Tubulobulbar complexes are intercellular podosome-like structures that internalize intact intercellular junctions during epithelial remodeling events in the rat testis

Tubulobulbar complexes are actin-related double-membrane projections that resemble podosomes in other systems and form at intercellular junctions in the seminiferous epithelium of the mammalian testis. They are proposed to internalize intact junctions during sperm release and during the translocation of spermatocytes through basal junction complexes between neighboring Sertoli cells. In this study we probe apical tubulobulbar complexes in fixed epithelial fragments and fixed frozen sections of rat and mouse testes for junction molecules reported to be present at apical sites of attachment (ectoplasmic specializations) between Sertoli cells and spermatids. The adhesion molecules nectin 2 (PVRL2), nectin 3 (PVRL3) and alpha 6 integrin (ITGA6) are present in the elongate parts of tubulobulbar complexes and concentrated at their distal ends. Tubulobulbar complexes contain cortactin (CTTN), a key component of podosomes, and vesicles at the distal ends of tubulobulbar complexes that contain junction molecules are related to early endosome antigen (EEA1). N-cadherin (CDH2), a protein reported to be present at ectoplasmic specializations, is not localized to these unique junctions or to tubulobulbar complexes but, rather, is primarily concentrated at desmosomes in basal regions of the epithelium. Our results are consistent with the conclusion that tubulobulbar complexes are podosome-like structures that are responsible for internalizing intact intercellular junctions during spermatogenesis.     

Evidence that tubulobulbar complexes in the seminiferous epithelium are involved with internalization of adhesion junctions

Tubulobulbar complexes may be part of the mechanism by which intercellular adhesion junctions are internalized by Sertoli cells during sperm release. These complexes develop in regions where Sertoli cells are attached to adjacent cells by intercellular adhesion junctions termed ectoplasmic specializations. At sites where Sertoli cells are attached to spermatid heads, tubulobulbar complexes consist of fingerlike processes of the spermatid plasma membrane, corresponding invaginations of the Sertoli cell plasma membrane, and a surrounding cuff of modified Sertoli cell cytoplasm. At the terminal ends of the complexes occur clusters of vesicles. Here we show that tubulobulbar complexes develop in regions previously occupied by ectoplasmic specializations and that the structures share similar molecular components. In addition, the adhesion molecules nectin 2 and nectin 3, found in the Sertoli cell and spermatid plasma membranes, respectively, are concentrated at the distal ends of tubulobulbar complexes. We also demonstrate that double membrane bounded vesicles are associated with the ends of tubulobulbar complexes and nectin 3 is present on spermatids, but is absent from spermatozoa released from the epithelium. These results are consistent with the conclusion that Sertoli cell and spermatid membrane adhesion domains are internalized together by tubulobulbar complexes. PKCalpha, a kinase associated with endocytosis of adhesion domains in other systems, is concentrated at tubulobulbar complexes, and antibodies to endosomal and lysosomal (LAMP1, SGP1) markers label the cluster of vesicles associated with the ends of tubulobulbar complexes. Our results are consistent with the conclusion that tubulobulbar complexes are involved with the disassembly of ectoplasmic specializations and with the internalization of intercellular membrane adhesion domains during sperm release.     

Non-muscle cofilin is a component of tubulobulbar complexes in the testis

Tubulobulbar complexes are finger-like structures that form at the interface between maturing spermatids and Sertoli cells prior to sperm release and at the interface between two Sertoli cells near the base of the seminiferous epithelium. They originate in areas previously occupied by actin filament-associated intercellular adhesion plaques known as ectoplasmic specializations. Actin filaments also are associated with tubulobulbar complexes where they appear to form a network, rather than the tightly packed bundles found in ectoplasmic specializations. Cofilin, a calcium-independent actin-depolymerizing protein, previously has been identified in the testis, but has not been localized to specific structures in the seminiferous epithelium. To determine if cofilin is found in Sertoli cells and is concentrated at actin-rich structures, we reacted fixed frozen sections of rat testis, fixed fragmented tissue, and blots of seminiferous epithelium with pan-specific and non-muscle cofilin antibodies. In addition, GeneChip microarrays (Affymetrix, Santa Clara, CA) were utilized to determine the abundance of mRNA for all cofilin isoforms in Sertoli cells. Using the monoclonal pan-specific cofilin antibody, we found specific labeling exclusively at tubulobulbar complexes and not at ectoplasmic specializations. On one-dimensional (1D) Western blots this antibody reacted monospecifically with one band, and on 2D blots reacted with two dots, which we interpret as phosphorylated and nonphosphorylated forms of a single cofilin isotype. Messenger RNA for non-muscle cofilin in Sertoli cells is about 8.5-fold higher than for muscle-type cofilin. To confirm that the non-muscle isoform of cofilin is present at tubulobulbar complexes, we used antibodies specific to non-muscle cofilin for immunofluorescent localization. As with the pan-specific antibody, we found that the non-muscle cofilin antibody exclusively labeled tubulobulbar complexes. Results presented here indicate that non-muscle cofilin is concentrated at tubulobulbar complexes. Our results also indicate that cofilin is not concentrated at ectoplasmic specializations.     

Actin-related intercellular junctions in the germinal compartment of the testis in Poecilia reticulata (Teleostei,Poeciliidae)

Summary In this paper we present evidence for the presence of actin-related junctions between neighboring Sertoli cells and between Sertoli cells and spermatids in the testis of the guppy (Poecilia reticulata). In the guppy, spermatogenesis occurs in spermatocysts that are lined by a simple squamous to cuboidal epithelium formed of Sertoli cells. At a certain stage of differentiation, elongate spermatids occur in Sertoli cell recesses in the apical surface of Sertoli cells. When evaluated by electron microscopy, junctions occur between Sertoli cells and spermatids situated in the recesses. In these regions, obvious linkages occur between the plasma membrane of Sertoli cell recesses and the adjacent spermatids. Moreover, large concentrations of microfilaments occur in the Sertoli cell cytoplasm immediately underlying the crypts. Also, junctional complexes are apparent between neighboring Sertoli cells near the apical surface of the epithelium. These complexes consist of microfilament-related components (probably contributing to both tight and adhesion junctions), which occur closest to the lumen, and intermediate-filament related desmosomes, which occur more basally. In fixed frozen sections of guppy testis, probes for filamentous actin (rhodamine phalloidin) and myosin II (polyclonal antisera raised against human platelet myosin II) react with function regions between neighboring Sertoli cells and between Sertoli cells and spermatids. We conclude that actin-related junctions occur at both these sites and that the actin networks have contractile properties because they contain myosin II.     

Observations on the inter-relationships of Sertoli cells at the level of the blood- testis barrier: evidence for formation and resorption of Sertoli-Sertoli tubulobulbar complexes during the spermatogenic cycle of the rat.

Structures termed tubulobulbar complexes are known to be formed by adjoining Sertoli cells at the level of the blood-testis barrier (Russell and Clermont, '76). Here, long (2-4 micrometer) tubular evaginations of one Sertoli cell, which end in bulbous dilations, are seen in corresponding invaginations of a neighboring Sertoli cell. In most regions of the tubular and bulbous portions of the complex, the Sertoli plasma membranes were found to be separated by a 4-5-nm intercellular space, but in some areas the membranes converged to form tight and gap junctions. The numbers, distribution and properties of tubulobulbar complexes were studied in relation to the cycle of the seminiferous epithelium. From the data obtained it was concluded that tubulobulbar complexes develop and undergo regressive changes during the spermatogenic cycle. Most complexes arise during the early stages of the cycle (Stages II-V) and develop large bulbous endings. Developing tubulobulbar complexes consist of short evaginations of one Sertoli cell which face a bristle-coated pit of the opposing Sertoli cell. At midcycle (Stages VI-VII) most show regressive changes and are eventually resorbed as a consequence of the action of nearby Sertoli lysosomes. Once resorbed, the probability of seeing a tubulobulbar complex in thin sections decreases from 4- to 8-fold. The few tubulobulbar complexes which remain past this period (Stages VII-XIV-I) usually lack bulbous endings and are fequently seen above type A spermatogonia. The data suggest that small fragments of cytoplasm and plasma membrane (including junctional surfaces) are lost from one Sertoli cell as a result of the degradative processes occurring in a neighboring Sertoli cell. Tubulobulbar resorption is discussed in relation to the impending breakdown of the blood-testis barrier above spermatocytes as these cells move upward. The possible significance of the cyclic resorption of tight and gap junctional sites between Sertoli cells is also discussed.     

Immunofluorescence localization of vinculin in ectoplasmic ("junctional") specializations of rat Sertoli cells

We have investigated, using indirect immunofluorescence techniques, the possibility that vinculin is a component of Sertoli cell ectoplasmic specializations. Affinity-purified polyclonal antibodies produced against human platelet vinculin were used to probe fixed frozen sections of rat testis. Specific fluorescence occurs in Sertoli cell regions adjacent to spermatids and to basally situated junctional complexes, sites at which ectoplasmic specializations are known to occur. Staining also occurs in Sertoli cell regions associated with tubulobulbar complexes. The antibody also labels focal contacts in cultured human dermal fibroblasts, apical junctional sites of rat epididymal epithelium, and dense plaques of smooth muscle. Our results are consistent with the prediction that vinculin is likely a component of ectoplasmic specializations and are also consistent with the hypothesis that these structures are a form of actin-associated adhesion complex.     

The consequences of actin disruption at Sertoli ectoplasmic specialization sites facing spermatids after in vivo exposure of rat testis to cytochalasin D

Cytochalasin D (CD) was used to perturb actin filaments of the Sertoli ectoplasmic specialization (ES)--a cytoskeletal complex of the Sertoli cell related to spermatids. CD (500 microM for 6 h) produced a loss of 88% of the ES facing the head region of early (Step 8) elongating spermatids as compared to vehicle (dimethylsulfoxide:saline) controls. Nitrobenzoxadiazole-phallacidin staining of F-actin revealed a CD-related loss of uniform fluorescence over the head of elongated spermatids. To examine for a possible relationship between the presence of actin and cell attachment at ES sites, hypertonic fixatives were introduced to provoke cell shrinkage and stress ES-associated junctions. After osmotic stress, cell-to-cell adhesion at ES sites remained intact in vehicle-treated animals. CD treatment caused Sertoli cells to separate from elongating spermatids at sites where ES had been lost from the Sertoli cell surface. It is suggested that actin of the ES plays a role in cell-to-cell interaction analogous to its possible role at the Sertoli cell barrier. In CD-treated animals, structures resembling tubulobulbar complexes frequently developed at sites where ES was lost, suggesting that the loss of ES has a facilitatory role in tubulobulbar complex formation. It is hypothesized that tubulobulbar complexes are devices that rid the cells of ES-associated junctional links to effect dissociation of the spermatid from the Sertoli cell during spermiation. Spermatids at Step 8 of development are known to become oriented with their acrosomes facing the base of the Sertoli cell. After CD treatment, a 5.8-fold increase in malorientation of Step 8 spermatids was noted. A role for the ES cytoskeletal complex in orienting the spermatid acrosome toward the basal aspect of the Sertoli cell is also suggested.     

The role of dynamin 3 in the testis

We report here that dynamin 3 in the testis is associated with structures termed tubulobulbar complexes that internalize intact intercellular junctions during sperm release and turnover of the blood-testis barrier. The protein lies adjacent to an actin-Arp2/3 network that cuffs the double plasma membrane tubular invagination at the core of each complex. To explore the possible relationship between dynamin 3 and nectin-based adhesion junctions, we transiently transfected DsRed-tagged dynamin 3 into MDCK cells stably transfected with eGFP-tagged nectin 2, one of the adhesion molecules known to be expressed in Sertoli cells at adhesion junctions. Cells transfected with the dynamin 3 construct had less uniformly distributed nectin 2 at intercellular contacts when compared to control cells expressing only nectin 2 or transfected with the DsRed plasmid alone. Significantly, tubular extensions positive for nectin 2 were visible projecting into the cells from regions of intercellular contact. Our findings are consistent with the conclusion that dynamin 3 is involved with tubulobulbar morphogenesis. Dynamin 3 also occurs in concentrated deposits around the capitulum and striated columns in the connecting piece of sperm tails suggesting that the protein in these cells may function to stabilize the base of the tail or serve as a reservoir for use during or after fertilization.     

Disorganization of actin bundles of ectoplasmic specialization

The degradation of ectoplasmic specialization consisting of bundles of actin sandwiched between the plasma membrane and endoplasmic reticulum of the Sertoli cell, occurs just before spermiation. For elucidation of the processes involved in this degradation, changes in fibrous actin of the rat testis were analyzed using BODIPY-phalloidin by fluorescence and electron microscopy.
Before step 17, the fluorescence of BODIPY-phalloidin was evenly distributed around the spermatid head. When the spermatids became positioned at the luminal surface, the fluorescence had condensed on the concave side of the spermatid head. At step 19, lines of fluorescence distributed at regular intervals projected at right angles from the head. Ultrastructural observation showed that the tubulobulbar complex was formed at step 19 and electron-dense material accumulated around thin tubules of the tubulobulbar complex. Immunohistochemical examination of BODIPY-phalloidin showed that the electron dense materials around the thin tubules of the tubulobulbar complex had the capacity to bind to phallotoxin. Therefore the pattern of fluorescence in the spermatid at step 19 corresponds to that of the tubulobulbar complex.
Actin bundles of the ectoplasmic specialization would thus appear to de-polymerize into actin monomers via electron dense materials around the thin tubules of the tubulobulbar complex. The tubulobulbar complex may contribute to the disorganization of actin bundles.     

Adhesion structures and their cytoskeleton-membrane interactions at podosomes of osteoclasts in culture

The organization of the cytoskeleton in the podosomes of osteoclasts was studied by use of cell shearing, rotary replication, and fluorescence cytochemical techniques. After shearing, clathrin plaques and particles associated with the cytoskeleton were left behind on the exposed cytoplasmic side of the membrane. The cytoskeleton of the podosomes was characterized by two types of actin filaments: relatively long filaments in the portion surrounding the podosome core, and highly branched short filaments in the core. Individual actin filaments radiating from the podosomes interacted with several membrane particles along the length of the filaments. Many lateral contacts with the membrane surface by the particles were made along the length of individual actin filaments. The polarity of actin filaments in podosomes became oriented such that their barbed ends were directed toward the core of podosomes. The actin cytoskeletons terminated or branched at the podosomes, where the membrane tightly adhered to the substratum. Microtubules were not usually present in the podosome structures; however, certain microtubules appeared to be morphologically in direct contact with the podosome core. Most of the larger clathrin plaques consisted of flat sheets of clathrin lattices that interconnected neighboring clathrin lattices to form an extensive clathrin area. However, the small deeply invaginated clathrin plaques and the podosomal cytoskeleton were located close together. Thus, the clathrin plaques on the ventral membrane of osteoclasts might be involved in both cell adhesion and the formation of receptor-ligand complexes, i.e., endocytosis. This work was supported by the following grants to T.A.: Grants-in-Aid for Scientific Research (C) (18592020) from the Ministry of Education, Science, and Culture of Japan and the Miyata Research Fund of Asahi University.     

Distribution of actin-filament bundles in myoid cells,Sertoli cells,and tunica albuginea of rat and mouse testes

Summary Frozen sections of the rat and mouse testes were stained with either FITC-phalloidin or NBD-phallacidin and viewed with conventional fluorescence and confocal laser microscopes in order to demonstrate the arrangment of actin-filament bundles in myoid cells, Sertoli cells and tunica albuginea. Myoid cells are rich in actin-filament bundles crossing at right angles. These bundles running in different directions can also be visualized by means of electron microscopy. Nerve fibers occur in the vicinity of myoid cells, suggesting a neural control of the cell. At Sertoli cell junctions actin filaments occur at the circumference of the cell, where they show a honeycomb pattern. The ratio of the number of Sertoli cells per myoid cell can be calculated by means of confocal microscopy; this technique may provide a new parameter for determining spermatogenic activity. In the tunica albuginea of the juvenile mouse testis, actin filaments are arranged in an alternate fashion.     

Crosstalk between Cell Adhesion Complexes in Regulation of Mechanotransduction

Physical forces regulate numerous biological processes during development, physiology, and pathology. Forces between the external environment and intracellular actin cytoskeleton are primarily transmitted through integrin-containing focal adhesions and cadherin-containing adherens junctions. Crosstalk between these complexes is well established and modulates the mechanical landscape of the cell. However, integrins and cadherins constitute large families of adhesion receptors and form multiple complexes by interacting with different ligands, adaptor proteins, and cytoskeletal filaments. Recent findings indicate that integrin-containing hemidesmosomes oppose force transduction and traction force generation by focal adhesions. The cytolinker plectin mediates this crosstalk by coupling intermediate filaments to the actin cytoskeleton. Similarly, cadherins in desmosomes might modulate force generation by adherens junctions. Moreover, mechanotransduction can be influenced by podosomes, clathrin lattices, and tetraspanin-enriched microdomains. This review discusses mechanotransduction by multiple integrin- and cadherin-based cell adhesion complexes, which together with the associated cytoskeleton form an integrated network that allows cells to sense, process, and respond to their physical environment.     

Ultrastructure of sustentacular (Sertoli) cells in the bovine testis

In the present communication, ultrastructural and cytochemical aspects of mature bovine Sertoli cells and their relationship to the different stages of germ cell development are described. As in other mammalian species, different types of junctional specializations exist between Sertoli and germ cells in the bovine seminiferous epithelium, including desmosome-like junctions, Sertoli cell ectoplasmic specializations and tubulobulbar complexes. The functional significance of the morphological results and the interactions of Sertoli and germ cells during spermatogenesis are discussed.     

Development of Sertoli cell junctional specializations and the distribution of the tight-junction-associated protein ZO-1 in the mouse testis

Basally located tight junctions between Sertoli cells in the postpubertal testis are the largest and most complex junctional complexes known. They form at puberty and are thought to be the major structural component of the "blood-testis" barrier. We have now examined the development of these structures in the immature mouse testis in conjunction with immunolocalization of the tight-junction-associated protein ZO-1 (zonula occludens 1). In testes from 5-day-old mice, tight junctional complexes are absent and ZO-1 is distributed generally over the apicolateral, but not basal, Sertoli cell membrane. As cytoskeletal and reticular elements characteristic of the mature junction are recruited to the developing junctions, between 7 and 14 days, ZO-1 becomes progressively restricted to tight junctional regions. Immunogold labeling of ZO-1 on Sertoli cell plasma membrane preparations revealed specific localization to the cytoplasmic surface of tight junctional regions. In the mature animal, ZO-1 is similarly associated with tight junctional complexes in the basal aspects of the epithelium. In addition, it is also localized to Sertoli cell ectoplasmic specializations adjacent to early elongating, but not late, spermatids just prior to sperm release. Although these structures are not tight junctions, they do have a similar cytoskeletal arrangement, suggesting that ZO-1 interacts with the submembrane cytoskeleton. These results show that, in the immature mouse testis, ZO-1 is present on the Sertoli cell plasma membrane in the absence of recognizable tight junctions. In the presence of tight junctions, however, ZO-1 is found only at the sites of junctional specializations associated with tight junctions and with elongating spermatids.     

Macrophage podosomes assemble at the leading lamella by growth and fragmentation

Podosomes are actin- and fimbrin-containing adhesions at the leading edge of macrophages. In cells transfected with beta-actin-ECFP and L-fimbrin-EYFP, quantitative four-dimensional microscopy of podosome assembly shows that new adhesions arise at the cell periphery by one of two mechanisms; de novo podosome assembly, or fission of a precursor podosome into daughter podosomes. The large podosome cluster precursor also appears to be an adhesion structure; it contains actin, fimbrin, integrin, and is in close apposition to the substratum. Microtubule inhibitors paclitaxel and demecolcine inhibit the turnover and polarized formation of podosomes, but not the turnover rate of actin in these structures. Because daughter podosomes and podosome cluster precursors are preferentially located at the leading edge, they may play a critical role in continually generating new sites of cell adhesion.     

Adaptors, junction dynamics, and spermatogenesis

Adaptors are component proteins of junctional complexes in all epithelia, including the seminiferous epithelium of the mammalian testis. They recruit other regulatory and structural proteins to the site of both anchoring junctions (such as cell-cell actin-based adherens junctions [AJs], e.g., ectoplasmic specialization [ES] and tubulobulbar complex, which are both testis-specific cell-cell actin-based AJ types, and cell-cell intermediate filament-based desmosome-like junctions) and tight junctions (TJ). Furthermore, adaptors per se can be substrates and/or activators of kinases or phosphatases. As such, the integrity of cell junctions and the regulation of junction dynamics during spermatogenesis rely on adaptors for their ability to recruit and link different junctional components to the same site and to tether transmembrane proteins at both anchoring and TJ sites to the underlying cytoskeletons, such as the actin filaments, intermediate filaments, and microtubules. These protein-protein interactions are possible because adaptors are composed of conserved protein binding domains, which allow them to link to more than one structural or signaling protein, recruiting multi-protein complexes to the same site. Herein, we provide a timely review of adaptors recently found at the sites of AJ (e.g., ES) and TJ. In addition, several in vivo models that can be used to delineate the function of adaptors in the testis are described, and the role of adaptors in regulating junction dynamics pertinent to spermatogenesis is discussed.     

Actin-related intercellular adhesion junctions in the germinal compartment of the testis in the hagfish (Eptatretus stouti) and lamprey (Lampetra tridentatus)

The germinal epithelium of male vertebrates consists of Sertoli cells and spermatogenic cells. Intercellular junctions formed by Sertoli cells assume critical roles in the normal functions of this epithelium. While Sertoli cell junctions have been well characterized in mammals, similar junctions in nonmammalian vertebrates have received little attention. We examined the intercellular junctions found within the germinal epithelium of the hagfish (Eptatretus stouti) and lamprey (Lampetra tridentatus). Ultrastructurally, Sertoli cells were seen to form filament-associated junctions in both species. Adjacent Sertoli cells formed microfilament-related junctions near their apices. Filaments of these junctions were arranged in loose networks and were not associated with cisterns of endoplasmic reticulum. In fixed, frozen sections of hagfish testis, similar areas labeled with rhodamine phalloidin, indicating the filament type is actin. In the lamprey, desmosomes were observed immediately below the microfilament-related junctions. In appearance and location, the Sertoli cell junctions observed in these species resembled those of the typical junctional complex of other epithelial cell types. No junctions were observed between Sertoli cells and elongating spermatids. In the hagfish, but not the lamprey, an additional zone of microfilaments occurred near the base of Sertoli cells in areas of association with the basal lamina. Our observations are consistent with the proposal that the unique forms of intercellular attachment found in the testes of higher vertebrates evolved from a typical epithelial form of intercellular junction.     

Regulation of intercellular adhesion strength in fibroblasts

The regulation of adherens junction formation in cells of mesenchymal lineage is of critical importance in tumorigenesis but is poorly characterized. As actin filaments are crucial components of adherens junction assembly, we studied the role of gelsolin, a calcium-dependent, actin severing protein, in the formation of N-cadherin-mediated intercellular adhesions. With a homotypic, donor-acceptor cell model and plates or beads coated with recombinant N-cadherin-Fc chimeric protein, we found that gelsolin spatially co-localizes to, and is transiently associated with, cadherin adhesion complexes. Fibroblasts from gelsolin-null mice exhibited marked reductions in kinetics and strengthening of N-cadherin-dependent junctions when compared with wild-type cells. Experiments with lanthanum chloride (250 microm) showed that adhesion strength was dependent on entry of calcium ions subsequent to N-cadherin ligation. Cadherin-associated gelsolin severing activity was required for localized actin assembly as determined by rhodamine actin monomer incorporation onto actin barbed ends at intercellular adhesion sites. Scanning electron microscopy showed that gelsolin was an important determinant of actin filament architecture of adherens junctions at nascent N-cadherin-mediated contacts. These data indicate that increased actin barbed end generation by the severing activity of gelsolin associated with N-cadherin regulates intercellular adhesion strength.     

细胞松弛素E对大鼠生精细胞发育的影响

本文采用微丝抑制剂——细胞松弛素E对大鼠生精细胞发育的影响作了形态学观察,特别对支持细胞骨架复合体的作用进行了较为详细的研究。结果表明睾丸内注射0.1ml,1000μmol/L～2000μmol/L,细胞松弛素E,6-14小时后,光镜下可见曲细精管上皮排列疏松,组合紊乱,有的生精上皮基底部出现双核和三核的圆形细胞和多核巨精子细胞,管腔内出现未成熟的精子;在第ⅤⅢ～Ⅸ期曲细精管上皮中,有许多第8、第9期的精子细胞顶体不指向基底方向,属定向不正的精子。电镜下,实验组动物可见一些面向第8-18期精子细胞顶体的支持细胞骨架复合体出现不同程度的缺如,有的断裂成小段;有的破坏仅发生在顶体上方;有的几乎全部丢失,并有类管球复合体的形成。另外,在高渗液处理下,可见精子细胞顶体和支持细胞间的间隙扩大。最后对微丝在精子发育中的作用进行了讨论。