Yixiu Peng

Background: Chicken Primordial Germ Cells (PGCs) are one of the few germ cells that can be cultured for a long time in vitro, but challenges remain such as low culture efficiency and unclear roles of nutrient factors and signaling pathways. Method: In this study, protein kinase B (AKT) pathway activator insulin-like growth factor 1 (IGF-1) was screened for its ability to promote cell proliferation by transcriptome results using various inhibitors of pathway activation. The effects of IGF-1 on PGCs were evaluated through EdU assays, qRT-PCR, flow cytometry, and migration experiments. Results: This study systematically examined the effects of insulin and IGF-1 on the proliferation, cell cycle, ferroptosis, migration capacity, and establishment efficiency of PGCs. The findings demonstrated that IGF-1 exhibited comparable effects to insulin and could effectively replace insulin in PGC culture systems. Conclusions: The research results are expected to provide a solid theoretical basis for optimizing the chicken PGC cultivation system and promoting its practical application.

Insulin is an important component of stem cell cultures; however, its role in the proliferation of avian primordial germ cells (PGCs) is unknown. The proliferation of PGCs in cultures varies and the growth factors and signaling pathways necessary to induce the proliferation of PGCs in chickens are unknown. Therefore, we conducted the present study to investigate the effect of insulin on the survival and proliferation of PGCs. In this study, we observed that under this culture system, PGCs proliferate in the presence of insulin, but do not proliferate in the absence of insulin. Furthermore, in insulin-lacking media, the expression of pluripotency genes, including LIN28, NANOG, POUV, and SOX2, was markedly decreased. Similarly, the expression of cell adhesion proteins ZO-1, Occludin, and JAM-A was significantly reduced. Elevated levels of ROS, GSSG, and MDA reduced the redox capacity of the cells and induced apoptosis. Subsequent transcriptome analyses revealed that insulin is one of the key factors in the proliferation of chicken PGCs through the regulation of downstream genes by PI3K/AKT, ECM–receptor interaction, Wnt, and P53 signaling, and that these downstream genes may be important for PGCs’ proliferation and survival.

Aromatase, a crucial enzyme for estrogen synthesis, plays a vital role in gender determination and differentiation. This study aimed to establish an inducible knockout model of the chicken CYP19A1 gene, which encodes aromatase, to support gender control in chickens. We selected the most efficient sgRNA target site and constructed an inducible knockout model based on the Tet-on system. The knockout efficiency reached 80% with 20 μg/mL DOX induction in vitro. The encapsulation of the plasmid with PEI and injection into eggs achieved a knockout efficiency of 45% in ovo. qRT-PCR analysis revealed a significant downregulation of female-related genes (CYP19A1, FOXL2, ESR1) and upregulation of male-related genes (DMRT1, SOX9, AMH) in female chicken embryos after induction. Western blotting showed decreased protein expression of CYP19A1 and FOXL2, and increased SOX9 expression in female embryos post-DOX induction. Elisa detection further confirmed lower estradiol levels in the gonads of induced female embryos compared to normal and non-induced females. These findings demonstrate the successful establishment of an inducible knockout system for the CYP19A1 gene in chickens, providing theoretical and technical support for the creation of new breeding materials for gender control.