DNA methylation is a prevalent epigenetic modification in vertebrates, and it has been shown to be involved the regulation of gene expression and embryo development. However, it remains unclear how DNA methylation regulates sexual development, especially in species without sex chromosomes. To determine this, we utilized zebrafish to investigate DNA methylation reprogramming during juvenile germ cell development and adult female-to-male sex transition. We reveal that primordial germ cells (PGCs) undergo significant DNA methylation reprogramming during germ cell development, and the methylome of PGCs is reset to an oocyte/ovary-like pattern at 9 days post fertilization (9 dpf). When DNA methyltransferase (DNMT) activity in juveniles was blocked after 9 dpf, the zebrafish developed into females. We also show that Tet3 is involved in PGC development. Notably, we find that DNA methylome reprogramming during adult zebrafish sex transition is similar to the reprogramming during the sex differentiation from 9 dpf PGCs to sperm. Furthermore, inhibiting DNMT activity can prevent the female-to-male sex transition, sug-gesting that methylation reprogramming is required for zebrafish sex transition. In summary, DNA methylation plays important roles in zebrafish germ cell development and sexual plasticity.

Objective To investigate the influence of interleukin-6(IL-6) on the expression and function of programmed death-1(PD-1) in patients with hepatocellular carcinoma (HCC) by measuring the plasma level of IL-6 and the expression of PD-1 in peripheral blood CD8 + T cells from HCC patients. Methods A total of 44 HCC patients who attended Shaanxi Provincial People's Hospital or The Second Affiliated Hospital of Air Force Medical University & Tangdu Hospital of Fourth Military Medical University from January to September 2019 were enrolled as HCC group, and 19 healthy controls, matched for age and sex, were enrolled as HC group. Peripheral blood was collected, and plasma and peripheral blood mononuclear cells were isolated to separate CD8 + T cells. ELISA was used to measure the plasma level of IL-6, and flow cytometry was used to measure the expression level of PD-1 in CD8 + T cells. The separated CD8 + T cells were stimulated with anti-IL-6 neutralizing antibody for 24 hours; CCK-8 assay was used to measure cell proliferation, ELISA was used to measure the levels of interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα) in supernatant, real-time PCR was used to measure the relative mRNA expression levels of perforin, granzyme B, and granulysin, and Western blot was used to measure the phosphorylation levels of STAT3 and Src. The t -test or the paired t -test was used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between groups. Results Compared with the HC group, the HCC group had a significant increase in the plasma level of IL-6 (99.67±20.92 pg/mL vs 81.05±16.76 pg/mL, t =3.427, P =0.001 1). There was no significant difference in the percentage of CD3 + CD8 + T cells between the HCC group and the HC group, while there was a significant increase in the percentage of PD-1 + CD8 + cells in HCC patients (3.79%±1.36% vs 2.20%±0.47%, t =5.335, P < 0.000 1). In the patients with HCC, although anti-IL-6 neutralizing antibody for inhibiting IL-6 in CD8 + T cells did not affect cell proliferation, it downregulated the expression of PD-1 (2.67%±0.91% vs 3.33%±1.12%, t =2.177, P =0.035) and increased the secretion of IFNγ (13.50±3.82 pg/mL vs 10.82±1.37 pg/mL, t =3.170, P =0.002 8), and there were also significant increases in the relative mRNA expression levels of perforin and granzyme B ( t =6.161 and 14.140, both P < 0.000 1) and a significant reduction in the level of phosphorylated STAT3 ( P < 0.000 1). Conclusion Anti-IL-6 neutralizing antibody can enhance the function of CD8 + T cells in HCC patients possibly by increasing the levels of perforin and granzyme B, improving the secretion of cytokines, and inhibiting the expression of PD-1.