the serologically defined H-Y (SD-H-Y) antigen at both low and high temperatures has been shown to be different in gonads and in blood : in gonads, it is closely associated with ovarian structure, whereas in blood it is independent of sexual phenotype and appears to indicate sexual genotype. Both sexes differentiate at 28.5 degrees C, suggesting that at this intermediate (threshold) temperature, sexual differentiation of gonads conforms with sexual genotype. To test this hypothesis, the expression of SD-H-Y antigen has been carried out in blood cells of Emys individuals raised from eggs incubated at the threshold temperature (28.5 degrees C). All phenotypic males typed SD-H-Y negative, whereas most phenotypic females typed SD-H-Y positive. From this concordance between sexual phenotype of gonads and SD-H-Y phenotype of blood, we postulate that a ZZ male/ZW female mechanism of genotypic sex determination is revealed at the threshold temperature for gonad differentiation in Emys.     

Increased H-Y antigen levels associated with behaviorally induced, female-to-male sex reversal in a coral-reef fish

It has been proposed that H-Y antigen is the synthetic product of sex-determining genes, and that H-Y antigen controls ontogenetic differentiation of the heterogametic sex throughout vertebrates. The coral-reef fish Anthias squamipinnis is a protogynous hermaphrodite in which all individuals mature initially as females. Males result when adult females change sex as a consequence of alterations in behavioral interactions within social groups. Three assay methods were used to measure H-Y antigen levels in the spleens, gonads, and epidermal tissue of 16 adult females and in 16 males that had been induced to change sex from a prior female phase by the removal of a pre-existing male from each of 16 social groups. In 15 male-female pairs, the H-Y antigen levels were higher in male than in female spleen, gonad, and epidermis tissues. The precise temporal relationship between the onset of sex change and the increase in the H-Y antigen level was not examined. If, as we strongly suspect, the temporal relationship proves to be close, the inference will be that the behavioral cues inducing sex change also influence the synthetic activity of genes controlling H-Y antigen production.     

Binding studies of H-Y antigen in rat tissues

Summary The binding capacity for H-Y antigen was studied in various rat tissues of both sexes. In nongonadal tissues (liver, kidney, brain, epidermis) binding could not be demonstrated. In contrast, the gonads are able to bind exogenously supplied H-Y antigen. In the ovary, the binding capacity remains unchanged in newborn and adult animals, while in the testis, this capacity decreases with age. A receptor like that of a proteohormone is assumed to exist in the gonads but not in other tissues. In nongonadal tissues, H-Y antigen apparently is present only if the cell itself synthesizes the antigen. The H-Y antigen receptor of the gonads is not sex-specific. Thus, the primary sex differentiation depends on whether H-Y antigen is synthesized in the organism.     

A study on the 3β- and 17β-hydroxysteroid dehydrogenase activities in the gonads of Monopterus albus (Pisces: Teleostei) at various sexual phases during natural sex reversal

Activities of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) in Monopterus gonads were studied at different sexual phases during natural sex reversal. Before sexual transformation, positive reactions for 3β-HSD in the follicular epithelium were found in the granulosa cells of some large, maturing follicles in some females during the breeding season. Weak reaction for this enzyme was also detected in some scattered interstitial cells found occasionally in some ovaries. At the intersexual and the male phases, intense 3β-HSD activities were demonstrated exclusively in the interstitial Leydig cells. No 17β-HSD activities were observable in the gonads at any stage of development. The reaction intensity of 3β-HSD in the interstitial cells exhibited a marked increase during the process of sex change from female to the intersexual and the male phases and there is a definite correlation with the density and nuclear size of these cells. It is concluded that in Monopterus , the granulosa cells in the ovary and the interstitial cells of the intersexual and male gonads are the major sites for the biosynthesis of oestrogens and androgens, respectively, and that the intensive development of interstitial tissue with increasing steroidogenic enzyme activities at the intersexual and male phases was directly related to the increase in androgen production in vitro reported previously. The occasional presence of some 3β-HSD positive interstitial cells in the ovary suggests that interstitial cell development might precede testicular lobule formation during natural sex reversal.     

Induction of female-to-male sex change in the honeycomb grouper (Epinephelus merra) by 11-ketotestosterone treatments

The honeycomb grouper, Epinephelus merra, is a protogynous hermaphrodite fish. Sex steroid hormones play key roles in sex change of this species. A significant drop in endogenous estradiol-17beta (E2) levels alone triggers female-to-male sex change, and the subsequent elevation of 11-ketotestosterone (11KT) levels correlates with the progression of spermatogenesis. To elucidate the role of an androgen in sex change, we attempted to induce female-to-male sex change by exogenous 11KT treatments. The 75-day 11KT treatment caused 100% masculinization of pre-spawning females. Ovaries of the control (vehicle-treated) fish had oocytes at various stages of oogenesis, while the gonads of the 11KT-treated fish had transformed into testes; these contained spermatogenic germ cells at various stages, including an accumulation of spermatozoa in the sperm duct. In the sex-changed fish, plasma levels of E2 were significantly low, while both testosterone (T) and 11KT were significantly increased. Our results suggest that 11KT plays an important role in sex change in the honeycomb grouper. Whether the mechanism of 11KT-induced female-to-male sex change acts through direct stimulation of spermatogenesis in the ovary or via the inhibition of estrogen synthesis remains to be clarified.     

A preliminary investigation of sex change inPseudotropheus lombardoi (Pisces: Cichlidae)

Synopsis Peters (1975) suggested the possibility of adult sex change in certain cichlids of Lake Malawi. When adultPseudotropheus lombardoi in male coloration were found mouthbrooding eggs under natural conditions in Lake Malawi, one of the possible explanations for this female-type behaviour was that sex change had occurred, but with the retention of male coloration. Behavioural investigations based on current models of social systems in sex-changing species were conducted in an attempt to substantiate this hypothesis. These observations were supplemented by an histological examination of the gonads of individuals of both sexes. A pronounced advantage in the mating success of dominant males over non-dominant males was noted. Similarly, large females had a greater reproductive success than smaller females. Thus, the possibility that sex change occurred inP. lombardoi following the size advantage model (Warner 1975) was investigated. However, histological studies did not provide conclusive evidence of sex change; only undeveloped oocytes were found in the testes of all males examined. It is postulated that gonads of maleP. lombardoi pass through an intersexual juvenile period. Later, testicular elements dominate within a gonad still containing immature oocyte tissues. It is further suggested that femaleP. lombardoi are dimorphic, some having male coloration and others having female coloration.     

From sneaker to parental male: change of reproductive traits in the black goby, Gobius niger (Teleostei, Gobiidae)

This study focuses on the consequences of the switch of tactic from parasitic to parental male in the black goby, Gobius niger (Teleostei: Gobiidae), a species showing two alternative male mating tactics. Older and larger males defend nests, court, and perform parental care on eggs, while younger and smaller ones behave as parasites, sneaking into nests while spawning occurs. Males adopting different tactics are known to present differences in primary and secondary sex traits. The social context of sneaker males was manipulated to induce a tactic switch. Sneakers were kept under two different experimental treatments with or without a female, and under exclusion of male-male competition. Males changed tactics, courting females, spawning, and performing parental care. All males showed substantial changes in primary sexual traits, such as a reduction in gonadal development and an increase in the investment in accessory structures. The experimental groups differed in the functionality of gonads and accessory organs and in the development of the secondary sex traits. These results demonstrate that the moment of switching is not genetically fixed in the black goby. Sneaker males are able to quickly reallocate energy in primary and secondary sex traits, in accordance with the adopted tactic. Several aspects of this flexible reproductive pattern resemble the socially controlled sex change found in sequential hermaphrodites.     

Testis-determining H-Y antigen in XO males of the mole-vole (Ellobius lutescens)

The XO sex chromosome constitution has been found in both sexes of the mole-vole (Ellobius lutescens) belonging to the rodent family Microtinae. This enigmatic species has apparently been enduring a 50% zygotic lethality. The current serological study revealed the presence in XO males and the absence from XO females of H-Y (histocompatibility Y) antigen. In all the mammalian species studied thus far, the expression of H-Y antigen strictly coincided with the presence of testicular tissue and not necessarily with the presence of the Y chromosome. The testis-organizing function of the H-Y gene appears to have been confirmed.In the mole-vole, X linkage of the testis-organizing H-Y gene is favored over its autosomal inheritance. Only X linkage of the H-Y gene creates a compelling evolutionary need to change the female sex chromosome constitution from XX to XO, and to abandon the dosage compensation by an X inactivation mechanism, so that the nonproductive X^H-YX zygote can be eliminated as an embryonic lethal. With regard to the electrophoretic mobilities of three X-linked marker enzymes, however, a genetic difference between the male-specific X^H-Y and the female-specific X was not detected. This might reflect a relatively recent speciation.     

Sexual dimorphism of gonadotropin-releasing hormone type-III (GnRH3) neurons and hormonal sex reversal of male reproductive behavior in Mozambique tilapia

In tilapia, hormone treatment during the period of sexual differentiation can alter the phenotype of the gonads, indicating that endocrine factors can cause gonadal sex reversal. However, the endocrine mechanism underlying sex reversal of reproductive behaviors remains unsolved. In the present study, we detected sexual dimorphism of gonadotropin-releasing hormone type III (GnRH3) neurons in Mozambique tilapia Oreochromis mossambicus. Our immunohistochemical observations showed sex differences in the number of GnRH3 immunoreactive neurons in mature tilapia; males had a greater number of GnRH3 neurons in the terminal ganglion than females. Treatment with androgen (11-ketotestosterone (11-KT) or methyltestosterone), but not that with 17β-estradiol, increased the number of GnRH3 neurons in females to a level similar to that in males. Furthermore, male-specific nest-building behavior was induced in 70% of females treated with 11-KT within two weeks after the onset of the treatment. These results indicate androgen-dependent regulation of GnRH3 neurons and nest-building behavior, suggesting that GnRH3 is importantly involved in sex reversal of male-specific reproductive behavior.     

Gonadal differentiation and hormonal sex reversal in Arctic charr (Salvelinus alpinus)

The purpose of this study was to develop a practical protocol for the production of female populations of Arctic charr (Salvelinus alpinus). Achieving this required knowledge of the timing of gonadal differentiation. Undifferentiated gonads were observed microscopically to be present by 194 degrees C-days post-hatch and definitive germ cells by 346 degrees C-days post-hatch, where " degrees C-days" denote acquired thermal units calculated as the product of temperature and days. Some of the gonads had developed a lumen by 510 degrees C-days post-hatch, and by 681 degrees C-days post-hatch anatomical divergence into two types of gonads was clear. Two protocols (immersion and feeding) were tested for hormonal sex reversal of genotypic females using the synthetic androgen 17alpha-methyldihydrotestosterone (MDHT). Six-hour MDHT immersions (0.5, 1, 3, 5 and 10 mg/L) were carried out weekly from hatch to first feeding (140 degrees C-days post-hatch), whereas daily feeding treatments (0.5 mg/kg) went from 140 to 600 degrees C-days post-hatch. The sex ratios of all immersion experimental groups were significantly different from the control, with the proportion of presumptive males increasing as MDHT concentration increased. The highest immersion treatment, 10 mg/L, yielded a population of 90% presumptive males and 10% with atypical gonads. However, the most effective treatment, yielding a population of 90% presumptive males and no fish with atypical gonads, was the feeding treatment. Given that female salmonid fishes are homogametic, sex-reversed (masculinized) genotypic females produced in this way can serve as broodstock for the creation of all-female charr populations for aquaculture.     

Gonadal sex reversal in triploid rainbow trout (Oncorhynchus mykiss).

Sex determination in salmonids is primarily governed by sex chromosomes; however, phenotypic expression and successful development of the gonads may be influenced by additional factors. Exposure to exogenous steroids during the critical period of gonadal differentiation will reverse the expected phenotypic sex of both female and male trout. Triploidy, a viable condition in rainbow trout (RBT), alters the degree of gonadal development in a gender-specific manner. Males produce testes with similar morphology and function as diploid fish, but females produce underdeveloped ovaries devoid of growing oocytes. One possible explanation for this observed gender difference is that the timing of meiotic initiation may influence ovarian/testicular development in triploid RBT. To determine whether the early entrance of germ cells into meiosis results in the lack of ovarian development in triploid females, the objective of this study was to sex-reverse genotypic triploid female RBT (XXX) into phenotypic males and genotypic triploid male RBT (XXY) into phenotypic females. Male fish were exposed to estradiol-17beta (E(2)) and females were exposed to the non-aromatizable androgen 17alpha-methyldihydrotestosterone (MDHT). Over 90% of the male fish treated with exogenous E(2) developed gonadal structures indistinguishable from the gonads of triploid females. Triploid female RBT treated with MDHT developed testes; however, not all fish treated with this androgen were completely sex reversed. The results of this investigation are consistent with the hypothesis that the failure of ovarian development in triploid RBT is due to the early onset of meiosis and does not appear to be due to genotypic sex. J. Exp. Zool. 284:466-472, 1999.     

Steroid enzyme gene expressions during natural and androgen-induced gonadal differentiation in the rainbow trout, Oncorhynchus mykiss.

In fish, according to Yamamoto's model, androgens would drive testis differentiation and estrogens ovarian differentiation. In order to study the implication of steroid enzymes in rainbow trout gonadal differentiation, we examined the expression of some steroid enzyme genes during natural differentiation (cholesterol side chain cleavage = P450scc, 17-hydroxylase/lyase = P450c17, 3beta-hydroxysteroid dehydrogenase = 3betaHSD) and androgen-induced differentiation (P450scc, P450c17, 3betaHSD, aromatase = P450aro, and 11beta-hydroxylase = P45011beta). Expressions of P450scc, 3betaHSD, and P450c17 were all detected in male and female gonads at 55 days post-fertilization (dpf), i.e., two weeks before histological differentiation. There were no differences in their expression level respective to the sex. The androgen treatment was carried out by administration of 11beta-hydroxyandrostenedione (11betaOHDelta4) in genetic all-female populations and the resulting sex ratios were found to be 100% male even at a low dosage of 1 mg/kg of food. Following 11betaOHDelta4 treatment, only the expression of P450c17 was found to be sustained when compared with the female untreated control. In contrast, P450scc was clearly up-regulated and 3betaHSD and P450aro down-regulated by the androgen treatment. P45011beta gene expression remained low in gonads of androgen-treated females, as it did in control untreated females. These results together demonstrate that steroidogenesis in rainbow trout is potentially active in pre-differentiating gonads of both sexes, and that one of the masculinizing actions of androgens in the species may be to down-regulate the female-specific gonadal P450aro gene expression. However, in vivo androgen treatment in genetic females does not induce the same pattern of steroid gene expression as in genetic males. These data suggest that exogenous androgens might induce a male differentiation process with P450aro inhibition being one of the steps required. However, this process would not involve endogenously produced 11-oxygenated androgens.     

Protogynous hermaphroditism in the half-banded sea perch, Hypoplectrodes maccullochi (Serranidae)

The half-banded sea perch, Hypoplectrodes maccullochi (Serranidae) exhibits protogynous hermaphroditism, based on the presence of transitional individuals collected from rocky reefs along the coast of New South Wales, Australia. Males dominated the larger size and age classes, whereas females predominated the smaller size and age groups. Histological preparations suggested that sex change occurs between 55–85 mm s.l. and 1 + to 4+ years of age. Three transitional phases were identified which varied according to the proportion of gametogenic tissue and remnant oocytes present within their gonads. All individuals greater than 85 mm s.l. were functional males containing well defined crypts of spermatids and spermatozoa. Remnant oocytes within these testes indicated prior female function. H. maccullochi were sexually monochromatic. The occurrence of some small males derived from pre-spawning females suggested pre-maturational sex change occurred in some individuals. Sex changing individuals were found in all the months of the year that gonads were examined in detail (January, February, April and June 1989). Fish were ripe in August and November. H. maccullochi conforms with the pattern of reproduction evident within the genus Hypopleclrodes .     

Immunohistochemical evidence for 11beta-hydroxylase (P45011beta) and androgen production in the gonad during sex differentiation and in adults in the protandrous anemonefish Amphiprion clarkii

To obtain basic information on the endocrine mechanisms underlying sex change in the protandrous anemonefish Amphiprion clarkii, we examined the immunolocalization of the steroidogenic enzyme cytochrome 11beta-hydroxylase (P45011beta), which is involved in 11-ketotestosterone (11-KT) production, and analyzed the ability of gonads to produce steroid hormones throughout the sex differentiation process and at the breeding stage. Immunopositive reactions against P45011beta appeared in sexually undifferentiated gonads at 30 days post hatching (dph). The number of immunopositive cells continued to increase during ovarian differentiation (from 60 to 180 dph) and throughout the formation of ambisexual gonads with both ovarian and testicular tissue until 270 dph. In the male phase, strongly immunopositive cells were observed in the cellular interstices of both testicular and ovarian tissues. P45011beta was localized only in the theca cells enclosing developed oocytes in the female phase. In-vitro 11-KT production in the gonads gradually increased with testicular differentiation (before, during, and after differentiation). Production of 11-KT in the gonads was higher in the male phase than during testicular differentiation or in the female phase. Our results suggest that androgen is involved in testicular differentiation during sex differentiation and spermatogenesis.     

Growth-related changes in color and sex inHalichoeres poecilopterus

Coloration and sex change were studied in a temperate wrasseHalichoeres poecilopterus in the central part of the Seto Inland Sea, Japan. 1,270 examples, 45–179 mm SL, were collected from May to December both in 1983 and 1984. The species is a diandric, protogynous hermaphrodite, and has three color patterns: pale color type (A), brilliant color type (B) and intermediate color type (AB). A-fish were less than 142 mm SL and consisted of primary males (42.6%), females (55.4%), secondary males (0.3%) and fish with transitional gonads (1.7%). A-females changed their color to B, through AB, in the size range 101–131 mm SL. A-primary males changed their color to B, through AB, in the size range 103–134 mm SL. B-fish consisted of primary males (38.6%), secondary males (54.6%) and fish with transitional gonads (6.8%). The majority of females changed their sex to male in the size range 98–131 mm SL.