miR‐34 regulates reproduction by inhibiting the expression of MIH, CHH, EcR, and FAMeT genes in mud crab Scylla paramamosain

Abstract

Mud crab Scylla paramamosain is a commercially important species widely cultured in China. It is well known that the eyestalk regulates reproductive activities in crustaceans. In our previous research, we found that the miR‐34 expression level in male eyestalk was significantly higher than that in females. Thus, we assumed that it may play an important role in regulating reproduction. In this study, we used bioinformatic tools to identify the target genes of miR‐34 in eyestalk. Six reproduction‐related genes with an intact 3′‐untranslated region (UTR), including molt‐inhibiting hormone (MIH), crustacean hyperglycemic hormone (CHH), vitellogenesis‐inhibiting hormone, red pigment concentrating hormone, ecdysone receptor (EcR), and farnesoic acid methyltransferase (FAMeT) were identified. When the 3′‐UTR plasmid vectors of the six genes were cotransfected with miR‐34 mimics into 293FT cells, respectively, the luciferase activities of four genes (MIH, CHH, EcR, and FAMeT) were significantly decreased compared with that in the control group; on the contrary, when the six plasmid vectors were cotransfected with the miR‐34 inhibitor respectively, the luciferase activities of four genes (MIH, CHH, EcR, and FAMeT) were significantly higher than that in the control group. When agomiR‐34 and antagomiR‐34 were injected into the eyestalk respectively in vivo, the expression levels of the MIH, CHH, EcR, and FAMeT genes were detected by a quantitative real‐time polymerase chain reaction. The results showed that agomiR‐34 suppressed the expression of the four genes, whereas antagomiR‐34 enhanced their expression. These experimental results confirmed our hypothesis that miR‐34 may indirectly regulate reproduction via binding to the 3′‐UTRs of MIH, CHH, EcR, and FAMeT genes and suppressing their expression.

The Regulatory Mechanism of Sexual Development in Decapod Crustaceans

“…Localization studies indicated that FAMeT is co-localized in the neurosecretory cells that produce CHH and MIH. Higher levels of FAMeT mRNA transcript and protein levels were reported along with higher levels of CHH and GIH (Zhou et al, 2019). MF biosynthesis is regulated by secreted neuropeptides from the X-organ which regulates the activity of FAMeT and thereby vitellogenesis (Gunawardene et al, 2002).…”

“…The major source of these neuropeptides is the X-organ located in the eyestalk ganglia. These neuropeptides involved in carbohydrate and lipid metabolism, moulting, and ovarian development (Böcking et al, 2002;Zhou et al, 2019). The RPCH is not only involved in the regulation of pigmentation but also the regulation of ovarian maturation in crustaceans (Fingerman, 1997).…”

“…miRNAs are also essential for ovulation and oocyte maturation in invertebrates (Kadener et al, 2009). In decapods, several sexrelated miRNAs have been identified in S. paramamosain (Jia et al, 2018;Waiho et al, 2019a;Zhou et al, 2019Zhou et al, , 2020, Portunus trituberculatus (Meng et al, 2018(Meng et al, , 2020, E. sinensis (Song et al, 2014;He et al, 2015), M. rosenbergii (Liu X. et al, 2019;Qiao et al, 2019) M. nipponense (Sb et al, 2015;Jin et al, 2017), P. monodon (Zhao et al, 2018), and Fenneropenaeus merguiensis (Saetan and Chotigeat, 2019). The roles of sex-related miRNAs in decapods are summarized in Table 3.…”

“…Transcriptomic analysis revealed that miRNAs may affect the sexual development of decapods through several pathways. The most important predicted pathways are ERK, MAPK, necroptosis pathway, GnRH signaling pathway, Wnt signaling pathway, ovarian steroidogenesis, TGF-beta signalling pathway, gluconeogenesis pathway, glycolysis pathway, estrogen signaling pathway, ovarian steroidogenesis, and insulin signaling pathway (He et al, 2015;Jia et al, 2018;Meng et al, 2018;Zhao et al, 2018;Liu X. et al, 2019;Zhou et al, 2019).…”

“…In males, miRNAs such as miR-17-3p, let-7c, and miR-21-5p demonstrated different expression levels during different stages of testicular development indicating their probable involvement in spermatogenesis (He et al, 2015). MiR-34 probably regulates the reproductive function via binding to the 3 -UTRs of MIH, EcR, FAMeT, and CHH genes (Zhou et al, 2019).…”