Expression and localization of myosin VI in developing mouse spermatids

Abstract

Myosin VI (MVI) is a versatile actin-based motor protein that has been implicated in a variety of different cellular processes, including endo- and exocytic vesicle trafficking, Golgi morphology, and actin structure stabilization. A role for MVI in crucial actin-based processes involved in sperm maturation was demonstrated in Drosophila. Because of the prominence and importance of actin structures in mammalian spermiogenesis, we investigated whether MVI was associated with actin-mediated maturation events in mammals. Both immunofluorescence and ultrastructural analyses using immunogold labeling showed that MVI was strongly linked with key structures involved in sperm development and maturation. During the early stage of spermiogenesis, MVI is associated with the Golgi and with coated and uncoated vesicles, which fuse to form the acrosome. Later, as the acrosome spreads to form a cap covering the sperm nucleus, MVI is localized to the acroplaxome, an actin-rich structure that anchors the acrosome to the nucleus. Finally, during the elongation/maturation phase, MVI is associated with the actin-rich structures involved in nuclear shaping: the acroplaxome, manchette, and Sertoli cell actin hoops. Since this is the first report of MVI expression and localization during mouse spermiogenesis and MVI partners in developing sperm have not yet been identified, we discuss some probable roles for MVI in this process. During early stages, MVI is hypothesized to play a role in Golgi morphology and function as well as in actin dynamics regulation important for attachment of developing acrosome to the nuclear envelope. Next, the protein might also play anchoring roles to help generate forces needed for spermatid head elongation. Moreover, association of MVI with actin that accumulates in the Sertoli cell ectoplasmic specialization and other actin structures in surrounding cells suggests additional MVI functions in spermatid movement across the seminiferous epithelium and in sperm release.

Keywords: Actin; Immunocytochemistry; Myosin VI splice variants; Spermiogenesis; Ultrastructure.

Fig. 1

Schematic representation of the main stages of spermiogenesis in mouse: the Golgi, acrosomal, and maturation phases. ag acrosomal granule, am acrosome membrane, av acrosome vesicle, ax acroplaxome, c centriole, cy cytoplasm, dp dense plaque, es apical ectoplasmic specialization, g Golgi apparatus, iam inner acrosomal membrane, if intermediate filaments, m mitochondria, mp midpiece, n spermatid nucleus, nm nuclear envelope, oam outer acrosomal membrane, pp principal piece

Fig. 2

Verification of MVI splice variants expressed in mouse testes and specificity of used commercial antibodies in these organs. a RT-PCR products obtained with primers designed to produce MVI fragments containing either a large insert (LI), a small insert (SI), both inserts (LI + SI) or no insert (NoI) from control plasmids (first four lanes) and mouse testis (last lane). b, c Immunoblotting of crude protein extracts from different mouse tissues with MVI PAb (b) and anti-actin JL20 MAb antibodies (c). Lane 1 testis, 2 liver, 3 kidney, 4 heart, 5 lung, 6 brain

Fig. 3

Toluidine blue staining (a–g) and immunofluorescence labeling of MVI (h) and actin (i) of mouse seminiferous tubules during spermatogenis. aSpT spermatid at the acrosome phase, BV blood vessel, gSpT spermatid at the Golgi phase, Lc Leydig cell, mSpT spermatid at the maturation phase, Sc Sertoli cell, SE seminiferous epithelium, SpC spermatocyte, SpG spermatogonium, SpZ spermatozoa, STL seminiferous tubule lumen. Arrows or double arrows show MVI (red) and actin (green) staining in spermatids at maturation or acrosome phase, respectively; stars in i show actin localization in basal ectoplasmic specialization; dashed lines basement membrane. Nuclei are stained with DAPI (blue). Bars 50 μm (a), 20 μm (g–i), 5 μm (b-f)

Fig. 4

Immunofluorescence localization of MVI (red) and actin (green) in mouse developing spermatids during the Golgi phase (gSpT), the acrosomal phase (aSpT), and the maturation phase (mSpT) as well as in spermatozoa (SpZ). All other indications are explained in the text (see “Results”). Nuclei are stained with DAPI (blue) or outlined with dashed line. Bars 5 μm

Fig. 5

Ultrastructural analysis (a–d, i–k) and immunogold localization (e–h, l–n) of MVI in developing mouse spermatids during the Golgi phase. ag acrosomal granule, av acrosome vesicle, cb chromatoid body, cy cytoplasm, g Golgi complex, gSpT spermatid at the Golgi phase, m mitochondria, n nucleus. Square brackets in k show the contact region between the nucleus and the chromatoid body. Dotted lines mark a boundary between the spermatid cytoplasm, the acrosomal vesicle and the nucleus (h) or between the spermatid cytoplasm and the nucleus (n). All other indications are explained in the text (see “Results”). Bars 1 μm (a, i), 500 nm (b–g, i–n), 250 nm (h)

Fig. 6

Ultrastructural analysis (a–c, g–i) and immunogold localization (d–f, j–m) of MVI in developing mouse spermatids during the acrosome phase. apx acroplaxome, aSpT spermatid at the acrosome phase, av acrosome vesicle, cy cytoplasm, er endoplasmic reticulum, g Golgi complex, iam inner acrosome membrane, m mitochondria, n nucleus, oam outer acrosome membrane, Sc Sertoli cell, Scm Sertoli cell membrane, SpTm spermatid membrane. Boxed regions in a and c include the acrosome–acroplaxome marginal ring regions. All other indications are explained in the text (see “Results”). Bars 1 μm (a), 500 nm (b, d, j–l), 250 nm (g–i, m), 200 nm (c, e, f)

Fig. 7

Ultrastructural analysis of developing mouse spermatids during the maturation phase. apx acroplaxome, av acrosome vesicle, ax axoneme, c centriole, dmp developing midpiece of the sperm tail, er endoplasmic reticulum, m mitochondria, mp midpiece of the sperm tail, mSpT spermatid at the maturation phase, mt manchette, n nucleus, pp principal piece of the sperm tail, pr perinuclear ring of the manchette, Sc Sertoli cell. Boxed region in a includes the spermatid head with spreading acrosome. All other indications are explained in the text (see “Results”). Bars 2 μm (a), 1 μm (b, c), 500 nm (g–h, l–n), 200 nm (d–f, i–k)

Fig. 8

Immunogold localization of MVI in developing mouse spermatids during the maturation phase. apx acroplaxome, av acrosome vesicle, ax axoneme, dmp developing midpiece of the sperm tail, ep end piece of the sperm tail, er endoplasmic reticulum, m mitochondria, mp midpiece of the sperm tail, mSpT spermatid at the maturation phase, mr marginal ring of the acroplaxome, mt manchette, n nucleus, pp principal piece of the sperm tail, pr perinuclear ring of the manchette, Sc Sertoli cell. All other indications are explained in the text (see “Results”). Bars 250 nm

Fig. 9

Schematic representation of MVI distribution (black dots) during mouse spermiogenesis. aSpT round spermatid at the acrosome phase, gSpT round spermatid at the Golgi phase, mSpT elongated spermatid at the maturation phase