Gonadal sex determination in vertebrates generally follows a sequence of genetically programmed events. In what is seemingly becoming a pattern, all confirmed or current candidate "master" sex-determining genes reported in this group, e.g., SRY in eutherian mammals, DMY/dmrt1bY in medaka, DM-W in the African clawed frog, and DMRT1 in chicken encode transcription factors. In contrast, here we show that a male-specific, duplicated copy of the anti-Mül-lerian hormone (amh) is implicated in testicular development of the teleost fish Patagonian pejerrey (Odontesthes hatcheri). The gene, termed amhy because it is found in a single metacentric/submetacentric chromosome of XY individuals, is expressed much earlier than the autosomal amh (6 d after fertilization vs. 12 wk after fertilization) and is localized to presumptive Sertoli cells of XY males during testicular differentiation. Moreover, amhy knockdown in XY embryos resulted in the up-regulation of foxl2 and cyp19a1a mRNAs and the development of ovaries. These results are evidence of a functional amh duplication in vertebrates and suggest that amhy may be the master sex-determining gene in this species. If confirmed, this would be a unique instance of a hormone-related gene, a member of the TGF-β superfamily, in such a role.T he sexual fate of the differentiating gonads in vertebrates is under the control of specific genes that initiate and direct the developmental pathway. A few genes have been already identified as master sex determiners, and they all encode transcription factors, e.g., SRY in eutherian mammals (1), DMY/ dmrt1bY in medaka (2, 3), DM-W in the African clawed frog (4), and DMRT1 in chicken (5). These findings might be construed as evidence that transcription factors always trigger gonadal sex determination in vertebrates. However, the molecular pathway of sex determination has been studied in relatively few nonmammalian species, and most of the details of this process remain elusive.We have recently identified a sex-linked locus in Odontesthes hatcheri (Atherinopsidae), a South American gonochoristic fish with an XX-XY sex determination system (6, 7). The existence of a sex-linked single nucleotide polymorphism (SNP) marker associated with this locus has allowed us to profile the expression of a series of genes involved in early sex differentiation of putative females (XX genotype) and males (XY genotype). Analyses performed during early stages of embryonic and larval development revealed a comparatively early mRNA expression of an anti-Müllerian hormone homolog [(amh); also known as Müllerian inhibitory substance/factor, or mis/mif (8)] in relation to other teleosts (9, 10) and showed that this unique feature was due to the up-regulation of a duplicated copy of this gene. AMH, a member of the TGF-β superfamily, is secreted by Sertoli cells and is responsible for the regression of Müllerian ducts during male fetal development in mammals, birds, and reptiles (11-13). Fish have amh even though they lack Müllerian ducts. However, as with mammals and bi...

BackgroundGonadal fate in many reptiles, fish, and amphibians is modulated by the temperature experienced during a critical period early in life (temperature-dependent sex determination; TSD). Several molecular processes involved in TSD have been described but how the animals “sense” environmental temperature remains unknown. We examined whether the stress-related hormone cortisol mediates between temperature and sex differentiation of pejerrey, a gonochoristic teleost fish with marked TSD, and the possibility that it involves glucocorticoid receptor- and/or steroid biosynthesis-modulation.Methodology/Principal FindingsLarvae maintained during the period of gonadal sex differentiation at a masculinizing temperature (29°C; 100% males) consistently had higher cortisol, 11-ketotestoterone (11-KT), and testosterone (T) titres than those at a feminizing temperature (17°C; 100% females). Cortisol-treated animals had elevated 11-KT and T, and showed a typical molecular signature of masculinization including amh upregulation, cyp19a1a downregulation, and higher incidence of gonadal apoptosis during sex differentiation. Administration of cortisol and a non-metabolizable glucocorticoid receptor (GR) agonist (Dexamethasone) to larvae at a “sexually neutral” temperature (24°C) caused significant increases in the proportion of males.Conclusions/SignificanceOur results suggest a role of cortisol in the masculinization of pejerrey and provide a possible link between stress and testicular differentiation in this gonochoristic TSD species. Cortisol role or roles during TSD of pejerrey seem(s) to involve both androgen biosynthesis- and GR-mediated processes. These findings and recent reports of cortisol effects on sex determination of sequential hermaphroditic fishes, TSD reptiles, and birds provide support to the notion that stress responses might be involved in various forms of environmental sex determination.

In this study, we examined whether a homolog of the master sex-determining gene amhy of Odontesthes hatcheri is present and plays any role in testis determination of pejerrey O. bonariensis, a species otherwise known for its strong temperature-dependent sex determination (TSD). Screening of wild and laboratory-reared pejerrey for amhy revealed a high, although not complete linkage with phenotypic sex. The sex ratio in an amhy +/−/amhy −/− full sibling progeny reared during the thermolabile period of sex determination at an intermediate temperature of 25°C was 68.7% male:31.3% female; all amhy +/− fish developed as males whereas about 2/3 and 1/3 of the amhy −/− were female and male, respectively. Expression analyses revealed that amhy transcription began during embryo stage and decreased by the end of sex determination period. The autosomal amha was present in all individuals regardless of amhy genotype; its expression increased significantly from the end of the same period in the gonads of all amhy+ /− but only in part of the amhy− /− animals. After histological gonadal differentiation, all gonads of amhy −/− animals with amha ISH signals were testes and those without it were ovaries. These results suggest that amhy is important for testicular differentiation in pejerrey, at least at intermediate temperatures. Thus, we hypothesize that amhy +/− animals differentiate as males by expression of either amhy alone or amhy and amha together whereas the amhy −/− probably rely solely on amha expression. These findings represent the first clear genomic evidence that genotypic and environmental sex determinants can coexist in species with marked TSD such as the pejerrey. The finding of amhy will make possible to monitor wild pejerrey populations for mismatches between genotypic and phenotypic sex and may prove instrumental for field studies addressing the effects of endocrine disruptors or abnormal temperatures on reproduction and the ecological relevance of TSD for this species.

The pejerrey Odontesthes bonariensis (Valenciennes, 1835) is an inland water ¢sh from the Pampas region comprising part of Argentina, Uruguay and the South of Brazil. Pejerrey is a very popular ¢sh in this region and has a long history of domestic and international introductions, which attests to the high quality and market value of its £esh, as well as its attractiveness as a game ¢sh. The desirable characteristics of pejerrey also make it a good candidate for aquaculture, and the ¢rst trials on pejerrey cultivation (atheriniculture) were started more than a century ago in Argentina. In spite of the considerable interest in its development, little progress has been made and atheriniculture is still restricted to propagation and stocking for sport ¢shing purposes. In this review, we summarize the history of atheriniculture and the biological, technological, scienti¢c, cultural and infrastructural constraints to pejerrey aquaculture development thus far. We also suggest possible scenarios of pejerrey aquaculture development compatible with the socio^economic conditions of South American countries. Our projections also take into consideration recent scienti¢c ¢ndings on the biology of pejerrey and technological advances in seafood processing, storage and transportation, as well as the latest trends of seafood consumption and international markets. The best production strategy remains to be determined by trial and error but it is likely that, initially, production should focus on a di¡erentiated, high-quality ¢sh for the premium or international markets rather than attempting to compete with the cheap ¢sh from natural sources. An international cooperation project with Japan has successfully demonstrated the feasibility of breeding pejerrey in captivity and mass producing seeds e⁄ciently in Argentina, and has provided the foundation for the development of intensive and extensive farming of this species.

Atherinidae is a family of fishes known to include at least three species with documented thermolabile sex determination. In Odontesthes bonariensis, fish exposed to about 17°C from hatching to juvenile stage become all-female, whereas groups exposed to about 25°C become male-biased. In contrast, sex in another atherinid, Patagonina hatcheri, appears to be under stronger genotypic control because the sex ratios are always balanced (1:1) within the same range of temperatures. The main purpose of this study was to determine the effect of temperature on the sex ratios of both species over their full range of viable temperatures. In addition, we determined the critical time of thermolabile sex determination in O. bonariensis at female-and male-forming temperatures. O. bonariensis larvae survived at temperatures between 13 and 29°C, but growth was negligible at 13 and 15°C. The proportions of females were 100% at 13-19°C, 95% at 21°C, 81.2% at 23°C, 29.4% at 25°C, 10% at 27°C, and 0% at 29°C. These results indicate a gradual transition from female-to male-forming temperatures evenly spread over the range of viable temperatures and hence the absence of any marked thermal threshold for thermolabile/ genotypic control of sex; they also suggest weak or no genotypic control of sex in this species. In addition, prolonged exposure to 29°C caused disappearance of germ cells. The critical time of thermolabile sex determination in O. bonariensis was estimated to be 3-5 wk, 2-4 wk, and 1-4 wk after hatching at 17, 19, and 27°C, respectively. Survival of P. hatcheri was limited to temperatures between 13 and 27°C (only 8.7% at the latter temperature) with slow but steady growth at 13 and 15°C. The proportions of females were 88.9 and 89.5% at 13 and 15°C, respectively, about 50% at temperatures between 17 and 23°C, and 30.8% at 25°C, hence, showing one clear threshold for thermolabile/genotypic control of sex at 15-17°C and possibly another at or around 25°C. These results confirmed that gonadal sex in this species is under genotypic control at intermediate temperatures but also showed that this control can be overridden at low (and possibly also at high) temperatures. J. Exp. Zool. 278:167-177, 1997. © 1997 Recent studies have shown that the phenotypic sex can be altered by the thermal conditions prevailing during the period of primary sex differentiation (a phenomenon termed thermolabile sex determination [TSD]) in two South American atherinids, Odontesthes bonariensis and O. argentinensis (Strüssmann et al., '96a,b). In O. bonariensis, for instance, thermal treatments of larvae and juveniles yielded 98-100% females at around 17°C, 85-100% females at around 21°C, and 14-83% females at around 25°C (Strüssmann et al., '96a). These results parallel those for another atherinid, the Atlantic silverside Menidia menidia (Conover and Kynard, '81), in that low temperatures favor the formation of females, whereas high temperatures yield more males. For this species, Conover and Heins ('87a) determined that the shape of the resp...

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In many ectotherm species the gonadal fate is modulated by temperature early in life [temperature-dependent sex determination (TSD)] but the transducer mechanism between temperature and gonadal differentiation is still elusive. We have recently shown that cortisol, the glucocorticoid stress-related hormone in vertebrates, is involved in the TSD process of pejerrey, Odontesthes bonariensis. Particularly, all larvae exposed to a male-producing temperature (MPT, 29 C) after hatching showed increased whole-body cortisol and 11-ketotestosterone (11-KT; the main bioactive androgen in fish) levels and developed as males. Moreover, cortisol administration at an intermediate, mixed sex-producing temperature (MixPT, 24 C) caused increases in 11-KT and in the frequency of males, suggesting a relation between this glucocorticoid and androgens during the masculinization process. In order to clarify the link between stress and masculinization, the expression of hydroxysteroid dehydrogenase (hsd)11b2, glucocorticoid receptors gr1 and gr2, and androgen receptors ar1 and ar2 was analyzed by quantitative real time PCR and in situ hybridization in larvae reared at MPT, MixPT, and female-producing temperature (FPT, 17 C) during the sex determination period. We also analyzed the effects of cortisol treatment in larvae reared at MixPT and in adult testicular explants incubated in vitro. MPT and cortisol treatment produced significant increases in hsd11b2 mRNA expression. Also, gonadal explants incubated in the presence of cortisol showed increases of 11-KT levels in the medium. Taken together these results suggest that cortisol promotes 11-KT production during high temperature-induced masculinization by modulation of hsd11b2 expression and thus drives the morphogenesis of the testes.

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