| Chinese mitten crab, Eriocheir sinensis, belongs to Crustacea, Decapoda, Eriocheir. It is an important kind of crab with high economical and nutritional value. It provides a good model for researching spermiogenesis especially for Decapoda. E.sinensis has high value to be researched in fields like developmental biology, evolutionary biology and reproductive biology. The current situation of cultivation in E.sinensis witnesses the increase in production as well as the decline in quality and promiscuity in species. To solve this problem, pivotal is to pay attention on the reproductive research of E.sinensis. The sperm in E.sinensis is special to other species as for its round spermatids, special structure with no flagellum and manchette. During spermatogenesis of E.sinensis, round spermatids undergo morphological, structural, and functional changes to become mature spermatozoa. The spermatogonium has an oval shape, in which mitochondria and endoplasm reticulum vesicles concentrate on one end of the cytoplasm to form a mitochondria zone. The spermatocyte is of polygonal shape, which contains a centriole appendage formed by the chromatoid body’s surrounding the centriole. In the early spermatid, many small dense particulates, proacrosomal granules which are produced by rough endoplasmic reticulum vesicles, aggregate to form a large proacrosomal vesicle. So at the late stage, the nucler transforms into a cup-like shape and surrounds the proacrosomal vesicle. Between the nuclear and the proacrosomal vesicle, it appears a layer of membrane complex. In the maturing phase, the proacrosomal vesicle differentiates into acrosome which is composed of apical cap, acrosomal tubule, fibrous layer, middle layer and some lamellar structure. Then in the mature sperm, the acrosome is surrounded by the nuclear cup with some radial arms.The cytoskeletons network in eukaryotic cells contain microtubule, microfilament, and intermediate filament. Multiple cellular events are tied to the cytoskeletons network. Kinesin, a group of highly related molecular motors, can transport various cargoes along microtubules through ATP hydrolysis. Many physiological regulations is related with kinesin. Kinesin superfamily proteins play an important part in intracellular transport and the physiological adjustment. KIF3A/3B, an N-terminal kinesin, can move along the microtubules with important roles in intraflagellar transport, intramanchette transport and the transport of other materials. So in the spermatogenesis of E.sinensis we can find the sperm shaping machinery, the exchange between nucleus and the cytoplasm, and the fertilization dependent on KIF3A and KIF3B.The cDNA of KIF3A and KIF3B were obtained by designing the degenerate primer,3’RACE, and5’RACE. 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, the protein presence of KIF3A in heart, muscle, gill, and testis reflects the content in protein level. Using situ hybridization and Immunofluorescence, we can track the dynamic location of KIF3A and KIF3B during different developmental phases of sperm. KIF3A and KIF3B expressed gradually around the nucleus in early spermatids. In intermediate spermatids, they expressed also around the nucleus at a high level and extended to the final phase. In the final spermatids with drastically reorganizing and nucleus shaping, KIF3A and KIF3B reached the maximum and located around the nucleus and concentrated in the acrosome to some extent. The results reveal the phenomenon that KIF3A and KIF3B are involved in the nuclear and cellular morphogenesis in the level of mRNA and protein. They can lead the intracellular transport organelles, proteins, and other cargoes. The results represent the functions of KIF3A and KIF3B in spermatogenesis in crustacean and clarified the connection between KIF3A and KIF3B. |
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