Transport of cholesterol to ovary via the Srb selective uptake pathway supports Star-mediated steroidogenesis in swimming crab (Portunus trituberculatus)

Cholesterol, essential for steroidogenesis, is crucial in ovarian development. In crustaceans, scavenger receptor class B member (Srb) and steroidogenic acute regulatory protein (Star) may be key proteins in cholesterol transport and steroidogenesis, respectively. This study aimed to explore cholesterol's role in steroidogenesis and ovarian development in female swimming crabs through in vivo and in vitro experiments. The control diet (Ctrl, without added cholesterol) and the cholesterol diet (CH) were fed swimming crabs and sampled first (30 d) and second ovarian development (60 d). Srb and Star functions were assessed via knockdown and overexpression experiments. Biochemical indices, histology, immunofluorescence, gene expression and Western blot were used to evaluate cholesterol transport, steroidogenesis and ovarian development. HEK293T cells were used for immunofluorescence and co-immunoprecipitation to explore mechanisms. Cholesterol supplementation significantly increased steroid hormone and vitellogenin (VTG) concentrations, as well as mRNA and protein expression levels of cholesterol transport, steroidogenesis, and vtg/Vtg. After interference and overexpression of Srb, the results indicated that Srb maintained cholesterol homeostasis by regulating cholesterol efflux gene abcg1, but not through compensatory regulation of ldlr. Similar patterns were found in vitro, Srb-mediated cholesterol transport promoted steroid hormone production. Moreover, star knockdown reduced steroidogenesis and vtg/Vtg expression, which was reversed by Star overexpression. The findings verified the roles of Srb in cholesterol transport and Star in steroidogenesis, and revealed that cholesterol supplementation promoted cholesterol transport via the Srb-selective uptake pathway, and supported Star-mediated steroidogenesis, thereby promoting ovarian development in swimming crab.