N⁶-methyladenosine (m⁶A), catalyzed by Mettl3 methyltransferase, is a highly conserved epigenetic modification in eukaryotic mRNA. Previous studies have implicated m⁶A modification in multiple biological processes, but the in vivo function of m⁶A has been difficult to study, because mettl3 mutants are embryonic lethal in both mammals and plants. In this study, we have used transcription activator-like effector nucleases and generated viable zygotic mettl3 mutant, Zmettl3^m/m, in zebrafish. We find that the oocytes in Zmettl3^m/m adult females are stalled in early development and the ratio of full grown stage (FG) follicles is significantly lower than that of wild type. Human chorionic gonadotropin-induced ovarian germinal vesicle breakdown in vitro and the numbers of eggs ovulated in vivo are both decreased as well, while the defects of oocyte maturation can be rescued by sex hormone in vitro and in vivo. In Zmettl3^m/m adult males, we find defects in sperm maturation and sperm motility is significantly reduced. Further study shows that 11-ketotestosterone (11-KT) and 17β-estradiol (E2) levels are significantly decreased in Zmettl3^m/m, and defective gamete maturation is accompanied by decreased overall m⁶A modification levels and disrupted expression of genes critical for sex hormone synthesis and gonadotropin signaling in Zmettl3^m/m. Thus, our study provides the first in vivo evidence that loss of Mettl3 leads to failed gamete maturation and significantly reduced fertility in zebrafish. Mettl3 and m⁶A modifications are essential for optimal reproduction in vertebrates

The luteinizing hormone surge is essential for fertility as it triggers ovulation in females and sperm release in males. We previously reported that secretoneurin-a, a neuropeptide derived from the processing of secretogranin-2a (Scg2a), stimulates luteinizing hormone release, suggesting a role in reproduction. Here we provide evidence that mutation of the scg2a and scg2b genes using TALENs in zebrafish reduces sexual behavior, ovulation, oviposition, and fertility. Large-scale spawning within-line crossings (n = 82 to 101) were conducted. Wild-type (WT) males paired with WT females successfully spawned in 62% of the breeding trials. Spawning success was reduced to 37% (P = 0.006), 44% (P = 0.0169), and 6% (P < 0.0001) for scg2a^−/−, scg2b^−/−, and scg2a^−/−;scg2b^−/− mutants, respectively. Comprehensive video analysis indicates that scg2a^−/−;scg2b^−/− mutation reduces all male courtship behaviors. Spawning success was 47% in saline-injected WT controls compared to 11% in saline-injected scg2a^−/−;scg2b^−/− double mutants. For these mutants, spawning success increased 3-fold following a single intraperitoneal (i.p.) injection of synthetic secretoneurin-a (P = 0.0403) and increased 3.5-fold with injection of human chorionic gonadotropin (hCG). Embryonic survival at 24 h remained on average lower in scg2a^−/−;scg2b^−/− fish compared to WT injected with secretoneurin-a (P < 0.001). Significant reductions in the expression of gonadotropin-releasing hormone 3 in the hypothalamus, and luteinizing hormone beta and glycoprotein alpha subunits in the pituitary provide evidence for disrupted hypothalamo-pituitary function in scg2a and scg2b mutant fish. Our results indicate that secretogranin-2 is required for optimal reproductive function and support the hypothesis that secretoneurin is a reproductive hormone. Significance The secretory granule protein secretogranin-2 is processed to the bioactive neuropeptide secretoneurin. While these are expressed in hypothalamic neurons and anterior pituitary cells, a role in hormonal control of reproduction is unknown. We provide in vivo evidence that secretogranin-2 plays critical roles in fertility regulation. Mutation of the secretogranin-2a and -2b genes in zebrafish leads to disrupted sexual behaviors, reduced ovulation and egg laying, and suboptimal fertility and embryo survival. Injection of a synthetic secretoneurin peptide enhanced reproductive outcomes in the mutant fish. Our secretogranin-2 mutants offer an amenable model system in which to screen potential injectable stimulators of reproduction, be they for spawning in cultured fish species, or to help with the search for new human infertility treatments.

Gonadotropin-releasing hormone (Gnrh) plays important roles in reproduction by stimulating luteinizing hormone release, and subsequently ovulation and sperm release, ultimately controlling reproduction in many species. Here we report on a new role for this decapeptide. Surprisingly, Gnrh3-null zebrafish was generated by CRISPR/Cas9 and exhibited a male-biased sex ratio. After the dome stage, the number of primordial germ cells (PGCs) in gnrh3^-/- fish was less than that in wild-type, an effect that was partially rescued by gnrh3 overexpression. A TUNEL analysis revealed no detectable apoptosis of PGCs in gnrh3^-/- embryos. Proliferating PGCs could be detected in wild-type embryos, while there was no detectable signal in gnrh3^-/- embryos. Compared to wild type, the phosphorylation of AKT was not significantly different in gnrh3^-/- embryos, but the phosphorylation of ERK1/2 decreased significantly. Treatment with a Gnrh analog (Alarelin) induced ERK1/2 phosphorylation and increased PGC numbers in both wild-type and gnrh3^-/- embryos, and this was blocked by the MEK inhibitor PD0325901. The relative expression of sox9a, amh and cyp11b were significantly up-regulated, while cyp19a1a was significantly down-regulated at 18 days post-fertilization in gnrh3^-/- zebrafish. Taken together, these results indicate that Gnrh3 plays an important role in early sex differentiation by regulating the proliferation of PGCs through a MAPK-dependent path.

γ-Aminobutyric acid (GABA) is a major amino acid neurotransmitter in the vertebrate brain. To provide detailed information on the distribution of the GABA in zebrafish (Danio rerio), neurons were labeled with mCherry driven by the glutamic acid decarboxylase 67 (gad67) promoter. In the transgenic line Tg(gad67:mCherry), mCherry-positive gad67 cell bodies were predominantly localized to the olfactory bulb, pallial zones, subpallium zones, parvocellular preoptic nucleus, periventricular gray zone of optic tectum, torus semicircularis, posterior tuberculum, medial longitudinal fascicle, caudal zone of periventricular hypothalamus, and oculomotor nucleus. mCherry-positive fibers were widely distributed in the olfactory bulbs, subpallium, thalamus, ventral hypothalamic zone, tectum opticum, mesencephalon, and rhombencephalon. mCherry-positive neurons were also observed in the retina and the spinal cord. The anatomical relationships between GABAergic and gonadotrophin-releasing hormone 3 (GnRH3) neurons were investigated by crossing Tg(gad67:mCherry) fish with the previously established Tg(gnrh3:EGFP) transgenic line. GnRH3 cell bodies and fibers were contacted by GABAergic fibers directly in the ventral telencephalon and anterior tuberal nucleus. A subpopulation of GnRH3 neurons in the ventral telencephalic area was also labeled with mCherry, so some GnRH3 neurons are also GABAergic. GABA_B receptor agonist (baclofen) and antagonist (CGP55845) treatments indicated that GABA_B receptor signaling inhibited gnrh3 expression in larval fish but was stimulatory in adult fish. The expression of pituitary lhβ and fshβ was stimulated by intraperitoneal injection of baclofen in adult fish. We conclude that GABA via GABA_B receptors regulates GnRH3 neurons in a developmentally dependent manner in zebrafish.

Growth and reproduction are closely related. Growth hormone (GH)-transgenic common carp exhibit accelerated growth and delayed reproductive development, which provides an amenable model to study hormone cross talk between the growth and reproductive axes. We analyzed the energy status and reproductive development in GH-transgenic common carp by using multi-tissue RNA sequencing, real-time-PCR, Western blotting, ELISA, immunofluorescence, and in vitro incubation. The expression of gys (glycogen synthase) and igfbp1 (insulin-like growth factor binding protein) as well as blood glucose concentrations are lower in GH-transgenic carp. Agrp1 (agouti-related protein 1) and sla (somatolactin a), which are related to appetite and lipid catabolism, are significantly higher in GH-transgenic carp. Low glucose content and increased appetite indicate disrupted metabolic and energy deprivation status in GH-transgenic carp. Meanwhile, the expression of genes, such as gnrhr2 (gonadotropin-releasing hormone receptor 2), gthα (gonadotropin hormone, alpha polypeptide), fshβ (follicle stimulating hormone, beta polypeptide), lhβ [luteinizing hormone, beta polypeptide] in the pituitary, cyp19a1a (aromatase A) in the gonad, and cyp19a1b (aromatase B) in the hypothalamus, are decreased in GH-transgenic carp. In contrast, pituitary gnih (gonadotropin inhibitory hormone), drd1 (dopamine receptor D1), drd3 (dopamine receptor D3), and drd4 (dopamine receptor D4) exhibit increased expression, which were associated with the retarded reproductive development. Leptin receptor mRNA was detected by fluorescence in situ hybridization in the pituitary including the pars intermedia and proximal pars distalis, suggesting a direct effect of leptin on LH. Recombinant carp Leptin protein was shown to stimulate pituitary gthα, fshβ, lhβ expression, and ovarian germinal vesicle breakdown in vitro. In addition to neuroendocrine factors, we suggest that reduced hepatic leptin signaling to the pituitary might be part of the response to overexpression of GH and the resulting delay in puberty onset.

Secretoneurin (SN) is a neuropeptide derived from specific proteolytic processing of the precursor secretogranin II (SgII). In zebrafish and other teleosts, there are two paralogs named sgIIa and sgIIb. Our results showed that neurons expressing sgIIb were aligned with central arteries in the hindbrain, demonstrating a close neurovascular association. Both sgIIb^−/− and sgIIa^−/−/sgIIb^−/− mutant embryos were defective in hindbrain central artery development due to impairment of migration and proliferation of central artery cells. Further study revealed that sgIIb is non-cell autonomous and required for central artery development. Hindbrain arterial and venous network identities were not affected in sgIIb^−/− mutant embryos, and the mRNA levels of Notch and VEGF pathway-related genes were not altered. However, the activation of MAPK and PI3K/AKT pathways was inhibited in sgIIb^−/− mutant embryos. Reactivation of MAPK or PI3K/AKT in endothelial cells could partially rescue the central artery developmental defects in the sgIIb mutants. This study provides the first in vivo evidence that sgIIb plays a critical role in neurovascular modeling of the hindbrain. Targeting the SgII system may, therefore, represent a new avenue for the treatment of vascular defects in the central nervous system.