A novel insulin-like growth factor (igf3), which is exclusively expressed in the gonads, has been widely identified in fish species. Recent studies have indicated that Igf3 regulates spermatogonia proliferation and differentiation in zebrafish; however, detailed information on the role of this Igf needs further in vivo investigation. Here, using Nile tilapia (Oreochromis niloticus) as an animal model, we report that igf3 is required for spermatogenesis and reproduction. Knockout of igf3 by CRISPR/Cas9 severely inhibited spermatogonial proliferation and differentiation at 90 days after hatching, the time critical for meiosis initiation, and resulted in less spermatocytes in the mutants. Although spermatogenesis continued to occur later, more spermatocytes and less spermatids were observed in the igf3^−/− testes when compared with wild type of testes at adults, indicating that Igf3 regulates spermatocyte to spermatid transition. Importantly, a significantly increased occurrence of apoptosis in spermatids was observed after loss of Igf3. Therefore, igf3^−/− males were subfertile with drastically reduced semen volume and sperm count. Conversely, the overexpression of Igf3 in XY tilapia enhanced spermatogenesis leading to more spermatids and sperm count. Transcriptomic analysis revealed that the absence of Igf3 resulted in dysregulation of many genes involved in cell cycle, meiosis and pluripotency regulators that are critical for spermatogenesis. In addition, in vitro gonadal culture with 17α-methyltetosterone (MT) and 11-ketotestosterone (11-KT) administration and in vivo knockout of cyp11c1 demonstrated that igf3 expression is regulated by androgens, suggesting that Igf3 acts downstream of androgens in fish spermatogenesis. Notably, the igf3 knockout did not affect body growth, indicating that this Igf specifically functions in reproduction. Taken together, our data provide genetic evidence for fish igf3 in the regulation of reproductive capacity by controlling spermatogenesis.

一种新型胰岛素样生长因子（ igf3 ）仅在性腺中表达，已在鱼类中广泛发现。最近的研究表明，Igf3 调节斑马鱼精原细胞的增殖和分化。然而，有关该 Igf 作用的详细信息需要进一步的体内研究。在这里，我们使用尼罗罗非鱼（ Oreochromis niloticus ）作为动物模型，报告说igf3是精子发生和繁殖所必需的。通过 CRISPR/Cas9 敲除igf3会严重抑制孵化后 90 天（减数分裂开始的关键时间）的精原细胞增殖和分化，并导致突变体中精母细胞减少。尽管精子发生继续发生，但与成人野生型睾丸相比，在igf3 ^-/−睾丸中观察到更多的精母细胞和更少的精细胞，表明 Igf3 调节精母细胞到精子细胞的转变。重要的是，在 Igf3 缺失后观察到精子细胞凋亡的发生率显着增加。因此， igf3 ^-/−雄性生育力低下，精液量和精子数量急剧减少。相反，XY 罗非鱼中 Igf3 的过度表达增强了精子发生，导致更多的精子细胞和精子数量。转录组分析显示，Igf3 的缺失导致许多参与细胞周期、减数分裂和多能性调节因子的基因失调，而这些基因对精子发生至关重要。 此外，使用17α-甲基睾酮（MT）和11-酮睾酮（11-KT）进行体外性腺培养以及体内敲除cyp11c1证明igf3表达受雄激素调节，表明Igf3在鱼类精子发生中作用于雄激素下游。值得注意的是， igf3敲除并不影响身体生长，这表明该 Igf 在生殖中具有特殊功能。总而言之，我们的数据为鱼类igf3通过控制精子发生来调节生殖能力提供了遗传证据。