Tubulobulbar Complexes Research Articles

Sarco/Endoplasmic Reticulum Ca2+‐ATPase 2 (SERCA2) is Present at Tubulobulbar Complexes

Endocytic structures known as tubulobulbar complexes (TBCs) internalize intact intercellular junction proteins at sites of intercellular attachment between spermatids and Sertoli cells prior to sperm release. TBCs are highly specialized clathrin‐based endocytosis machines. They also resemble podosomes which have an elongated membrane tube and an associated dendritic actin network. However, unlike podosomes that have a membrane core of a single plasma membrane, the core of TBCs consists of two plasma membranes: the membrane of the Sertoli cell which the structure is invaginated into and the membrane of a spermatid. TBCs have a bulb region in the distal third part of the complex which does not have the cuff of actin but is closely associated with a cisterna of endoplasmic reticulum (ER). We hypothesize that the plasma membrane/ER junction is involved in Ca2+ signaling. If this is true then we would expect to find molecular machinery involved in calcium signaling mechanisms in other systems. In this study, we used immunofluorescence to probe for the calcium pump, sarco/endoplasmic reticulum Ca2+ ‐ATPase 2 (SERCA2), which pumps Ca2+ into the ER. Cryosections and epithelial fragments from perfusion‐fixed rat testes were probed with antibodies against SERCA2 and an appropriate fluorescent secondary antibody. The SERCA2 antibody has a labeling pattern that extends from the base of the epithelium to the apex which is consistent with a Sertoli cell pattern. As seen in both epithelial fragments and cryosections, the SERCA2 antibody labels regions where we would expect TBCs to be. Our results show that one possible component of a calcium signaling mechanism is present in regions containing TBCs.Support or Funding InformationSupported by the Natural Sciences and Engineering Research Council of Canada Discovery Grants program.

The Non‐Receptor Tyrosine Kinase, Src, and its Regulator PTP1B are Present at Tubulobulbar Complexes

During spermiation, the intercellular junctions between Sertoli cells and the spermatids must be internalized before the spermatid can be released into the lumen of the seminiferous tubule. Prior to release from the epithelium, the spermatids are engulfed in protrusions of Sertoli cell cytoplasm which are referred to as apical processes. Unique endocytic structures termed, tubulobulbar complexes (TBCs), internalize these intercellular junctions during sperm release. TBCs have three regions: a proximal tubule, a bulbar region and a distal tubule ending in a clathrin‐coated pit. At sites of intercellular junctions between Sertoli and sperm cells, TBCs form by invaginating into the Sertoli cell and dragging the spermatid's membrane along with it through its intercellular attachments. This results in the entire structure consisting of a double membrane. The tubular regions are surrounded by a dendritic actin network, which is absent at the bulbar region. The bulbar region is a swelling in the distal third part of the TBC and is closely associated with a cisterna of endoplasmic reticulum. Eventually the bulb region of the TBC vesiculates and enters the endosomal pathway of the Sertoli cell. It has been shown by immunoprecipitation that Src, a non‐receptor tyrosine kinase, associates with components of the intercellular junctions between Sertoli cells and spermatids. One of the substrates that Src is able to phosphorylate is cortactin, a component of the dendritic actin networks at TBCs. We suspect that Src is involved in regulating the actin network at TBCs. In this study, we used immunofluorescence to evaluate whether or not Src and its phosphatase regulator, protein tyrosine phosphatase 1b (PTP1B), are associated with TBCs. Rat testes were perfusion‐fixed and cryosectioned or fragmented to get epithelial fragments. Sections and fragments were probed with antibodies against Src or PTP1B and an appropriate fluorescent secondary antibody. The Src antibody labels linear tracts in apical processes that could represent the tubular regions of TBCs. In cryosections, the PTP1B antibody has a Sertoli cell‐labeling pattern that extends from the base of the seminiferous epithelium to the apex. In fragments and cryosections, the PTP1B antibody labels regions of the Sertoli cell where TBCs would be expected to form. This evidence suggests that Src and its regulator PTP1B are present at TBCs.Support or Funding InformationSupported by the Natural Sciences and Engineering Research Council of Canada Discovery Grants program.

Immunoelectron Microscopy of Endocytosis and Junction Proteins at Tubulobulbar Complexes in the Mammalian Testis

The seminiferous epithelium is the site of spermatogenesis, the cellular basis for male fertility. Large junction complexes separate the epithelium into basal and adluminal compartments, as well as attach morphologically differentiating spermatids to apical regions of Sertoli cells. During spermatogenesis, turnover of these large junction complexes enables both the next generation of spermatogenic cells to move from the basal into adluminal regions of the epithelium, and mature spermatozoa to be released into the duct system of the reproductive tract. We have proposed that unique structures termed tubulobulbar complexes (TBCs) are subcellular clathrin‐based endocytosis machines that internalize intact intercellular junctions during spermatid translocation and sperm release. If this is true, then proteins associated with endocytosis in general should be associated with TBCs. Previous immunofluorescence results have indicated that Rab5 and EEA1, together with junction proteins, are present in areas occupied by TBCs. However, association with specific compartments of TBCs at the ultrastructural level has not been done. Conventional methods of immunoelectron microscopy do not provide adequate preservation of membranes to clearly localize these proteins to TBCs. We have applied a recently published pre‐embedding protocol (Melo et al. 2014, Nature Protocols 9:2382‐2394) to the rat testis and show that Rab5 associates with the bulb region of TBCs. EEA1 is associated mainly with regions after the bulbs have been internalized. Nectin‐2 associates with junctions and with TBCs. This data supports the hypothesis that TBCs are involved with endocytosis of junction proteins.Support or Funding InformationNSERC Discovery Grant to A. Wayne Vogl

Differential roles of estrogen receptors, ESR1 and ESR2, in adult rat spermatogenesis

Estrogens, through their receptors, play an important role in regulation of spermatogenesis. However, the precise role of the estrogen receptors (ESR1 and ESR2) has been difficult to determine as in vivo estradiol treatment would signal through both the ESRs. Hence we had developed in vivo selective ESR agonist administration models in adult male rats to decipher the individual roles of the ESRs. Treatment with both ESR1 and ESR2 agonists decreased sperm counts after 60 days of treatment. The present study aimed to delineate the precise causes of decreased sperm counts following treatment with the two ESR agonists. Treatment with ESR1 agonist causes an arrest in differentiation of round spermatids into elongated spermatids, mainly due to down-regulation of genes involved in spermiogenesis. ESR2 agonist administration reduces sperm counts due to spermiation failure and spermatocyte apoptosis. Spermiation failure observed is due to defects in tubulobulbar complex formation because of decrease in expression of genes involved in actin remodelling. The increase in spermatocyte apoptosis could be due to increase in oxidative stress and decrease in transcripts of anti-apoptotic genes. Our results suggest that the two ESRs regulate distinct aspects of spermatogenesis. ESR1 is mainly involved with regulation of spermiogenesis, while ESR2 regulates spermatocyte apoptosis and spermiation. Activation of estrogen signaling through either of the receptors can affect their respective processes during spermatogenesis and lead to low sperm output. Since many environmental estrogens can bind to the two ESRs with different affinities, these observations can be useful in understanding their potential effects on spermatogenesis.

An Alternative Model of Tubulobulbar Complex Internalization During Junction Remodeling in the Seminiferous Epithelium of the Rat Testis.

Tubulobulbar complexes (TBCs) are elongate subcellular machines responsible for internalizing intercellular junctions during sperm release. Each complex consists of a double-membrane tubular core terminating in a clathrin-coated pit. The core is surrounded by a network of actin filaments, and a distinct swelling or bulb, which lacks an association with actin, develops in the distal third of the structure. The bulb eventually buds from the complex and enters endocytic compartments of the Sertoli cell. The relationship of the actin cuff to the formation and budding of the bulb is not known. To gain insight into this relationship, we perturbed the actin networks of TBCs with cytochalasin D. When isolated testes were perfused with a physiological buffer containing cytochalasin D, apical TBCs at stage VII of spermatogenesis were associated with lower levels of actin compared to controls. At the ultrastructural level, the actin networks in cytochalasin D-treated testes appeared patchy, and ectopic bulbs and swollen tubular regions occurred. When normal untreated samples at early stage VII were analyzed, large elongate bulbs and short tubular sections were observed. Together, these results suggest a new model for TBC vesiculation in which the actin network begins to disassemble and the tubular region begins to swell into a bulb. As actin disassembly continues, the coated pit and most of the tubular region are incorporated into the enlarging bulb. The remaining short neck of the bulb near the base of the complex undergoes scission, and the bulb is internalized.

A New Model of Tubulobulbar Complex Budding in the Seminiferous Epithelium of the Testis

Endocytic structures known as tubulobulbar complexes internalize intercellular attachments during sperm release. They are elongate structures capped with a clathrin‐coated pit. Their membranous core is a double‐membrane tube which is surrounded by a network of actin filaments. A bulbous region develops in the distal region which lacks the associated actin network but has a close association with cisternae of endoplasmic reticulum. The bulb eventually buds from the complex and enters endocytic compartments of the Sertoli cell. When rat testes were perfused for one hour ex vivo with oxygenated Krebs Henseleit buffer (with BSA) at 33°C containing either cytochalasin D (10, 20 or 40 µM) or the drug carrier (DMSO), apical TBCs at stage VII of spermatogenesis were associated with lower levels of actin in treated epithelia relative to controls. When evaluated at the ultrastructural level, the actin networks appeared patchy. In addition, ectopic bulbs and swollen tubular regions occurred. When thin sections of normal untreated samples were analyzed, large elongate bulbs and short tubular sections occasionally were observed. Together, these results suggest a new model for TBC vesiculation in which the actin network begins to disassemble and the tubular region begins to swell into a bulb. As the actin network continues to disassemble, the coated pit and most of the tubular region are incorporated into the enlarging bulb. Then the remaining short ‘neck’ of the bulb undergoes scission. Supported by a NSERC Discovery Grant to AWV.

Estrogen and androgen regulate actin-remodeling and endocytosis-related genes during rat spermiation

Spermiation, the sperm release process, is imperative to male fertility and reproduction. Morphologically, it is characterized by removal of atypical adherens junctions called ectoplasmic specializations, and formation of transient endocytic devices called tubulobulbar complexes requiring cytoskeleton remodeling and recruitment of proteins needed for endocytosis. Earlier, estrogen administration to adult male rats was seen to cause spermiation failure due to disruption of tubulobulbar complexes. This was accompanied by reduction in intratesticular testosterone levels and increase in intratesticular estrogen along with deregulation of genes involved in cytoskeleton remodeling (Arpc1b, Evl and Capg) and endocytosis (Picalm, Eea1 and Stx5a). In the present study, we aim to understand the role of estrogen and androgen in regulating these genes independently using seminiferous tubule culture system treated with estrogen, androgen or agonists and antagonists of estrogen receptors. We find that transcripts of Arpc1b, Evl and Picalm are responsive to estrogen while those of Picalm, Eea1 and Stx5a are responsive to androgen. We also find that the estrogen regulation of Arpc1b and Evl is mediated through estrogen receptor β and that of Picalm occurs through estrogen receptors α and β. Localization of these proteins at or in the vicinity of tubulobulbar complexes reveals that ARPC1B, EVL, PICALM, EEA1 and STX5A seem to be involved in spermiation. Thus, estrogen and androgen regulate specific genes in seminiferous tubules that could play a role in spermiation.

PRKRA Localizes to Nuage Structures and the Ectoplasmic Specialization and Tubulobulbar Complexes in Rat and Mouse Testis

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including PRKRA, TRBP, and Dicer. RISC localizes to P-bodies. The nuage of the spermatogenic cells has function similar to the P-bodies. We study whether PRKRA localizes to nuage of spermatogenic cells of rat and mouse. PRKRA localized to four types of nuage structures, including aggregates of 60–90 nm particles, irregularly-shaped perinuclear granules, and intermitochondrial cement of pachytene spermatocytes, and chromatoid bodies of round spermatids. In addition, PRKRA is associated with dense material surrounding tubulobulbar complexes and with the ectoplasmic specialization. The results suggest that PRKRA functions in the nuage as an element of RNA silencing system and plays unknown role in the ectoplasmic specialization and at the tubulobulbar complexes of Sertoli cells attaching the head of late spermatids.

Sertolin mediates blood-testis barrier restructuring.

Two important events that occur during mammalian spermatogenesis are the release of elongated spermatids at late stage VIII of the seminiferous epithelial cycle and the restructuring of the blood-testis barrier (BTB) during stages VIII-XI. Still, it is not completely understood how these cellular events are accomplished within the seminiferous epithelium. In the present study, we investigate how sertolin, a protein that was initially identified, cloned, and partially characterized by our laboratory, functions in these critical events. Sertolin was found at the BTB, as well as at the apical ectoplasmic specialization and apical tubulobulbar complex, where it colocalized with epidermal growth factor receptor kinase substrate 8 and actin-related protein 3, two actin-regulatory proteins. Knockdown of sertolin by RNA interference showed Sertoli cell barrier function to be enhanced when assessed by transepithelial electrical resistance measurements and immunolocalization experiments. By contrast, the integrity of the BTB was disrupted when sertolin was overexpressed in vitro and in vivo. Sertolin overexpression also prompted germ cell loss from the seminiferous epithelium. Taken collectively, these results suggest that sertolin may be involved in coordinating spermatid release and BTB restructuring during spermatogenesis in the rat.

Localization of proteins involved in endocytosis at tubulobulbar complexes in rat testes

Tubulobulbar Complexes (TBCs) are actin-rich structures formed between Sertoli-cells and spermatids at the time of sperm release. The main functions of the TBCs are to remove excess spermatid cytoplasm and acrosomal contents, internalize and recycle junctional complexes by endocytosis prior to spermiation. However, in addition to recycling some of the molecules undergo lysosomal degradation. The molecular machinery involved in endocytosis at the TBCs is not well understood. To bridge this gap localization of various proteins, involved at various steps of endocytosis studied in other systems, was demonstrated in TBCs using testicular fragmented material or sections by immunoblotting and immunofluroscence. The presence of key molecules like Vamp-2, syntaxin and Lamp-2 indicates occurrence of lysosomal degradation in addition to junctional recycling at the TBCs present at the time of sperm release. TBCs are endocytic devices functioning to recycle junctional molecules or remove spermatid cytoplasm that were present between spermatids and Sertoli-cells all through the process of spermatid maturation and in turn regulate male fertility.

Estrogen Effects on Actin Cytoskeletal and Endocytic Proteins Associated With Tubulobulbar Complex Disruption in Rat Testes

Tubulobulbar complexes (TBCs), evaginations of mature spermatids, penetrate into the surrounding Sertoli cell cytoplasm of testis seminiferous epithelium during rat spermatogenesis. These structures prepare mature spermatids for their release into the seminiferous tubular lumen via a process called spermiation. Based on their functions of transient attachment and endocytosis, many actin-regulatory and endocytic proteins are associated with TBCs. Previously, exogenous 17β-estradiol administration to adult male rats showed spermiation failure that was attributed to TBC disruption. To determine the molecular basis of estrogen-induced TBC disruption, we examined the expressions and localizations of actin-regulatory proteins, endocytic proteins, Rho-GTPases, and phosphorylation in TBCs during sperm release. Results demonstrated absence of neural Wiscott Aldrich syndrome protein, cortactin, adaptor-related protein complex 2 sigma-1 subunit, dynamin 2, cell division control protein 42, and phosphocortactin in the concavity of spermatid head where TBCs are present without change in their protein expression levels. Absence of these proteins could have led to collapse of the TBC structure which is involved in its formation and function.

A network of spectrin and plectin surrounds the actin cuffs of apical tubulobulbar complexes in the rat

Tubulobulbar complexes (TBCs) are actin-related endocytic structures that internalize intercellular junctions in the seminiferous epithelium. The structures consist of elongate tubular projections of the attached plasma membranes of two adjacent cells that project into Sertoli cells. This double membrane core is cuffed by a dentritic actin network and is capped at its end by a clathrin-coated pit. Here we explore the possibility that elements of the spectrin cytoskeleton are associated with clusters of tubulobulbar complexes that develop at adhesion junctions between late spermatids and Sertoli cells at the apex of the epithelium, and extend what is known about the distribution of plectin at the sites.Cryo-sections of perfusion-fixed testes and apical processes of Sertoli cells mechanically dissociated from perfusion-fixed testes were probed for spectrin, EPB41, and actin and analyzed using conventional fluorescence microscopy and confocal microscopy. Data sets from confocal microscopy were analyzed further in three-dimensional reconstructions using computer software. Additional apical Sertoli cell processes were probed for plectin and analyzed using conventional fluorescence microscopy. Antibodies generated against elements of the spectrin cytoskeleton react with material around and between the actin cuffs of tubulobulbar complexes, but appear excluded from the actin cuffs themselves. A similar staining pattern occurs with a probe for plectin. Immunoelectron microscopy confirmed the staining patterns observed by fluourescence microscopy. Based on our results, we suggest that a network of spectrin and plectin forms a scaffold around tubulobulbar complexes that may provide support for the actin network that cuffs each complex and also link adjacent complexes together.

A Novel Subcellular Machine Contributes to Basal Junction Remodeling in the Seminiferous Epithelium1

Tubulobulbar complexes are cytoskeleton-related membrane structures that develop at sites of intercellular attachment in mammalian seminiferous epithelium. At apical junctions between Sertoli cells and spermatids, the structures internalize adhesion junctions and are a component of the sperm release mechanism. Here we explore the possibility that tubulobulbar complexes that form at the blood-testis barrier are subcellular machines that internalize basal junction complexes. Using electron microscopy, we confirmed that morphologically identifiable tight and gap junctions are present in basal tubulobulbar complexes in rats. In addition, immunological probes for claudin-11 (CLDN11), connexin-43 (GJA1), and nectin-2 (PVRL2) react with linear structures at the light level that we interpret as tubulobulbar complexes, and probes for early endosome antigen 1 (EEA1) and Rab5 (RAB5A) react in similar locations. Significantly, fluorescence patterns for actin and claudin-11 indicate that the amount of junction present is dramatically reduced over the time period that tubulobulbar complexes are known to be most prevalent during spermatogenesis. We also demonstrated, using electron microscopy and fluorescence microscopy, that tubulobulbar complexes develop at basal junctions in primary cultures of Sertoli cells and that like their in vivo counterparts, the structures contain junction proteins. We use this culture system together with transfection techniques to show that junction proteins from one transfected cell occur in structures that project into adjacent nontransfected cells as predicted by the junction internalization hypothesis. On the basis of our findings, we present a new model for basal junction remodeling as it relates to spermatocyte translocation in the seminiferous epithelium.

Polyubiquitination and Proteasome Signals in Tubulobulbar Complexes of Rat Late Spermatids

To illustrate the involvement of tubulobulbar complexes (TBC) in ubiquitin-proteasome degradation of unnecessary proteins in the head cytoplasm of late spermatids, the localization of polyubiquitin and proteasome was studied by immunofluorescence and immunoelectron microscopy. Polyubiquitin localized to TBC and proteasome subunit α to dense materials surrounding the TBC in the cytoplasm of Sertoli cell enwrapping sickle-shaped spermatid heads. The results suggest that the TBC is a structural device for ubiquin-proteasome degradation of unnecessary proteins in the cytoplasm of spermatid head during rapid reduction of the head cytoplasm and nuclear compaction of late spermatids.

Differential Proteomic and Phospho-proteomic Analysis of Normal versus Failed Spermiation in Adult Rats by Label-Free LC-MS/MS

Spermiation is the final step of spermatogenesis and involves release of mature spermatids from the Sertoli cells. It is a complex process involving displacement and removal of spermatid cytoplasm, formation and degradation of tubulobulbar complexes and progressive loss of adhesive junctions, including ectoplasmic specializations and subsequent phagocytosis of residual bodies by the Sertoli cell. Spermiation occurs in stage VIII of the rat seminiferous epithelium cycle. In spite of the important role played by spermiation process in sperm output to the epididymis, very little is known about this process. To enhance our knowledge of the sperm release biology, we sought to understand the molecular events occurring at the time of spermiation. Towards this aim, we induced spermiation failure condition by 17β estradiol treatment to adult male rats and compared with the normal spermiation. On comparing both the groups, we identified a total of 104 differentially expressed proteins and 23 differentially expressed phosphoproteins by LC-MS/MS analysis and quantitation. Localization and expression of some of the validated differential proteins in the testes highlight their importance during the sperm release. The present study represents an initial step to understand the molecular basis of the process of spermiation.

Cortactin depletion results in short tubulobulbar complexes and spermiation failure in rat testes

SummaryTubulobulbar complexes are actin-related endocytic structures that form at sites of intercellular attachment in the seminiferous epithelium and are proposed to internalize intact junctions. In this study, we test the prediction that altering the structure/function of tubulobulbar complexes results in failure to release mature spermatids from Sertoli cells. We used an in vivo knockdown strategy to target cortactin, a component of tubulobulbar complexes, in Sprague Dawley rats. In each animal, one testis was surgically injected with cortactin siRNA reagents and the other testis was injected with non-targeting siRNA. After three days, experimental and control testes were processed for immunoblotting, electron microscopy or immunofluorescence microscopy. In testis sections immunostained for cortactin or labeled for filamentous actin, fluorescence microscopy revealed that tubulobulbar complexes were shorter in siRNA-treated testes relative to controls. Significantly, in the knockdown testes, spermiation was delayed in some tubules and had failed in others. When evaluated by electron microscopy, adhesion complexes (ectoplasmic specializations) remained associated with mature spermatids that failed to be released from Sertoli cells. Immunoblots both of whole testis lysates and of isolated seminiferous epithelial lysates confirmed that cortactin expression was knocked-down in experimental testes and in the seminiferous epithelium respectively, relative to controls. Moreover, in testes injected with siRNA reagents with a dye modification on one of the four targeting siRNA sequences, dye clusters were detected at the base of the epithelium confirming that the reagents entered Sertoli cells. Our results are consistent with the hypothesis that tubulobulbar complexes internalize intercellular junctions and that they are a significant component of the sperm release mechanism.

Testis Expressed Actin-Like 7b (Actl7b) Is Required for Mouse Spermatid Morphogenesis and Male Fertility.

During spermiogenesis round spermatids differentiate into elongated and then condensed spermatids followed by spermiation. Although the structural components and morphological changes in this complex process have been described in detail, relatively little is known about the mechanisms that drive these structural changes. The involvement of filamentous actin (F-actin) has been suggested for several aspects of spermatid differentiation, including acrosome formation and attachment to the nucleus, formation of tubulobulbar complexes and cytoplasmic removal, and spermiation. In addition, human male infertility is often associated with a high incidence of abnormally shaped sperm heads, suggesting that cytoskeletal regulation is important for male fertility. Actin-like 7b (Actl7b) is an intronless gene expressed in spermatids and conserved in mammals. Immunohistochemistry and indirect immunofluorescence were utilized to investigate the expression of ACTL7B. It is localized in the cytoplasm of round and elongating spermatids and co-localizes with phalloidin labeled F-actin) in or around the forming acrosome, suggesting a role in this process. To determine the functional relevance of (Actl7b), knockout mice were generated from targeted ES cells in which the (Actl7b) coding region was replaced with a LacZ reporter sequence, obtained from the KnockOut Mouse Project (KOMP) Repository. X-gal staining of tissues from heterozygous animals revealed that (Actl7b) is expressed in the testis and, unexpectedly, the brain. While (Actl7b) knockout mice develop to adulthood and appear normal, breeding studies revealed that (Actl7b) knockout males mate and produce vaginal plugs, but are infertile. Testis and epididymal weights and sperm counts are lower in knockout males than in wild type males. In addition, sperm heads are misshapen with a rounded appearance. Most of these sperm are immotile, with less than three percent showing minimal flagellar movement. These results indicate that ACTL7B is required for spermatid morphogenesis, sperm motility, and male fertility. They also suggest that ACTL7B is either the F-actin recognized by phalloidin or is required for F-actin assembly in spermatids. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (EME).

Tubulobulbar complex: cytoskeletal remodeling to release spermatozoa.

Tubulobulbar complexes (TBCs) are actin-based structures that help establish close contact between Sertoli–Sertoli cells or Sertoli–mature germ cells (spermatids) in the seminiferous tubules of the testes. They are actin-rich push-through devices that eliminate excess spermatid cytoplasm and prepare mature spermatids for release into the tubular lumen. Just prior to spermiation, the elongated spermatid interacts with the Sertoli cell via an extensive structure comprising various adhesion molecules called the apical ectoplasmic specialization which is partially replaced by the apical TBC, on the concave surface of the spermatid head. The sperm release process involves extensive restructuring, namely the disassembly and reassembly of junctions at the Sertoli–spermatid interface in the seminiferous epithelium. Based on the presence of different classes of molecules in the TBCs or the defects observed in the absence of TBCs, the main functions attributed to TBCs are elimination of excess spermatid cytoplasm, endocytosis and recycling of junctional molecules, shaping of the spermatid acrosome, and forming transient anchoring devices for mature spermatids before they are released. This review summarizes the recent findings that focus on the role of TBCs in cell cytoskeleton restructuring during sperm release in the testes and the molecular mechanism involved.

Tubulobulbar Complexes Formed In Sertoli Cells In Primary Cultures Associate With Intercellular Junction Elements

In the seminiferous epithelium, unique double membrane structures termed tubulobulbar complexes (TBCs) develop in association with basal junction complexes. These actin‐related structures are proposed to internalize intercellular junctions during the movement of spermatocytes from basal to adluminal compartments of the epithelium. To study the role of basal TBCs in junction turnover, we use a primary Sertoli cell culture system in which TBCs develop in the vicinity of junction complexes between Sertoli cells. We immunoprobed cultured Sertoli cells for the intercellular junction elements Claudin‐11, Connexin‐43 and Nectin‐2 and discovered rod‐like structures that protrude into Sertoli cells from the cell periphery. These protrusions resemble TBCs that form in the intact seminiferous epithelium. These protrusions also co‐localize with F‐actin, which is known to associate with the proximal tubular part of TBCs and can be used as a marker for TBCs. Significantly, cultured Sertoli cells that express GFP‐tagged Claudin‐11 and Connexin‐43 extend similar protrusions into neighboring non‐transfected Sertoli cells. Our results are consistent both with the conclusion that junction elements from neighboring Sertoli cells are found together within TBCs, and with the hypothesis that tubulobulbar complexes may play a role in the turnover of intercellular junctions. Supported by an NSERC Discovery Grant to AWV.Grant Funding Source : NSERC

Focal adhesion proteins Zyxin and Vinculin are co-distributed at tubulobulbar complexes

Tubulobulbar complexes (TBCs) are actin-related double-membrane invaginations formed at intercellular junctions in the seminiferous epithelium of mammalian testis. They occur at basal junction complexes between neighboring Sertoli cells and at apical junctions between Sertoli cells and spermatids. They are proposed to internalize intercellular junctions during the translocation of spermatocytes from basal to adluminal compartments of the seminiferous epithelium, and during sperm release from Sertoli cells. Although TBCs are specific to the seminiferous epithelium, they morphologically resemble podosomes in osteoclasts. Previously, we have reported that a key group of proteins consisting of N-WASp, Arp2/3, cortactin and dynamin that occur at podosomes also is present at TBCs. Here we explore the prediction that zyxin, a focal adhesion protein known to be present at podosomes, also is present at apical TBCs. A rabbit polyclonal anti-zyxin antibody (B71) was used to label fixed fragments and frozen sections of testis. In both fragments and sections, B71 labeled tubular regions of TBCs at apical sites of attachment between Sertoli cells and spermatids, in addition to being localized at actin related intercellular adhesion junctions termed ectoplasmic specializations. Although the function of zyxin at TBCs has yet to be determined, the protein is known to interact with the cytoplasmic domain of integrins at focal adhesions, and integrins are known to be present in TBCs.