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.

Maternal investment in fish oocytes and eggs: The molecular cargo and its contributions to fertility and early development

Growth factors modulate germ line stem cell self-renewal and differentiation behavior. We investigate the effects of Igf3, a fish-specific member of the igf family. Fsh increased in a steroid-independent manner the number and mitotic index of single type A undifferentiated spermatogonia and of clones of type A differentiating spermatogonia in adult zebrafish testis. All 4 igf gene family members in zebrafish are expressed in the testis but in tissue culture only igf3 transcript levels increased in response to recombinant zebrafish Fsh. This occurred in a cAMP/protein kinase A-dependent manner, in line with the results of studies on the igf3 gene promoter. Igf3 protein was detected in Sertoli cells. Recombinant zebrafish Igf3 increased the mitotic index of type A undifferentiated and type A differentiating spermatogonia and up-regulated the expression of genes related to spermatogonial differentiation and entry into meiosis, but Igf3 did not modulate testicular androgen release. An Igf receptor inhibitor blocked these effects of Igf3. Importantly, the Igf receptor inhibitor also blocked Fsh-induced spermatogonial proliferation. We conclude that Fsh stimulated Sertoli cell production of Igf3, which promoted via Igf receptor signaling spermatogonial proliferation and differentiation and their entry into meiosis. Because previous work showed that Fsh also released spermatogonia from an inhibitory signal by down-regulating anti-Mullerian hormone and by stimulating androgen production, we can now present a model, in which Fsh orchestrates the activity of stimulatory (Igf3, androgens) and inhibitory (anti-Mullerian hormone) signals to promote spermatogenesis.

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Fsh Stimulates Spermatogonial Proliferation and Differentiation in Zebrafish via Igf3.

Gonadal soma-derived factor (gsdf) is critical for testicular differentiation in teleosts, yet detailed analysis of Gsdf on testicular differentiation is lacking. In the present study, we knocked out tilapia gsdf using CRISPR/Cas9. F0 gsdf-deficient XY fish with high mutation rate (≥58%) developed as intersex, with ovotestes 90 days after hatching (dah), and become completely sex-reversed with ovaries at 180 and 240 dah. Those individuals with a low mutation rate (<58%) and XY gsdf(+/-) fish developed as males with normal testes. In F2 XY gsdf(-/-) fish, the gonads first expressed Dmrt1, which initiated the male pathway at 10 dah, then both male and female pathways were activated, as reflected by the simultaneous expression of Dmrt1 and Cyp19a1a in different cell populations at 18 dah, shifted to the female pathway expressing only Cyp19a1a at 36 dah, and finally developed into functional ovaries as adults. The male pathway and Dmrt1 expression was initiated, but failed to be maintained, in the absence of Gsdf. Aromatase-inhibitor treatment from 10 to 35 dah, however, rescued the phenotype, resulting in XY gsdf(-/-) with normal testes that expressed Dmrt1 and Cyp11b2. In vitro promoter analyses demonstrated that Dmrt1 activated gsdf expression in a dose-dependent manner in the presence of Sf1, even though Dmrt1 alone could not. Taken together, our results demonstrated that gsdf is a downstream gene of dmrt1. Gsdf probably inhibits estrogen production to trigger testicular differentiation. Mol. Reprod. Dev. 83: 497-508, 2016. © 2016 Wiley Periodicals, Inc.

gsdf is a downstream gene of dmrt1 that functions in the male sex determination pathway of the Nile tilapia

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.

Genome editing in fishes and their applications.

Folliculogenesis is controlled by intimate communications between oocytes and surrounding follicle cells. Epidermal growth factor (EGF/Egf) is an important paracrine/autocrine factor in vertebrate ovary, and it is well known for its stimulation of oocyte maturation. However, the role of EGF signaling through its receptor (EGFR/Egfr) in ovarian folliculogenesis is poorly understood, especially at early stages of follicle development. In this study, we created zebrafish mutants for Egf (egf −/− ) and Egfr (egfra −/− and egfrb −/− ) by CRISPR/Cas9 technique. Surprisingly, these mutants all survived well with little abnormality in growth and development. Spermatogenesis and folliculogenesis were both normal in egf −/− males and females. Their fecundity was comparable to that of the wildtype fish at 4 months post-fertilization (mpf); however, the fertilization rate of mutant eggs (egf −/− ) decreased significantly at 7 mpf. Interestingly, disruption of egfra (egfra −/− ) led to failed follicle activation with folliculogenesis being blocked at primary–secondary growth transition (PG-SG transition), leading to female infertility, whereas the mutant males remained fertile. The mutant ovary (egfra −/− ) showed abnormal expression of a substantial number of genes involved in oxidative metabolism, gene transcription, cytomembrane transport, steroid hormone biosynthesis, and immune response. The stunted PG oocytes in egfra −/− ovary eventually underwent degeneration after 6 months followed by sex reversal to males with functional testes. No abnormal phenotypes were found in the mutant of truncated form of EGFR (egfrb). In summary, our data revealed critical roles for EGFR signaling in early folliculogenesis, especially at the PG-SG transition or follicle activation.

Disruption of Epidermal Growth Factor Receptor but Not EGF Blocks Follicle Activation in Zebrafish Ovary

As in mammals, ovarian folliculogenesis in teleosts also consists of two phases: the primary growth (PG) and secondary growth (SG) phases, which are analogous to the preantral and antral phases respectively in mammals. In this study, we performed a proteomic analysis on zebrafish follicles undergoing the PG-SG transition aiming to identify factors involved in the event. Numerous proteins showed significant changes, and the most prominent one was Y-box binding protein 1 (YB-1; Ybx1/ybx1), a transcription factor and mRNA-binding protein. YB-1 belongs to the Y-box binding protein family, which also includes the gonad-specific YB-2. Interestingly, phylogenetic analysis showed no YB-2 homologue in zebrafish. Although ybx1 mRNA was expressed in various tissues, its protein Ybx1 was primarily produced in the gonads, similar to YB-2 in other species. In the ovary, Ybx1 protein started to appear in early follicles newly emerged from the germ cell cysts, reached the highest level in late PG oocytes, but decreased precipitously when the follicles entered the SG phase. In PG follicles, Ybx1 might function as a key component of the messenger ribonucleoprotein particles (mRNP) in association with other RNA-binding proteins. Similar to mammalian YB-1, zebrafish Ybx1 also contains functional signals that determine its intracellular localization. In conclusion, Ybx1 may play dual roles of YB-1 and YB-2 in zebrafish. In the ovary, Ybx1 binds mRNAs to stabilize them while preventing their translation. At PG-SG transition, Ybx1 is removed to release the masked mRNAs for translation into functional proteins, leading to follicle activation.

Proteomic analysis of zebrafish Folliculogenesis identifies YB-1 (Ybx1/ybx1) as a potential gatekeeping molecule controlling early ovarian Folliculogenesis.

Bo Zhu

Papers

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

Genome editing in fishes and their applications.

Disruption of Epidermal Growth Factor Receptor but Not EGF Blocks Follicle Activation in Zebrafish Ovary

Proteomic analysis of zebrafish Folliculogenesis identifies YB-1 (Ybx1/ybx1) as a potential gatekeeping molecule controlling early ovarian Folliculogenesis.