We report herein structural details and the partial molecular characterization of the acroplaxome, an attachment plate present in the subacrosomal space of mammalian spermatids (including mouse, rat, and human) and linking the inner acrosomal membrane to the nuclear envelope. The acroplaxome contains F-actin and K5 and presumably other actin-associated proteins and motor proteins, which may be responsible for tethering the primary proacrosomal vesicle during the early stages of acrosomal biogenesis. A specific feature of the acroplaxome is its marginal ring housed in a shallow circular indentation of the spermatid nucleus. An attribute of the marginal ring is a conspicuous bundle of 10-nm-thick intermediate filaments attached to the dense plaque associated with the inner acrosomal membrane. The finding of intermediate filaments in the acroplaxome is not surprising. In fact, intermediate filament-like structures in the subacrosomal space were reported previously (Russell et al., 1986
). However, neither their biochemical nature nor regular organization at the marginal ring of the acroplaxome was described. Opposite to the intermediate filament–plaque complex is another thin plaque spanning across a shallow indentation in the spermatid nuclear envelope and linked to a nuclear lamina (see for a summary diagram). The subacroplaxome position of the nuclear lamina correlates with the propidium iodide dense chromatin staining seen in . The band-like edge of the chromatin density correlates with the collar-like arrangement of the plaque housing the marginal ring of the acroplaxome. The marginal ring and associated plaques are reminiscent of the zonula adherens belt conjoining the plasma membranes of adjacent epithelial cells. During spermiogenesis, the F-actin/K5-containing marginal ring trails the descending leading edge of the acrosomal sac and then seems to disassemble when nuclear elongation reaches completion (). This steady association implies that the acroplaxome is expected to adjust its diameter and shape without compromising the relationship of the marginal ring with both the descending acrosomal edge and the elongating spermatid nucleus. In this regard, the insertion plaques, whose molecular components are presently unknown, may play a pivotal role in stabilizing the position of the acroplaxome. Because gentle hypotonic and mild Triton X-100 treatment cannot dislodge the acrosome–acroplaxome complex from its nuclear attachment site, we postulate that the acroplaxome secures the acrosome at the corresponding nuclear pole during the elongation of the spermatid head. Several observations in mouse mutants with sperm head abnormalities determined by deficient acrosome development (Hrb mutant, Kang-Decker et al., 2001
; GOPC mutant, Yao et al., 2002
) make this an attractive hypothesis. As a corollary, a defect in acrosome biogenesis is likely to hamper the assembly and function of the acroplaxome in nuclear shaping.
Figure 9. Proposed mechanism of spermatid nuclear elongation in rodents. (A) The apical pole of a round spermatid is encircled by parallel F-actin hoops originated in the ectoplasmic region of a Sertoli cell. In the ectoplasmic margin, actin-containing bundles (more ...)
There are two aspects of this work that merit further discussion. First, the expression of classical keratins and outer dense fibers (Odfs) during spermatogenesis and spermiogenesis in particular. Second, the potential mechanical contribution of the acroplaxome to the nuclear shaping of the male gamete. Both nucleotide sequence analysis and deduced amino acid sequence demonstrate that testicular K5 has a 89% amino acid homology with K5, a type II keratin abundant in the basal layers of the epidermis (Fuchs, 1996
). K5 forms a cortical cytoplasmic shell in primary spermatocytes, is present in the centrosomal region during late male meiotic prophase I, is a component of the cytoplamic bridges conjoining cohorts of spermatocytes (Tres et al., 1996
), and is associated first with the manchette and later with the Odfs and fibrous sheath of the developing tail during rat (Kierszenbaum et al., 1996
; Tres and Kierszenbaum, 1996
) and mouse spermiogenesis (Akutsu et al., 2001
). A role of K5 in spermatogenesis is supported by the occurrence of signifi-cant apoptosis during meiotic prophase and spermatid nuclear and tail abnormalities in selected seminiferous tubules of K5+/- mutant mice (testis samples provided by Thomas M. Magin and Lu Hong, University of Bonn, Germany). Spermiogenesis in K5 null mice cannot be evaluated because they exhibit neonatal lethality (Peters et al., 2001
). Another keratin, K9, is one of the components of the perinuclear ring of the manchette (Mochida et al., 2000
). K9 is a unique keratin because its expression is predominant in the epidermis of palm and sole (human) and footpad of rodents and other species. K9 and K5 are examples of type I keratin (which includes K9 to K20) and type II keratin (which includes K1 to K8), respectively, thus far detected during spermatogenesis. At least one member of each family of keratins is required to form heterodimeric intermediate filament proteins. In fact, we have found that keratins 14 and 2e, the common partners of K5 and K9, respectively, are also expressed in testis (our unpublished data).
It is significant to point out that Odfs are cytoskeletal proteins that are heavily disulfide-linked, contain repeats of the Cys-X-Promotif, and surround the axoneme of the sperm tail (Calvin and Bedford, 1971
; Olson and Sammons, 1980
; Vera et al., 1984
; Oko, 1988
). Three Odfs have been so far reported: Odf1 (27 kDa, van der Hoorn et al., 1990
; Burfeind and Hoyer-Fender, 1991
), Odf2 (84 kDa, Brohmann et al., 1997
), and Odf3 (110 kDa, Petersen et al., 2002
). An Odf2-like protein was found to be a component of the centrosome scaffold in somatic cells (Nakagawa et al., 2001
). The widespread distribution of an Odf2-like protein as a centrosome scaffold component, together with the presence of K5 in the centrosome region of pachytene spermatocytes (Tres et al., 1996
), and in the acroplaxome (this article) are a demonstration that keratins are used throughout spermatogenesis. Together, these observations strengthen the view that classical keratins (initially identified in the epidermis) and Odf proteins (initially ascribed as sperm-specific proteins) can coexist in the sperm tail to provide structural and mechanical support for stabilizing long flagella while minimizing the risk of kinking or breaking.
The second aspect of the discussion relates to the mechanism of assembly of the acroplaxome and its potential function during spermiogenesis. Concerning the assembly of the acroplaxome, F-actin precedes the appearance of K5 (), thus suggesting that it may provide a template for its association with keratin filaments, an event known to occur in vitro (Weber and Bement, 2002
). Because the diameter of the acroplaxome increases in parallel with the corresponding spreading of the acrosome over the elongating spermatid nucleus (), it is conceivable that this process may occur at the expense of actin polymerization. F-Actin is present in the subacrosomal space during most of spermiogenesis before depolymerizing to G-actin in late spermatids and sperm (reviewed by Vogl, 1989
A significant question is whether F-actin in the acroplaxome can provide a binding scaffold for motor proteins and contain actin regulatory proteins. The motor protein myosin-Va is present in the acroplaxome (our unpublished observation), and there are precedents for actin regulatory proteins. It is known that binding of the actin-depolymerizing factor cofilin to actin promotes the transition of polymeric F-actin into oligomeric or monomeric G-actin (reviewed in Ayscough, 1998
). Phosphorylation of cofilin by LIMK (LIM kinase) prevents cofilin binding and depolymerization of actin, leading to the accumulation of filamentous actin. In this respect, the testicular isoform of LIMK2 is associated with spermatids and targeted Limk2
gene disruption affects the progression of spermatogenesis (Takahashi et al., 2002
). Furthermore, a number of actin-binding proteins have been reported at the acrosomal region (for example, actin-capping proteins, Hurst et al., 1998
; calicin, Lécuyer et al., 2000
; profilin-3, Braun et al., 2002
; Arc, Maier et al., 2003
). A variant of Wiskott-Aldrich syndrome protein-interacting src homology 3 protein (designated WISH) was exclusively found in testis (Fukuoka et al., 2001
). WISH strongly enhances Wiskott-Aldrich syndrome protein-induced actin-related protein 2/3 (Arp2/3) complex activation, resulting in rapid actin nucleation and polymerization. Given the existence of a number of testis-specific actin binding proteins, an analysis of their role in the regulation of actin assembly and disassembly in the acroplaxome would contribute greatly to advance an understanding of the mechanistic complexity of this structure. An experimental approach to addressing these questions is to sequester actin monomers resulting in rapid depolymerization of F-actin. Unfortunately, the physiological consequences of impairing the function of the acroplaxome by disruptors of the actin cytoskeleton (such as latrunculin A) cannot be assessed in cultured spermatids. First, the developmental time of the acrosome-acroplaxome complex is long (days; Clermont et al., 1993
). Second, actin-disrupting agents can target the supporting Sertoli cells, which are required for spermatogenic cell viability and differentiation in vivo and in vitro (Kierszenbaum, 1994
). Because of these reasons, mouse mutants provide at present a more feasible approach to determine how the acroplaxome might work.
How can the acroplaxome contribute to spermatid nuclear shaping? It is likely that a stress-resistant acroplaxome can transmit gentle and sustained clutching forces generated by Sertoli cell ectoplasmic F-actin bundles to the elongating spermatid nucleus (). The spatial arrangement of stacked F-actin hoops embracing the apical one-third of the spermatid nucleus seems to be kept in place by the actin-linked afadin–nectin complex, which may ensure constant Sertoli-spermatid contact (Ozaki-Kuroda et al., 2002
). In addition to the force exerted by Sertoli cell F-actin hoops, the perinuclear ring of the manchette can provide an endogenous clutch applied at the caudal two-thirds of the spermatid nucleus. As the manchette descends along the elongating spermatid nucleus, its perinuclear ring gradually reduces its diameter and, in a sleeve-like manner, may apply forces steering the elongation of the spermatid nucleus. In fact, fractionated manchette perinuclear rings of various diameters have been demonstrated (Mochida et al., 1998
), and a nuclear deforming constriction at the level of the perinuclear ring of azh mutant mice spermatids can be seen (). In summary, we have shown that the F-actin–keratin-containing acroplaxome provides a planar scaffold, which maintains the acrosome at the nuclear anchoring site during nuclear elongation. This study provides a novel model for spermatid nuclear shaping, which can be extended by the analysis of a number of mutant mice in which the development of the acrosome and integrity of the acroplaxome are defective.