Mvosin Va Participates In Acrosomal Formation And Nuclear Morphogenesis During Spermatogenesis Of Chinese Mitten Crab Eriocheir Sinensis

Spermatogenesis is a highly organized process which diploid spermatogonium develops into haploid mature spermatozoon. It can be divided into three phases:the mitotic phase, meiotic phase, and spermiogenesis phase. The Chinese mitten crab Eriocheir sinensis belongs to the Class Crustacea, Decapoda, Brachyura. The spermatozoon of this species is of aflagellated type, it has a spherical acrosome surrounded by the cup-shaped nucleus, which is unique to the brachyuran. Myosin V a, a member of Class Ⅴ myosin, has been implicated in acrosome biogenesis and vesicle transport during spermatogenesis in mammals. Given the disparity of spermatozoon, we determine to study the role of myosin V a during E. sinensis spermatogenesis.We have cloned myosin Va cDNA sequence using degenerated PCR and rapid amplification of cDNA end (RACE). Analysis of the sequence revealed that myosin Va cDNA contains6046bp with an open reading frame containing5331bp. It encodes a protein of1776amino acids. Myosin V a contains the head domain, the neck domain and the tail domain. The tissue expression suggested that myosin Va mRNA is expressed in the testes, hepatopancreas, gill, heart and muscle. In situ hybridization result showed that abundant myosin Va mRNA concentrates in the nucleus at the spermatogonia and the spermatocytes. At the early spermatids, myosin Va mRNA distributed in the nucleus and the cytoplasm. At the mid and late spermatids, myosin Va mRNA predominates in the proacrosomal vesicle. myosin Va mRNA localizes in the acrosomal tubule. Real time PCR indicated that the relative expression level of myosin Va at the mitosis stage and the meiosis stage is higher than the spermiogenesis. Western blot demonstrated that myosin V a is expressed during spermatogenesis. Immunocytochemical and ultrastructural analyses showed that myosin V a mainly localizes in the cytoplasm in spermatocytes. At the early stage of spermiogenesis, myosin V a binds to the endoplasmic reticulum vesicle and proacrosomal granule. Subsequently, myosin Va localizes within the proacrosomal vesicle formed by endoplasmic reticulum vesicle and proacrosomal granule fusion and locates in the membrane complex at the mid spermatid stage. At the late spermatid stage, myosin Va is associated with the shaping nucleus and mitochondria. In mature spermatozoa, myosin V a predominates in acrosomal tubule and nucleus.Our study demonstrates that myosin Va transports the proacrosomal vesicle and endoplasmic reticulum vesicle, and then facilitates them to form the proacrosomal vesicle. Subsequently, myosin Va may serve as an anchor that links the proacrosomal vesicle to the nucleus during nuclear shaping. Additionally, we also propose a hypothesis of acrosomal tubule formation in this species. It is the first time to uncover the role of myosin V a in crustacean spermatogenesis.