Functional Analysis of KIF3A and KIF3B during Spermiogenesis of Chinese Mitten Crab Eriocheir sinensis

et al. (2014) Functional Analysis of KIF3A and KIF3B during Spermiogenesis of Chinese Mitten Crab Eriocheir sinensis. PLoS ONE 9(5): e97645. doi:10.1371/journal.pone.0097645 Functional Analysis of KIF3A and KIF3B during Spermiogenesis of Chinese Mitten Crab Eriocheir sinensis Yang Lu 0 Qi Wang 0 Da-Hui Wang 0 Hong Zhou 0 Yan-Jun Hu 0 Wan-Xi Yang 0 Sue Cotterill, St. Georges University of London, United Kingdom 0 1 The Sperm Laboratory, College of Life Sciences, Zhejiang University , Hangzhou , China , 2 Department of Reproductive Endocrinology, Women's Hospital, School of Medicine, Zhejiang University , Hangzhou , China Background: Spermatogenesis represents the transformation process at the level of cellular development. KIF3A and KIF3B are believed to play some roles in the assembly and maintenance of flagella, intracellular transport of materials including organelles and proteins, and other unknown functions during this process. During spermatogenesis in Eriocheir sinensis, if the sperm shaping machinery is dependent on KIF3A and KIF3B remains unknown. Methodology/Principal Findings: The cDNA of KIF3A and KIF3B were obtained by designing degenerate primers, 39RACE, and 59RACE. We detected the genetic presence of kif3a and kif3b in the heart, muscle, liver, gill, and testis of E. sinensis through RT-PCR. By western blot analysis, the protein presence of KIF3A and KIF3B in heart, muscle, gill, and testis reflected the content in protein level. Using in situ hybridization and immunofluorescence, we could track the dynamic location of KIF3A and KIF3B during different developmental phases of sperm. KIF3A and KIF3B were found surrounding the nucleus in early spermatids. In intermediate spermatids, these proteins expressed at high levels around the nucleus and extended to the final phase. During the nuclear shaping period, KIF3A and KIF3B reached their maximum in the late spermatids and were located around the nucleus and concentrated in the acrosome to some extent. Conclusions/Significance: Our results revealed that KIF3A and KIF3B were involved in the nuclear and cellular morphogenesis at the levels of mRNA and protein. These proteins can potentially facilitate the intracellular transport of organelles, proteins, and other cargoes. The results represent the functions of KIF3A and KIF3B in the spermatogenesis of Crustacea and clarify phylogenetic relationships among the Decapoda. - Spermiogenesis represents one of the most complicated morphological transformation procedures and is divided into three main phases along with proliferation and differentiation from diploid spermatogonia to haploid spermatozoa [1,6,12,14,17]. Firstly, mitosis induces the spermatogonia to develop into two identical primary spermatocytes. Then through meiosis each primary spermatocyte develops into the secondary spermatocyte [11,12,16]. Subsequently, the spermatids develop into mature spermatozoa with nucleus elongation and condensation, the formation of a mid-piece, the remodeling of the acrosome, and the reorganization of the intracellular organelle [1,8,14]. But the mature sperm of various species may differ from each other. Eriocheir sinensis, also called Chinese mitten crab, is a species of the class Crustacea, Decapoda, Brachyura [1,2]. Compared to other species, the spermatozoon of E. sinensis is of very peculiar shape. The nucleus of the spermatozoon looks just like a cap that surrounds the oval acrosome [1,7]. The acrosome consists of the acrosomal tubule, the apical cap (AC), and the acrosomal vesicle [7]. The mature spermatozoon has no flagellar tail that is mainly responsible for the movement of the spermatozoa of other species. However, there are about 20 radial arms extruding from the outside of the cup-shaped nucleus. Whether the content of radial arms belongs to the microfilament or the microtubule is still a matter of debate. E. sinensis is a commercially important seafood crab due to its delicious taste and rich nutrition. Studies of spermatogenesis in E. sinensis are essential for the maintenance and improvement of reproduction in general. E. sinensis has been used as a suitable model for general studies of spermatogenesis [1,7]. The morphological variations of the nucleus during spermatogenesis are regarded as evidences for analyzing evolution of Decapoda, and the phylogenetic status of this species can be inferred as well [10,11]. However, the specific molecular functions of the motor proteins are as yet less known than the morphology [9]. An example is given by the gap junctions between cells that improve spermatogenesis, but the molecular mechanisms are still far from being fully understood [9]. Most striking morphological transformations occur with the microtubules (MTs), microfilaments, and the motor proteins associating with them. The transport and sorting of cargoes in tail and manchette, the specific structure surrounding the nucleus of the spermatid, are all dependent on the kinesin superfamily of proteins (KIFs) [13,17]. KIFs can make use of ATP hydrolysis to produce energy for transporting a series of organelles, protein complexes, and vesicles [18]. Kinesin-2 motors are mainly heterotrimeric proteins consisting of two different motor subunits and one accessory subunit [19]. The motor subunit is composed of the N-terminal domain, a rod domain, and the C-terminal globular domain [17,20,21]. KIF3A can assemble with KIF3B or KIF3C, while KIF3B can not assemble with KIF3C [22]. The heterodimer and the accessory subunit, KAP3, can combine as a heterotrimeric motor protein, KIF3. KAP3 has the location to associate with cargoes through small G proteins, whereas KIF3A/ KIF3B bond to the MTs [23]. The homolog of KIF3 in sea urchin has been reported to be a heterotrimer composed of SpKRP85, SpKRP95 and SpKAP115 [2426]. KIF3 is responsible for the formation and elongation of cilia along with the central pair of MTs [19]. OSM-3 functions in the anterograde transport of cargoes that can mediate sensory ciliary growth in sensory neurons and inner labial neurons [27]. It is speculated that KIF3B promotes the aggregation of mitochondria with the formation of IFT and IMT [28]. There is evidence showing that the link between kinesin-2 and IFT depends on IFT20 and KIF3B. IFT20 connects directly with IFT57 of the IFT complex consisting of IFT57, IFT88, and IFT52 [29]. Mice with kif3a knockdown are lethal to embryos before any differential organogenesis takes place [30,31]. The abnormal primary cilia caused by kif3a deficiency in mice develop to polycystic kidney disease (PKD), the rapid formation of kidney cysts, and false planar cell polarity [30,31]. KIF3A associates with b-catenin to mediate the epithelia from some reports. NEK1 is another protein related to the formation of kidney cysts and PKD. It is the first time to report that KIF3A is the interacting protein partner of NEK1 during the genesis of PKD [32,33]. KIF3A, therefore, links the development of cilia to regulate t (...truncated)