Tubulobulbar Complexes Research Articles

Implication of Amphiphysin 1 and Dynamin 2 in Tubulobulbar Complex Formation and Spermatid Release

Tubulobulbar complexes (TBCs) are composed of several tubular invaginations formed at the plasma membrane of testicular Sertoli cells. TBCs are transiently formed at the contact region with spermatids at spermatogenic stage VII in rat and mouse, and such TBC formation is prerequisite for spermatid release. Since the characteristic structure of TBCs suggests that the molecules implicated in endocytosis could be involved in TBC formation, we here investigated the localization and physiological roles of endocytic proteins, amphiphysin 1 and dynamin 2, at TBCs. We demonstrated by immunofluorescence that the endocytic proteins were concentrated at TBCs, where they colocalized with cytoskeletal proteins, such as actin and vinculin. Immunoelectron microscopy disclosed that both amphiphysin 1 and dynamin 2 were localized on TBC membrane. Next, we histologically examined the testis from amphiphysin 1 deficient {Amph(-/-)} mice. Morphometric analysis revealed that the number of TBCs was significantly reduced in Amph(-/-). The ratio of stage VIII seminiferous tubules was increased, and the ratio of stage IX was conversely decreased in Amph(-/-). Moreover, unreleased spermatids in stage VIII seminiferous tubules were increased in Amph(-/-), indicating that spermatid release and the following transition from stage VIII to IX was prolonged in Amph(-/-) mice. These results suggest that amphiphysin 1 and dynamin 2 are involved in TBC formation and spermatid release at Sertoli cells.

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.

Transfection of Sertoli cells ex vivo; Opening the door to the in vitro study of junction turnover in the seminiferous epithelium

Sertoli cells interact with adjacent Sertoli cells and with spermatids through elaborate intercellular adhesion junctions termed ectoplasmic specializations (ESs). Among adhesion molecules present at the sites is Nectin-2. Tubulobulbar complexes have been proposed to play a role in junction internalization. Here we establish a culture model for exploring the role of tubulobulbar complexes during junction turnover. Cell clumps enriched for Sertoli cells were isolated from ten day-old rat pups and cultured on Matrigel coated Transwells. These cells established a columnar morphology and formed identifiable basal ESs. Significantly, structures resembling tubulobulbar complexes developed at the junction sites. We generated eGFP-tagged nectin-2 constructs and transfected them first into Hela cells (cells that do not normally form adherens junctions) and then into our primary cultures of morphologically differentiated Sertoli cells. In Hela cells, eGFP-Nectin-2 localized to sites of intercellular contact between adjacent cells indicating that the protein was expressed and targeted to the plasma membrane where it appeared to establish adherens junctions. In Sertoli cells, the protein was localized to areas consistent with the position of ESs and to tubular structures associated with them. Primary Sertoli cell cultures may be a model for studying the role of tubulobulbar complexes in junction turnover. CIHR MOP 62728

Cell–cell interactions at the ectoplasmic specialization in the testis

During spermatogenesis, the movement of developing germ cells across the seminiferous epithelium involves the restructuring of adherens junctions that form between Sertoli cells and between Sertoli and germ cells such as the ectoplasmic specialization (ES). At the ultrastructural level, the ES has been thoroughly studied for the past three decades. Until recently, however, relatively little has been known about the molecular architecture, not to mention the mechanism, that regulates the ES. Recent findings in the field have highlighted several areas of research that deserve attention in future studies. For example, proteins that constitute the ES can be targeted to compromise cell adhesion. This approach will not only provide a better understanding of ES dynamics, but also will yield innovative approaches for the development of male contraceptives. During spermatogenesis, the movement of developing germ cells across the seminiferous epithelium involves the restructuring of adherens junctions that form between Sertoli cells and between Sertoli and germ cells such as the ectoplasmic specialization (ES). At the ultrastructural level, the ES has been thoroughly studied for the past three decades. Until recently, however, relatively little has been known about the molecular architecture, not to mention the mechanism, that regulates the ES. Recent findings in the field have highlighted several areas of research that deserve attention in future studies. For example, proteins that constitute the ES can be targeted to compromise cell adhesion. This approach will not only provide a better understanding of ES dynamics, but also will yield innovative approaches for the development of male contraceptives. An anchoring junction between epithelial cells in which the cytoplasmic face of the plasma membrane is linked to actin filaments. The ES and tubulobulbar complex are testis-specific AJ types. A pathological condition in which no semen is produced. A pathological condition in which there is no sperm in the semen. A transmembrane protein belonging to the immunoglobulin superfamily. A barrier consisting of the tight junction and basal ES that divides the seminiferous epithelium into basal and apical compartments. An anchoring junction between epithelial cells in which the cytoplasmic face of the plasma membrane is linked to intermediate filaments. A testis-specific anchoring junction between Sertoli cells at the BTB (basal ES) and between Sertoli and elongate and elongating spermatids (apical ES) that uses actin filaments as its attachment site. An anchoring junction between epithelial cells and the extracellular matrix in which the cytoplasmic face of the plasma membrane is linked to actin. A member of the Src family of non-receptor protein tyrosine kinases. Cells in the seminiferous epithelium, also known as spermatogenic cells, that undergo mitosis and meiosis to give rise to haploid germ cells in which the genetic material is restricted to the head region. Somatic cells that extend from the base to the apex of the seminiferous epithelium and interact directly with developing germ cells throughout spermatogenesis; they are also known to nourish developing germ cells via their secretory products. A process consisting of biochemical, molecular and cellular events that occur simultaneously and result in one spermatogonium becoming 256 spermatozoa, one of which will fertilize an ovum. The process by which mature spermatids are released from the seminiferous epithelium at late stage VIII of the epithelial cycle. A specialized junction localized at the BTB that seals adjacent epithelial cells, preventing the passage of almost all molecules from one side of the epithelial sheet to another. A testis-specific anchoring junction found between Sertoli cells and elongated spermatids that uses actin filaments as its attachment site.

Sertoli-Sertoli and Sertoli-germ cell interactions and their significance in germ cell movement in the seminiferous epithelium during spermatogenesis.

Spermatogenesis is the process by which a single spermatogonium develops into 256 spermatozoa, one of which will fertilize the ovum. Since the 1950s when the stages of the epithelial cycle were first described, reproductive biologists have been in pursuit of one question: How can a spermatogonium traverse the epithelium, while at the same time differentiating into elongate spermatids that remain attached to the Sertoli cell throughout their development? Although it was generally agreed upon that junction restructuring was involved, at that time the types of junctions present in the testis were not even discerned. Today, it is known that tight, anchoring, and gap junctions are found in the testis. The testis also has two unique anchoring junction types, the ectoplasmic specialization and tubulobulbar complex. However, attention has recently shifted on identifying the regulatory molecules that "open" and "close" junctions, because this information will be useful in elucidating the mechanism of germ cell movement. For instance, cytokines have been shown to induce Sertoli cell tight junction disassembly by shutting down the production of tight junction proteins. Other factors such as proteases, protease inhibitors, GTPases, kinases, and phosphatases also come into play. In this review, we focus on this cellular phenomenon, recapping recent developments in the field.

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.

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.

Zyxin, axin, and Wiskott-Aldrich syndrome protein are adaptors that link the cadherin/catenin protein complex to the cytoskeleton at adherens junctions in the seminiferous epithelium of the rat testis.

During spermatogenesis, the movement of germ cells across the seminiferous epithelium is associated with extensive junction restructuring. Yet the underlying mechanism (or mechanisms) that regulates these events is largely unknown. If the molecular architecture of the cell-cell actin-based adherens junction (AJ), such as ectoplasmic specialization (ES) and tubulobulbar complex- two testis-specific AJ types, is known, many functional mechanistic studies can be designed. We thus undertook an investigation to study 3 adaptors in the seminiferous epithelium: zyxin, axin, and Wiskott-Aldrich syndrome protein (WASP). All 3 adaptors were shown to be products of Sertoli and germ cells. Zyxin was shown to be a stage-specific protein that was most prominent during stages V-VII and restricted mostly to pachytene spermatocytes, but it could also be detected at the site of basal and apical ectoplasmic specialization (ES). Zyxin, axin, and WASP were shown to be structurally linked to the N-cadherin/beta-catenin/alpha-actinin/actin complex but not to the nectin-3/afadin or the beta 1-integrin-mediated protein complexes. Interestingly, zyxin, axin, and WASP are also structurally linked to vimentin (an intermediate filament protein) and alpha-tubulin (the subunit of a microtubule), which suggests that they have a role (or roles) in the regulation of the dynamics of the desmosome-like junction and microtubule. These results illustrate that zyxin, axin, and WASP are adaptors in both AJs and intermediate filament-based desmosome-like junctions. This raises the possibility that classic cadherins are also associated with vimentin-based intermediate filaments via these adaptors in the testis. While virtually no N-cadherin was found to associate with vimentin in the seminiferous tubules, it did associate with vimentin when testis lysates were used. Interestingly, about 5% of the E-cadherin associated with vimentin in isolated seminiferous tubules, and about 50% of the E-cadherin in the testis used vimentin as its attachment site. These data suggest that cadherins in the testis, unlike those in other epithelia, use different attachment sites to anchor the cadherin/catenin complex to the cytoskeleton. The levels of zyxin, axin, and WASP were also assessed during AF-2364-mediated AJ disruption of the testis, which illustrated a time-dependent protein reduction that was similar to the trends observed in nectin-3 and afadin but was the opposite of those observed for N-cadherin and beta-catenin, which were induced. Collectively, these results illustrate that while these adaptors are structurally associated with the cadherin/catenin complex in the testis, they are regulated differently.

Calponin is expressed by Sertoli cells within rat testes and is associated with actin-enriched cytoskeleton.

Within seminiferous tubules, Sertoli cells form three types of actin-filament-containing cell junctions, viz., tight junctions, ectoplasmic specializations, and tubulobulbar complexes. These Sertoli cell junctions are involved in the formation of the blood-testis barrier, germ cell translocation, and the release of spermatozoa. Actin and actin-binding proteins are important for these functions. In the present study, a monoclonal antibody against human smooth-muscle-cell calponin detected a 38-kDa protein in a total protein extract of rat testis. The protein has a molecular weight identical to that of aorta calponin and binds calmodulin. Calponin mRNA was detected in the testis and cultured rat Sertoli cells by the reverse transcription/polymerase chain reaction method. Thus, the 38-kDa protein present in the testes is a basic isoform of calponin. In adult rats (70 days of age or older), calponin was detected within seminiferous tubules at sites of Sertoli cell junctions. Cultured Sertoli cells express calponin, whereas cultured spermatogonia do not. When rat testicular tissue was fractionated into cytosol, membrane, and cytoskeleton fractions, calponin was detected in all three fractions. Calcium, potassium, and okadaic acid reduced the amount of calponin associated with the cytoskeleton. These data suggest that Sertoli cells express calponin. The function of calponin within the testis is as yet unknown.

The acrosome-acroplaxome-manchette complex and the shaping of the spermatid head

A combination of exogenous contractile forces generated by a stack of F-actin-containing hoops embracing the apical region of the elongating spermatid nucleus and an endogenous modulating mechanism dependent on the spermatid-containing acrosome-acroplaxome-manchette complex may play a cooperative role in the shaping of the spermatid head. In addition, the manchette is a key element in the transport of vesicles and macromolecules to the centrosome and developing spermatid tails as well as in nucleocytoplasmic transport. The proposed model of spermatid head shaping is based on: 1) currently known structural and molecular components of the F-actin hoops, the main cytoskeletal element of the Sertoli cell ectoplasmic specializations; 2) the molecular features of acrosome biogenesis; 3) the assembly of a subacrosomal cytoskeletal plate called the acroplaxome; and 4) the spatial relationship of the acrosome-acroplaxome complex with the manchette, a transient microtubular/actin-containing structure. During acrosome biogenesis, the acroplaxome becomes the nucleation site to which Golgi-derived proacrosomal vescicles tether and fuse. The acroplaxome has at least two functions: it anchors the developing acrosome to the elongating spermatid head. It may also provide a mechanical scaffolding plate during the shaping of the spermatid nucleus. The plate is stabilized by a marginal ring with junctional complex characteristics, adjusting to exogenous clutching forces generated by the stack of Sertoli cell F-actin-containing hoops applied to the elongating spermatid head. A tubulobulbar complex, formed by cytoplasmic processes protruding from the elongating spermatid head extending into the adjacent Sertoli cell, is located at the concave side of the spermatid head. The tubulobulbar complex might provide stabilizing conditions, together with the actin-afadin-nectin-2/nectin-3 adhesion unit, to enable sustained and balanced clutching exogenous forces applied during the elongation of the spermatid head.

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.

Synopsis: 2003 Annual Meeting of the American Society of Andrology.

Synopsis: 2003 Annual Meeting of the American Society of Andrology.

Spermiogenesis and spermiation in a monotreme mammal, the platypus, Ornithorhynchus anatinus.

Spermatogenesis in the platypus (Ornithorhynchus anatinus) is of considerable biological interest as the structure of its gametes more closely resemble that of reptiles and birds than marsupial or eutherian mammals. The ultrastructure of 16 steps of spermatid development is described and provides a basis for determining the kinetics of spermatogenesis. Steps 1-3 correspond to the Golgi phase of spermatid development, steps 4-8 correspond to the cap phase, steps 9-12 are the acrosomal phase, and steps 13-16 are the maturation phase. Acrosomal development follows the reptilian model and no acrosomal granule is formed. Most other features of spermiogenesis are similar to processes in reptiles and birds. However, some are unique to mammals. For example, a thin, lateral margin of the acrosome of platypus sperm expands over the nucleus as in other mammals, and more than in reptiles and birds. Also, a tubulobulbar complex develops around the spermatid head, a feature which appears to be unique to mammals. Further, during spermiation the residual body is released from the caudal end of the nucleus of platypus sperm leaving a cytoplasmic droplet located at the proximal end of the middle piece as in marsupial and eutherian mammals. Other features of spermiogenesis in platypus appear to be unique to monotremes. For example, nuclear condensation involves the formation of a layer of chromatin granules under the nucleolemma, and development of the fibrous sheath of the principal piece starts much later in the platypus than in birds or eutherian mammals.

Morphogenesis of the acrosome during the final steps of rat spermiogenesis with special reference to tubulobulbar complexes

We report the ultrastructural changes in acrosome morphology during the final steps of rat spermiogenesis, focusing on the relationship between the acrosome morphogenesis and the tubulobulbar complexes (TBC) development. During steps 18-19, the electron-lucent area in the dorsal cortex of the anterior acrosome gradually diminished, and finally, the acrosome became condensed and reduced its volume. Simultaneously with this tightening up of the acrosome, TBC developed from the head portion of late spermatids, protruding into the surrounding Sertoli cells. To investigate the incorporation of acrosomal contents into TBC, step 19 spermatids were stained by periodic acid-Schiff (PAS) reaction and by using the anti-acrosomal monoclonal antibody mMN7. Both PAS-reactivity and the mMN7-immunoreactivity were found in the TBC, as well as in the acrosome. In addition, the acrosome projected into the TBC-like structure, and materials of a density similar to that of the acrosome were observed in the core of the TBC. These results suggest that the TBC eliminate excess acrosomal contents prior to spermiation.

Morphogenesis of the acrosome during the final steps of rat spermiogenesis with special reference to tubulobulbar complexes.

We report the ultrastructural changes in acrosome morphology during the final steps of rat spermiogenesis, focusing on the relationship between the acrosome morphogenesis and the tubulobulbar complexes (TBC) development. During steps 18-19, the electron-lucent area in the dorsal cortex of the anterior acrosome gradually diminished, and finally, the acrosome became condensed and reduced its volume. Simultaneously with this tightening up of the acrosome, TBC developed from the head portion of late spermatids, protruding into the surrounding Sertoli cells. To investigate the incorporation of acrosomal contents into TBC, step 19 spermatids were stained by periodic acid-Schiff (PAS) reaction and by using the anti-acrosomal monoclonal antibody mMN7. Both PAS-reactivity and the mMN7-immunoreactivity were found in the TBC, as well as in the acrosome. In addition, the acrosome projected into the TBC-like structure, and materials of a density similar to that of the acrosome were observed in the core of the TBC. These results suggest that the TBC eliminate excess acrosomal contents prior to spermiation.

Morphogenesis of the acrosome during the final steps of rat spermiogenesis with special reference to tubulobulbar complexes

Morphogenesis of the acrosome during the final steps of rat spermiogenesis with special reference to tubulobulbar complexes

Spermiogenesis And the Cycle of The Seminiferous Epithelium in The Platypus, Ornithorhynchus Anatinus.

Department of Biological Sciences, University of Newcastle, New South Wales 2308. Ultrastructural studies of spermiogenesis in platypuses indicate that they display some unique features. The origin and morphology of the perforatorium are different than in sperm from birds and therian mammals, and development of the fibrous sheath of the principal piece starts much later in the platypus. However, some features of spermiogenesis in the platypus are similar to birds, but different from therian mammals. For example, the dense granules in the developing acrosome of therian mammals are absent in platypus spermatids. Other features of spermiogenesis in the platypus are similar to processes described in therian mammals, but not birds. For example, the acrosome of platypus sperm is not confined to the rostral surface of the nucleus as in avian sperm, but extends laterally over the nucleus as in sperm from therian mammals. Further, during spermiation the tubulobulbar complexes were also observed in the platypus. Sixteen steps of spermatid development were identified and used to classify the cycle of the seminiferous epithelium into 12 stages. The area of the wall of a seminiferous tubule, which is occupied by a stage of the cycle of the seminiferous epithelium in the platypus (about 6-8 stages of the cycle per tubular cross-section), is between that of therian mammals (1-4 stages) and birds (up to 12 stages). The cycle ofseminiferous epithelium will provide a basis to determine the rate of sperm production in the platypus.

Sertoli cell-spermatid tubulobulbar complexes in the ram.

The fine structure of the tubulobulbar complex (TBC) of the ram seminiferous epithelium was studied in immersion-fixed testicular samples obtained in autumn (sheep's mating period). The TBC was visible during the last two stages of the seminiferous epithelium cycle. It originated as a tubular invagination of the Sertoli cytoplasm harbouring a complementary evagination for the spermatid head. Later, it expanded into a bulbous structure, finally becoming detached from the spermatid and apparently phagocytosed by the Sertoli cell. The significance of this transient structure is discussed and compared with previous reports for other eutherian mammals.

Site of Actin Fiber Depolymerization within Rat Sertori Cells as Detected by Localization of .BETA.-thymosin.

The localization of actin sequestering peptide thymosin β10 in rat testis was analyzed by light and electron microscopic immunohistochemistry using the anti-thymosin β10 antibody. A positive reaction of thymosin β10 was detected in a closely associated portion within the developing spermatid head. In step 15 spermatids, localized deep in the seminiferous epithelium, a prominent positive reaction was detected in the Sertoli cell cytoplasm adjacent to the spermatids. In step 19 spermatids, just before spermiation, thymosin β10 was highly concentrated in the Sertoli cell cytoplasm situated at the concave side of the sickle shaped spermatid head, where the tubulobulbar complex is formed. According to previous ultrastructural observations, actin bundles of ectoplasmic specialization change to electron dense material just prior to spermiation. The localization of thymosin β10 suggests that large amounts of actin monomers accumulate in the region of the tubulobulbar complex just prior to spermiation. These findings suggest that thymosin β10 accumulation is correlated with fragmentation of the ectoplasmic specialization, and strongly support the hypothesis that the tubulobulbar complex is an actin-disorganizing organelle of the Sertoli cell.

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.