Transcriptional Inhibition of Sp-IAG by Crustacean Female Sex Hormone in the Mud Crab, Scylla paramamosain

Abstract

In crustaceans, the regulation of sex differentiation is mediated by insulin-like androgenic hormone (IAG) and crustacean female sex hormone (CFSH). CFSH is reported to inhibit IAG gene (Sp-IAG) expression in the mud crab Scylla paramamosain, but the regulatory mechanism is not well understood. A 2674 bp 5′ flanking Sp-IAG contains many potential transcription factor binding sites. In this study, analysis of serially deleted 5′ flanking Sp-IAG and site-directed mutation (SDM) of transcription factor binding sites of the same gene showed that the promoter activity of reporter vectors with Sox-5-binding site, signal transducers and activators of transcription (STAT)-binding site and activator protein 1 (AP-1)-binding site were significantly higher than that of vectors without these regions, suggesting that they were involved in transcriptional regulation of Sp-IAG expression. The expression analysis of these transcription factor showed that there was no difference in the level of mRNA in Sox-5 and AP-1 in androgenic gland treated with recombinant CFSH, but expression of Sp-STAT was significantly reduced, suggesting that CFSH regulates the expression of Sp-STAT, inhibiting its function to regulate Sp-IAG. Further experiment revealed that RNAi mediated Sp-STAT gene knockdown reduced the expression of Sp-IAG. These results suggested that Sp-CFSH regulates Sp-IAG by inhibiting STAT. This is a pioneering finding on the transcriptional mechanism of IAG gene in crustaceans.

The IAG-Switch and Further Transcriptomic Insights Into Sexual Differentiation of a Protandric Shrimp

“…Even in isopods, in which an elevated bacterial dosage of Wolbachia resulted in feminization, it was postulated that Wolbachia feminized males through intervention in the IAGswitch by instigating dysfunctional insulin receptors (Herran et al, 2020). However, while few other genes were found to be associated with the expression of IAG [MIH, GIH, CHH, CFSH, DMRT, and GEM (Li et al, 2015;Jin et al, 2019;Zhong et al, 2019;Jiang et al, 2020)], genes (other than the IAG itself) that act directly within the core of the IAG-switch cascade, have yet to be found.…”

The Regulatory Mechanism of Sexual Development in Decapod Crustaceans

“…CFSH from the eyestalk is a negative regulator and inhibits AG development in decapods. Jiang et al (2020b) found no difference in the expression level of Sox-5 and AP-1 in AG exposed to rCFSH, however, STAT significantly declined, indicating that CFSH controls the expression of STAT, inhibiting its function to control IAG in the mud crab. Further experiment showed that RNAi mediated STAT gene knockdown lowered IAG expression indicating that CFSH regulates IAG by inhibiting STAT.…”

“…Further experiment showed that RNAi mediated STAT gene knockdown lowered IAG expression indicating that CFSH regulates IAG by inhibiting STAT. In a study by Jiang et al (2020b), promoter activity was significantly increased in pGL3-pSp-IAG1 (Sox-5binding site) and pGL3-pSp-IAG6 (Sox-5-binding site) than in previous plasmids (pGL3-basic and pGL3-pSp-IAG5), indicating that the Sox-5-binding site is probably involved in enhancing this activity, consistent with site-directed mutation (SDM) results of decreased promoter activity in pGL3-pSp-IAG6-Mut (Jiang et al, 2020b).…”

“…The Sox (Sry-related high-mobility group (HMG) box) family of transcription factors is extensively distributed in different animal groups, and plays crucial roles in development processes including cell pluripotency, neurogenesis, and sex determination. To date, the Sox gene has been isolated from several decapods including Macrobrachium nipponense (Hu et al, 2020), Penaeus Ventura et al, 2014;Zmora and Chung, 2014;Veenstra, 2015;Nguyen et al, 2016;Toullec et al, 2017;Kotaka and Ohira, 2018;Liu C. et al, 2018;Jiang et al, 2020b Vitellogenin (Vg) and vitellogenin receptor (VgR) vannamei (Peng et al, 2015), Palaemon serratus (González-Castellano et al, 2019), E. sinensis (Liu et al, 2016), and S. paramamosain (Liao et al, 2020;Lin et al, 2020). SoxE1 is involved in testis development in M. nipponense (Hu et al, 2020).…”

Sex Determination and Differentiation in Decapod and Cladoceran Crustaceans: An Overview of Endocrine Regulation

“…This discovery of CFSH has resulted in a major research trend for exploring its homologs from other decapod species. To date, eyestalk transcriptome and peptidome approaches have successfully identified CFSH orthologs in several other brachyuran crabs, such as the swimming crab Portunus trituberculatus [ 85 ], the Chinese mitten crab Eriocheir sinensis [ 86 ], the green shore crab C. maenas [ 87 ], and the mud crab Scylla paramamosain [ 88 , 89 , 90 ], as well as in the kuruma prawn M. japonicus [ 91 ], the Pacific white shrimp L. vannamei [ 86 ], the banana shrimp Fenneropenaeus merguiensis [ 92 ], the Antarctic shrimp Chorismus antarcticus [ 93 ], the Eastern rock lobster S. verreauxi [ 94 ], the giant freshwater prawn M. rosenbergii [ 86 , 95 , 96 ], the red swamp crayfish P. clarkii [ 85 ], and the Australian crayfish C. quadricarinatus [ 97 ]. Despite the growing amount of CFSH sequence information, little is known about its physiological functions.…”