Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
Characterization of PHB in the gonadal development of the swimming crab Portunus trituberculatus
Graphical abstract
Introduction
Prohibitin (PHB) is an evolutionarily highly conserved protein found in the plasma membrane, nucleus, and mainly in the inner membranes of mitochondria (Ande and Mishra, 2010; Snedden and Fromm, 1997; Sun et al., 2004). It was first identified by its anti-proliferation function in mouse liver (Mcclung et al., 1989), and later reported in human (Sato et al., 1992), plant (Ahn et al., 2006), yeast (Mcclung et al., 1995) and Drosophila (Eveleth and Marsh, 1986). Two highly homologous PHB proteins, PHB1 (32kD) and PHB2 (34kD), were characterized by their prohibitin-like domains (PHB domains) (Mishra et al., 2006). PHB1 has been reported to act as a tumor suppressor in normal human fibroblasts (Jupe et al., 1996) and PHB2 has been identified as a binding partner for the IgM isotype of the B-cell receptor in plasma membrane (Terashima et al., 1994). Furthermore, PHB1 and PHB2 are known to form heterodimers as subunits of the mitochondrial PHB complex (Back et al., 2002).
PHB has been demonstrated to be important for reproduction in various species, including rat (Thompson et al., 1999), yeast (Piper et al., 2003) and Caenorhabditis elegans (Sanz et al., 2003). PHB was shown to regulate human sperm motility by increasing low mitochondrial membrane potentials (MMP) and lipid peroxidation (Chai et al., 2017). In Charybdis japonica, PHB was found to participate actively in spermatogenic cell anti-apoptosis, cell nucleus distortion and acrosome morphogenesis by maintaining mitochondrial functions (Xu et al., 2017). Similar phenomenon have been observed in the Chinese mitten crab Eriocheir sinensis (Mao et al., 2012) and the crayfish Procambarus clarkia (Dong et al., 2015). The ubiquitinated PHB and its function in spermatogenesis have been investigated in various species, such as rat (Thompson et al., 2003), P. clarkia (Dong et al., 2015), E. sinensis (Mao et al., 2012), and C. japonica (Xu et al., 2017). These studies indicated that PHB and its ubiquitination may mediate mitochondrial function and nucleus distortion during spermatogenesis as an inner membrane protein. However, there are few studies considering PHB and its ubiquitination in the ovary. PHB was involved in the proliferation and differentiation of granulosa cells during oogenesis of the pig and rat (Thompson et al., 2004). In addition, in the oocytes of mouse and pig, the proteolysis of securin, which was dependent on the ubiquitin proteasome pathway (UPP), was necessary during meiosis (Huo et al., 2006). At present, PHB and its ubiquitination in crustacean ovaries have not been reported.
The Swimming crab Portunus trituberculatus is an important marine economic species widely distributed from Japan, Korea to China (Hamasaki et al., 2006). With the advancement of artificial breeding techniques, the P. trituberculatus aquaculture industry have rapidly expanded. Although studies of PHBs have been carried out in many other crustaceans, the characterization of PHB and its ubiquitination during the spermatogenesis and oogenesis in P. trituberculatus remain unknown. We aim to explore whether the characterization of PHB in spermatogenesis is similar to that of other crustaceans and provide new information of PHB in crustacean ovaries.
Accordingly, in this study, the PHB gene was cloned and the expression level of phb mRNA in P. trituberculatus larvae and different tissues (including the testis and ovaries at different developmental stages) were investigated by qRT-PCR. The expression level of PHB protein in the testis, ovaries and other tissues were analyzed by Western blot and enzyme-linked immunosorbent assay (ELISA). Furthermore, in order to investigate the characterization of PHB and its ubiquitination in the gonadal development of P. trituberculatus, the mitochondria membrane protein identity and the ubiquitination of PHB in the testis and ovaries were clarified by immunofluorescence (IF) staining.
Section snippets
Animals and tissues preparation
Larvae and adult crabs were purchased from an aquaculture company. Individual live male and female crab (gonad development in stage V) were dissected on ice. Tissues of testis, ovary, hepatopancreas, heart, intestine, gill and muscle were dissected and immediately stored at −80 °C for RNA and protein extraction. The testis and ovaries at different developmental stages were sampled at regular intervals (every other month commencing in July). Six crabs were taken for each type of tissue. The
Molecular characterization of PtPHB
The nucleotide and corresponding amino acid sequence was shown in Fig. 1. The ORF encodes 275 amino acids, including a signal peptide of 26 amino acids. The predicted theoretical pI and molecular weight of PHB are 8.23 and 18.4 kDa respectively. SMART program analysis revealed a PHB domain ranging from Ala29 to Gln190. The amino acid sequence alignment demonstrated the similarity of P. trituberculatus PHB with that of other species (Fig. 2). PtPHB shared high similarity with that from Scylla
Discussion
PHB was expressed in cells that depend heavily on mitochondrial function, such as muscle, heart, liver, brown adipocytes and pancreatic islet cells (Coates et al., 2001). Tissues distribution of PtPHB protein and phb mRNA were similar to that on Charybdis japonica (Xu et al., 2017), Eriocheir sinensis (Hou et al., 2017) and Procambarus clarkia (Dong et al., 2015), which indicated that these tissues were often especially susceptible to mitochondrial dysfunction (Wallace, 2000). Meanwhile, PHB
Conclusion
In this study, the functions of PHB during spermatogenesis and oogenesis in P. trituberculatus were investigated. The results indicated that PtPHB might play important roles during spermatogenesis and oogenesis by regulating mitochondrial activities. Though this study was the first time to reveal the characterization of PHB and ubiquitin during oogenesis of crustacean, the specific mechanism of how PHB and ubiquitin play their roles in oogenesis is not clear yet. Further investigations are
Declaration of Competing Interest
We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
Acknowledgements
The authors wish to express their gratitude to all the members of the lab for their technical advice and helpful discussions. This work was supported by the National Natural Science Foundation of China (41676140), Major Agriculture Program of Ningbo (2017C110007), Modern Technology System Of Agricultural Industry (CARS-46), the Scientific Research Foundation of Graduate School of Ningbo University, and the K C Wong Magana Fund in Ningbo University.
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