Aquaculture has made an enormous contribution to the world food production, especially to the sustainable supply of animal proteins. The utility of diverse reproduction strategies in fish, such as the exploiting use of unisexual gynogenesis, has created a typical case of fish genetic breeding. A number of fish species show substantial sexual dimorphism that is closely linked to multiple economic traits including growth rate and body size, and the efficient development of sex-linked genetic markers and sex control biotechnologies has provided significant approaches to increase the production and value for commercial purposes. Along with the rapid development of genomics and molecular genetic techniques, the genetic basis of sexual dimorphism has been gradually deciphered, and great progress has been made in the mechanisms of fish sex determination and identification of sex-determining genes. This review summarizes the progress to provide some directive and objective thinking for further research in this field.

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Genetic basis and biotechnological manipulation of sexual dimorphism and sex determination in fish

Vertebrate reproduction is controlled by two gonadotropins (FSH and LH) from the pituitary. Despite numerous studies on FSH and LH in fish species, their functions in reproduction still remain poorly defined. This is partly due to the lack of powerful genetic approaches for functional studies in adult fish. This situation is now changing with the emergence of genome-editing technologies, especially Transcription Activator-Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). In this study, we deleted the hormone-specific β-genes of both FSH and LH in the zebrafish using TALEN. This was followed by a phenotype analysis for key reproductive events, including gonadal differentiation, puberty onset, gametogenesis, final maturation, and fertility. FSH-deficient zebrafish (fshb−/−) were surprisingly fertile in both sexes; however, the development of both the ovary and testis was significantly delayed. In contrast, LH-deficient zebrafish (lhb−/−) showed normal gon...

Genetic analysis of zebrafish gonadotropin (FSH and LH) functions by TALEN-mediated gene disruption.

Gonadotropins are primary hormones that control vertebrate reproduction. In a recent study, we analyzed the impacts of FSH and LH on zebrafish reproduction by disrupting FSH and LH-β genes (fshb and lhb) using transcription activator-like effector nuclease (TALEN) technology. Using the same approach, we successfully deleted FSH and LH receptor genes (fshr and lhcgr) in the present study. In contrast to the deficiency of its cognate ligand FSH, the fshr-deficient females showed a complete failure of follicle activation with all ovarian follicles arrested at the primary growth-previtellogenic transition, which is the marker for puberty onset in females. Interestingly, after blockade at the primary growth stage for varying times, all females reversed to males, and all these males were fertile. In fshr-deficient males, spermatogenesis was normal in adults, but the initiation of spermatogenesis in juveniles was retarded. In contrast to fshr, the deletion of the lhcgr gene alone caused no obvious phenotypes in both males and females; however, double mutation of fshr and lhcgr resulted in infertile males. In summary, our results in the present study showed that Fshr was indispensable to folliculogenesis and the disruption of the fshr gene resulted in a complete failure of follicle activation followed by masculinization into males. In contrast, lhcgr does not seem to be essential to zebrafish reproduction in both males and females. Neither Fshr nor Lhcgr deficiency could phenocopy the deficiency of their cognate ligands FSH and LH, which is likely due to the fact that Fshr can be activated by both FSH and LH in the zebrafish.

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Disruption of Zebrafish Follicle-Stimulating Hormone Receptor (fshr) But Not Luteinizing Hormone Receptor (lhcgr) Gene by TALEN Leads to Failed Follicle Activation in Females Followed by Sexual Reversal to Males.

Sexual or gonadal differentiation is a complex event and its mechanism remains elusive in teleosts. Despite its complexity and plasticity, the process of ovarian differentiation is believed to involve gonadal aromatase (cyp19a1a) in nearly all species studied. However, most data concerning the role of aromatase have come from gene expression analysis or studies involving pharmacological approaches. There has been a lack of genetic evidence for the importance of aromatase in gonadal differentiation, especially the timing when the enzyme starts to exert its effect. This is due to the lack of appropriate loss-of-function approaches in fish models for studying gene functions. This situation has changed recently with the development of genome editing technologies, namely TALEN and CRISPR/Cas9. Using both TALEN and CRISPR/Cas9, we successfully established three mutant zebrafish lines lacking the ovarian aromatase. As expected, all mutant fish were males, supporting the view that aromatase plays a critical role in directing ovarian differentiation and development. Further analysis showed that the ovarian aromatase did not seem to affect the formation of so-called juvenile ovary and oocyte-like germ cells; however, it was essential for further differentiation of the juvenile ovary into the true ovary.

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Knockout of Zebrafish Ovarian Aromatase Gene (cyp19a1a) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation.

Fish sex reversal is a means to understand sex determination and differentiation, but it is also used to control sex in aquaculture. This review discusses sex reversal in gonochoristic fish, with the

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The reversible sex of gonochoristic fish: Insights and consequences

This study investigated whether undifferentiated germ and/or somatic stem cells remain in the differentiated ovary of a species that does not undergo sex changes under natural conditions and retain their sexual plasticity. The effect of aromatase inhibitor (AI)-treatment on sexually mature female zebrafish was examined. A 5-month AI treatment caused retraction of the ovaries after which testes-like organs appeared, and cyst structures filled with spermatozoa-like cells were observed in sections of these tissues. Electron microscopic observations revealed that these cells appeared as large sperm heads without tails. Sperm formation was re-examined after changing the diet to an AI-free food. A large number of normal sperm were obtained after eight weeks, and no formation of ovarian tissue was observed. Artificial fertilization using sperm from the sex-changed females was successful. These results demonstrated that sex plasticity remains in the mature ovaries of this species.

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Induction of Female-to-Male Sex Change in Adult Zebrafish by Aromatase Inhibitor Treatment

Temperature-dependent sex determination (TSD) is a mechanism in which environmental temperature, rather than innate zygotic genotype, determines the fate of sexual differentiation during embryonic development. Reeves' turtle (also known as the Chinese three-keeled pond turtle, Mauremys reevesii) exhibits TSD and is the only species whose genome has been determined in Geoemydidae to date. Thus, M. reevesii occupy phylogenetically important position for the study of TSD and can be compared to other TSD species to elucidate the underlying molecular mechanism of this process. Nevertheless, neither embryogenesis nor gonadogenesis has been described in this species. Therefore, herein, we investigated the chronology of normal embryonic development and gonadal structures in M. reevesii under both female- and male-producing incubation temperatures (FPT 31 °C or MPT 26 °C, respectively). External morphology remains indistinct between the two temperature regimes throughout the studied embryonic stages. However, the gonadal ridges present on the mesonephros at stage 16 develop and sexually differentiate at FPT and MPT. Ovarian and testicular structures begin to develop at stages 18-19 at FPT and stages 20-21 at MPT, respectively, and thus, the sexual differentiation of gonadal structures began earlier in the embryos at FPT than at MPT. Our results suggest that temperature sensitive period, at which the gonadal structures remain sexually undifferentiated, spans from stage 16 (or earlier) to stages 18-19 at FPT and to stages 20-21 at MPT. Understanding the temperature-dependent differentiation in gonadal structures during embryonic development is a prerequisite for investigating molecular basis underlying TSD. Thus, the result of the present study will facilitate further developmental studies on TSD in M. reevesii. 

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Chronology of embryonic and gonadal development in the Reeves’ turtle, Mauremys reevesii


 Temperature-dependent sex determination (TSD) is a mechanism in which environmental temperature rather than innate zygotic genotype determines the fate of sexual differentiation during embryonic development. The Chinese three-keeled pond turtle, Mauremys reevesii, has TSD and can thus be compared to other TSD species to elucidate the molecular basis underlying this mechanism. Nevertheless, neither embryogenesis nor gonadogenesis has been described in this species. Here, we investigated the chronology of normal embryonic development and gonadal structures in M. reevesii under both female- and male-producing incubation temperatures (FPT or MPT, respectively). External morphology remained indistinct between the two temperature regimes throughout the studied embryonic stages. However, the sexual differentiation of gonadal structures began earlier in the embryos at FPT than at MPT. Moreover, the offset of the thermosensitive period, at which the gonadal structures often remain sexually undifferentiated, did not occur at the same stage at FPT and MPT. In ovo experiments are particularly important for understanding the functional roles of genes involved in TSD, and the present study provides the basis required for designing such developmental studies in M. reevesii.

Kaori Miyaoku

Papers

Induction of Female-to-Male Sex Change in Adult Zebrafish by Aromatase Inhibitor Treatment

Chronology of embryonic and gonadal development in the Reeves’ turtle, Mauremys reevesii

Embryonic and Gonadal Development of the Chinese Three-keeled Pond Turtle, Mauremys Reevesii