The significant sex-biased expression pattern of Sp-Wnt4 provides novel insights into the ovarian development of mud crab (Scylla Paramamosain)

The wingless-type MMTV integration site family member-4 (Wnt4), a member of the wingless-related integration site (Wnt) family, is widely accepted as a key regulator of ovarian development in mammals. In this study, a full-length cDNA of Wnt4 (designated as Sp-Wnt4) was cloned, characterized, and functionally studied in mud crab (Scylla paramamosain). The full-length cDNA of Sp-Wnt4 consists of 2659 bp with an open reading frame (ORF) encoding 359 amino acids, a 907 bp 5 '-UTR and a 672 bp 3 '-UTR. Sp-Wnt4 contains 25 cysteine (Cys) residues and three potential N-glycosylation sites. Sp-Wnt4 protein shared the highest identity (98.9%) to the Wnt4 protein of Portunus trituberculatus. The phylogenetic tree showed that Sp-Wnt4 and Wnt4 protein of Malacostracan crustaceans clustered together, indicating that they had a close genetic distance. Sp-Wnt4 was expressed at a higher level in the ovary compared to other tissues, with the highest expression level at the third stage (O-III) of the ovarian development (P < 0.05). A downward trend was observed in the expression level of Sp-Wnt4 from the embryo stage to crablet stages (P < 0.05). After unilateral eyestalk ablation, the expression level of Sp-Wnt4 significantly increased in testis (14-fold) and downregulated (3.1-fold) in the gill (P < 0.05) of females. In situ hybridization (ISH) assay revealed that Sp-Wnt4 transcripts were mainly localized in the cytoplasm of oocyte cells. These findings showed that Sp-Wnt4 play crucial roles in the ovarian development of S. paramamosain. In conclusion, our study provides novel insights into the evolution and roles of the Wnt4 gene. (c) 2021 Elsevier B.V. All rights reserved.

Automated summary

In this study, the wingless-type MMTV integration site family member-4 (Wnt4) gene was cloned and functionally studied in mud crab (Scylla paramamosain). The expression of Sp-Wnt4 was found to play crucial roles in ovarian development, with differential expression levels in different tissues and developmental stages. Unilateral eyestalk ablation also affected the expression of Sp-Wnt4 in testis and gill of female crabs. In situ hybridization assay revealed the localization of Sp-Wnt4 transcripts in the cytoplasm of oocyte cells.