Myosin Va participates in acrosomal formation and nuclear morphogenesis during spermatogenesis of Chinese mitten crab Eriocheir sinensis

Abstract

Background: 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 are unique to brachyurans. For the past several decades, studies on the spermatogenesis of the mitten crab mainly focus on the morphology. Compared with the extensive study of molecular mechanism of spermatogenesis in mammals, relatively less information is available in crustacean species. Myosin Va, a member of Class V myosin, has been implicated in acrosome biogenesis and vesicle transport during spermatogenesis in mammals. In the present study we demonstrate the expression and cellular localization of myosin Va during spermatogenesis in E. sinensis.

Methodology/principal findings: Western blot demonstrated that myosin Va is expressed during spermatogenesis. Immunocytochemical and ultrastructural analyses showed that myosin Va mainly localizes in the cytoplasm in spermatocytes. At the early stage of spermiogenesis, myosin Va binds to the endoplasmic reticulum vesicle (EV) and proacrosomal granule (PG). Subsequently, myosin Va localizes within the proacrosomal vesicle (PV) formed by PG and EV fusion and locates in the membrane complex (MC) at the mid spermatid stage. At the late spermatid stage, myosin Va is associated with the shaping nucleus and mitochondria. In mature spermatozoon, myosin Va predominates in acrosomal tubule (AT) and nucleus.

Conclusions/significance: Our study demonstrates that myosin Va may be involved in acrosome biogenesis and nuclear morphogenesis during spermatogenesis in E. sinensis. Considering the distribution and molecular characteristics of myosin Va, we also propose a hypothesis of AT formation in this species. It is the first time to uncover the role of myosin Va in crustacean spermatogenesis.

Figure 1. A model of spermiogenesis in Chinese mitten crab E. sinensis.

(A) At early stage proacrosomal granule (PG) and the endoplasmic reticulum vesicle (EV) distribute in the spermatid cytoplasm around the nucleus (N). PM indicates the plasma membrane. (B–C) At mid stage, proacrosomal granule (PG) and the endoplasmic reticulum vesicle (EV) aggregate into proacrosomal vesicle (PV) (B), subsequently, the nucleus (N) initiates to wrap up proacrosomal vesicle (PV) and the membrane complex (MC) emerges between proacrosomal vesicle (PV) and the nucleus (N) (C). The spermatid discards most of the cytoplasm (C). (D–F) At late stage, the proacrosomal vesicle (PV) invaginates to form the acrosomal tubule (AT) (D, E). The mature spermatozoon consists of acrosome with apical cap (AC), acrosomal tubule (AT) and three layers (fibrous layer FL, middle layer ML and lamellar structures LS), centriole (C) at the base of acrosomal tubule (AT), the nuclear cup with several radial arms (RA) and mitochondria (M) (F).

Figure 2. Myosin Va is expressed in the testis of E. sinensis.

Immunoblots of myosin Va in E. sinensis testis and brain extracts of ICR male mice probed with the anti-myosin Va polyclonal antibody. Brain extracts of mice known to contain myosin Va serve as positive control. A similar band was observed in extracts of E. sinensis testis. β-actin was used as a loading control. The molecular weight marker is shown at right.

Figure 3. Immunofluorescent localization of myosin Va in spermatocytes of E. sinensis.

Actin (green) is visualized with FITC conjugated monoclonal anti-actin, myosin Va (red), and DNA with DAPI (blue). Myosin Va mainly localizes in the cytoplasm (arrows in B, D) in spermatocytes; actin mainly localizes in the nucleus, while the cytoplasm presents weak diffuse staining. The chromatin granules dispersed in the nucleus can be seen (C).

Figure 4. The localization of myosin Va at the early stage during E. sinensis spermiogenesis.

(A–D) Immunofluorescent localization of myosin Va in early spermatids. The cytoplasm presents myosin Va staining (arrows in B, D), the chromatin lumps attach to the nuclear membrane (C). (e) Ultrastructural localization of myosin Va. Myosin Va binds to the endoplasmic reticulum vesicle (EV) membrane (arrow) and proacrosomal granule (PG) (arrowhead). The proacrosomal granule (PG) is characterized by high density while the endoplasmic reticulum vesicle (EV) has low density.

Figure 5. Localization of myosin Va at the mid stage during E. sinensis spermiogenesis.

(A–D) Immunofluorescent localization of myosin Va in early mid stage. Myosin Va staining can be seen at the proacrosomal vesicle (PV) (arrows in B, D). Scale bar = 10 µm. (E–F) Immunogold labeling of myosin Va is present in the membrane complex (MC) and the proacrosomal vesicle (PV) membrane at late mid stage (arrows in E, F). E shows longitudinal section and F shows cross section of mid stage spermatid.

Figure 6. Localization of myosin Va at the beginning of late stage during E. sinensis spermiogenesis.

(A–D) Immunofluorescent localization of myosin Va. Myosin Va concentrates in the nucleus (arrows in B and D). (E–F) Immunogold labeling of myosin Va. Prominent myosin Va labeling is present in the nucleus (N), and some associates with the nuclear membrane and the membrane complex (MC) (arrows in E, F), as well as mitochondria (M) (arrowhead in E). E. longitudinal section and F. cross section of the initiation of late stage.

Figure 7. Localization of myosin Va at the late stage during E. sinensis spermiogenesis.

(A–D) Immunofluorescent localization of myosin Va. Myosin Va distributes in the nucleus (N) (arrowhead in B,D) and acrosomal tubule (AT) (arrow in B,D), actin staining is present in the nucleus (N) (arrowhead in A,D) and acrosomal tubule (AT) (arrow in A,D), myosin Va colocalizes with actin (D). (E–G) Immunoelectron microscopy analyses of myosin Va. (E) When the acrosomal tubule (AT) begin to form, myosin Va associates with the nuclear membrane and the membrane complex (MC) (arrow in E). (F) After the acrosomal tubule (AT) formation, myosin Va bounds to the nuclear membrane and localizes in the membrane complex (MC) (arrows in F) and acrosomal tubule (AT). (G) Myosin Va localization in mature spermatozoon. Myosin Va distributes in the nucleus (N) and acrosomal tubule (AT). Mitochondria (M) are decorated with myosin Va labeling (arrowheads in E–G).

Figure 8. A model of myosin Va localization and function during E. sinensis spermiogenesis.

The black point indicates myosin Va and the purple point indicates KIFC1. (A) In early stage, myosin Va is associate with the proacrosomal granule (PG) and the endoplasmic reticulum vesicle (EV) (black point myosin Va). (B, C) In mid stage, myosin Va localizes in the proacrosomal vesicle (PV) (B), as the nucleus (N) begin to wrap proacrosomal vesicle (PV), some myosin Va localizes to the membrane complex (MC) (C). (D–H) A hypothesis of the acrosomal tubule (AT) formation at late stage. At the initiation of late stage, myosin Va and KIFC1 exert a clutching force on the nuclear membrane at the bottom of the nuclear cup to push the membrane complex (MC) forward (red arrows in D, E); meanwhile, myosin Va at the other part of the membrane complex (MC) anchors proacrosomal vesicle (PV) to the nucleus and it also associates with the mitochondria (M) (D, E). When the acrosomal tubule (AT) reaches to the apical cap (AC), the evaginated nucleus (N) initiates to contract by pulling of myosin Va and KIFC1 (red arrows in F, G), some myosin Va interacts with actin in acrosomal tubule (AT), part of KIFC1 translocate to the apical cap (AC) (F, G). In mature spermatozoon, myosin Va localizes in the nucleus (N), membrane complex (MC) and acrosomal tubule (AT), while KIFC1 localizes in the apical cap (AC), nucleus (N) and acrosomal tubule (AT) (H).