High temperature causes masculinization of genetically female olive flounder (Paralichthys olivaceus) accompanied by primordial germ cell proliferation detention

Sex determination is not only resort to a single genetic cascade but also regulated along a continuum of environmental and heritable factors in many fish species. Temperature is one of the most important non-genetic sex differentiation elements. Previous studies regarding high temperature masculinization of flatfish typically focused on sex ratios and reproductive endocrinology, but little was known about germ cells during the process. In this study, we investigated the dynamic changes of germ cells in terms of morphology and number employing serial-sectioning and the molecular investigation of expression of dnd and amh genes by qRT-PCR for qualitative and quantitative analysis of germ cell proliferation during sexual differentiation. Moreover, the proliferation activity of primordial germ cells was investigated using PCNA (proliferating cell nuclear antigen) immunohistochemistry.Experimental results revealed that high temperatures caused juvenile olive flounder masculinization (male ratio, 95.24%, 27.5°C±0.5°C) in comparison with controls (male ratio 5.56%, 18°C±0.5°C). The proliferation pattern of germ cells presented sexual dimorphism, from 42 to 66days post-hatching (dph). The experimental group presented specifically slow proliferation and greatly reduced cell numbers. No cyst clusters were apparent in comparison to the control group. Consistently, the proliferation activity of germ cells in the experimental group was significantly reduced in comparison to that observed in the control group at 50dph by PCNA immunohistochemistry. Furthermore, the expression levels of poamh during the process of temperature-induced masculinization gradually increased and were significantly higher than that in the control group at the stage gonads have been considered still undifferentiated in particular from a histological point of view. Thus, elevated temperatures result in masculinization, poamh up-regulation, accompanied by germ cell detention proliferation.