Role of the steroid hormone synthesis pathway-related gene StAR-like in gonadal development of mud crab (Scylla paramamosain).

In vertebrates, the canonical steroidogenic acute regulatory (StAR) proteins, which lack N-terminal MENTAL domains, transport cholesterol into mitochondria to initiate steroidogenesis. However, whether a vertebrate-like steroidogenic pathway exists in crustaceans remains controversial. In this study, the complete ORF of SpStAR-like was identified from the transcriptome data of the mud crab Scylla paramamosain and subsequently cloned from testicular cDNA. Bioinformatic analysis showed that the SpStAR-like protein, consistent with other crustacean StAR-like homologs, contains an extra N-terminal MENTAL domain and exhibits higher structural homology with StAR-related lipid transfer proteins (StARLTPs) than with canonical vertebrate StAR proteins. qRT-PCR analysis revealed ubiquitous SpStAR-like expression across all mud crab tissues, with transcript levels in testes significantly higher than those in ovaries and other tissues. The gene was expressed throughout gonadal development in both sexes, peaking at testicular stage T2 and ovarian stage O3, respectively. Functional investigations were performed via RNA interference as well as in vitro and in vivo cholesterol supplementation assays. SpStAR-like knockdown significantly repressed the transcription of steroidogenic genes (Hsd3b, Hsd17b, PGMRC1, ERR, GPCR89) and gonadal development-related genes (IAG, vtg, vtgR, CYP3A24, foxl-2). Exogenous cholesterol treatment elevated ovarian pregnenolone concentrations, as determined by ELISA, and up-regulated all aforementioned target genes. In vivo cholesterol injection increased hemolymph pregnenolone levels and promoted ovarian morphological maturation. This study provides preliminary molecular evidence for steroidogenic regulatory cascades in mud crabs and advances our understanding of crustacean reproductive endocrinology.