Ovarian development is a complex process controlled by precise coordination of multiple factors. The targeted gene knockout technique is a powerful tool to study the functions of these factors. The successful application of this technique in mice in the past three decades has significantly enhanced our understanding on the molecular mechanism of ovarian development. Recently, with the advent of genome editing techniques, targeted gene knockout research can be carried out in many species. Zebrafish has emerged as an excellent model system to study the control of ovarian development. Dozens of genes related to ovarian development have been knocked out in zebrafish in recent years. Much new information and perspectives on the molecular mechanism of ovarian development have been obtained from these mutant zebrafish. Some findings have challenged conventional views. Several genes have been identified for the first time in vertebrates to control ovarian development. Focusing on ovarian development, the purpose of this review is to briefly summarize recent findings using these gene knockout zebrafish models, and compare these findings with mammalian models. These established mutants and rapid development of gene knockout techniques have prompted zebrafish as an ideal animal model for studying ovarian development.

As in other vertebrates, fish reproduction is tightly controlled by gonadotropin signaling. One of the most perplexing aspects of gonadotropin action on germ cell biology is the restricted expression of gonadotropin receptors in somatic cells of the gonads. Therefore, the identification of factors conveying the action of gonadotropins on germ cells is particularly important for understanding the mechanism of reproduction. Insulin-like growth factors (Igfs) are recognized as key factors in regulating reproduction by triggering a series of physiological processes in vertebrates. Recently, a novel member of Igfs called Igf3 has been identified in teleost. Different from the conventional Igf1 and Igf2 that are ubiquitously expressed in a majority of tissues, Igf3 is solely or highly expressed in the fish gonads. The role of Igf3 in mediating the action of gonadotropin through Igf type 1 receptor on several aspects of oogenesis and spermatogenesis have been demonstrated in several fish species. In this review, we will summarize existing data on Igf3. This new information obtained from Igf3 provides insight into elucidating the molecular mechanism of fish reproduction, and also highlights the importance of Igf system in mediating the action of gonadotropin signaling on animal reproduction.

Fish require abundant nutrients to generate a large number of eggs for spawning. Based on the evolutionary conservation of human FBN2 and its C-terminal placensin-like sequences in fish, we identified a peptide hormone gonacin (GONAdal Cell placensIN) and found its high expression in early-stage germ cells in the ovary and testis of zebrafish. We demonstrated that gonacin is essential for food intake, glucose release, and ovarian development in zebrafish. Similar expression patterns and functions of gonacin were also demonstrated in rainbow trout. Gonacin represents the first hormone secreted by germ cells with endocrine functions in vertebrates, bridging the energy homeostasis and reproduction.

Asprosin, a fasting-induced glucogenic hormone, plays a crucial role in maintaining glucose homeostasis; however, its receptor remains elusive. This study identifies protein tyrosine phosphatase receptor F (Ptprf) as the receptor mediating Asprosin's metabolic functions. Using zebrafish models, we demonstrate the evolutionary conservation of Asprosin's role in glucose metabolism. Through binding assays, we determined Ptprf as Asprosin's interacting receptor in zebrafish liver. Zebrafish possess two Ptprf paralogs (Ptprfa/b), both hepatically expressed and binding Asprosin with high affinity. The genetic ablation of ptprfa/b reduced basal glucose levels and eliminated Asprosin-induced hyperglycemia. Conversely, the overexpression of soluble Ptprf ligand-binding domains neutralized Asprosin's glucogenic effects. These findings were validated in mammals: Asprosin binds PTPRF in mice and humans, and Ptprf knockout mice showed a blunted response to Asprosin. Our results establish Ptprf as an evolutionarily conserved Asprosin receptor across vertebrates, providing mechanistic insights for developing therapies targeting this pathway for diabetes and obesity.

Here, we present a protocol for conditional mutagenesis in zebrafish germ cells using Tol2 transposon and a CRISPR-Cas9-based plasmid system. We describe steps for conditional mutagenesis plasmid construction, zebrafish embryo microinjection, and screening for green fluorescence in the heart. This protocol is simple to execute, time efficient, and multifunctional, enabling the disruption of genes in zebrafish germ cells to be conducted with ease. For complete details on the use and execution of this protocol, please refer to Hu et al.¹.