Abstract
Recently, the crustacean female sex hormone (CFSH), which is considered a female-specific hormone, has been shown to play a crucial role in female phenotypes in crustaceans. In this study, two transcripts (Sp-CFSH1 and Sp-CFSH2) encoding the same CFSH precursor were cloned from the mud crab Scylla paramamosain. Homology and phylogenetic analysis showed that CFSHs were homologous to interleukin-17 and highly conserved among brachyuran crabs. PCR analysis revealed that Sp-CFSH was expressed exclusively in the eyestalk ganglion of both prepubertal males and females, and surprisingly, the abundance of Sp-CFSH transcripts detected in the males were not significantly different from that of the females (P > 0.05). In addition, mRNA in situ hybridization showed that Sp-CFSH was localized in the X-organ of the male eyestalk ganglion. During the development of the androgenic gland (AG), the level of Sp-IAG mRNA in AG remained at low levels from stages I to II (early stage) but had a significant increase at stage III (mature stage). In contrast, the level of Sp-CFSH transcripts in the eyestalk ganglion was high in the early stage but extremely low in the mature stage. To investigate the potential function of CFSH in male S. paramamosain, the recombinant protein (∼20 kDa) was expressed in Escherichia coli and was subsequently added to AG explants in vitro. It was demonstrated that recombinant Sp-CFSH protein significantly reduced the expression of Sp-IAG in the AG explants at a concentration of 10−6 M (P < 0.05). In conclusion, our study provides the first piece of evidence that shows CFSH from the eyestalk ganglion acts as a negative regulator inhibiting the development of AG in crustaceans.
Highlights
In the animal kingdom, most species have separate genders, which often can be distinguished by different sexual characteristics relating to male and female in gonads, the genital tract, or the external genitalia (Nagaraju, 2011)
It was found that the nucleotide sequences of Sp-CFSH1 (1032 bp) and Sp-CFSH2 (916 bp) were identical in 5 -UTR and open reading frame (ORF) but different in 3 -UTR (Figure 1A), which could be translated into a same hormone (223-aa)
The recent discovery that crustacean female sex hormone (CFSH) was responsible for regulating the development of female phenotypes in the blue crab C. sapidus is a major breakthrough (Zmora and Chung, 2013)
Summary
Most species have separate genders, which often can be distinguished by different sexual characteristics relating to male and female in gonads, the genital tract, or the external genitalia (Nagaraju, 2011). Male sex differentiation is largely controlled by the androgenic gland (AG), an organ unique to males (Charniaux-Cotton, 1992). The function of this gland was first reported in the amphipod Orchestia gammarellus (CharniauxCotton, 1954). By removing AG or by the silencing of IAG gene from males, as well as by AG implantation or injection of AG extracts into females, the influence of AG/IAG on primary and secondary sex characteristics has been verified in various crustacean species (reviewed by Khalaila et al, 2001; Sagi and Khalaila, 2001; Barki et al, 2003; Manor et al, 2004; Rosen et al, 2010; Ventura et al, 2011b). It has been shown that the expression of Mr-IAG could be detected at the postlarval stage; during the subsequent development, the pubertal males still required IAG to develop into reproductivecompetent adult males (Ventura et al, 2011a)
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