Tubulobulbar Complexes Research Articles

Internalization of adhesion junction proteins and their association with recycling endosome marker proteins in rat seminiferous epithelium

Tubulobulbar complexes (TBCs) are elaborate cytoskeleton-related structures that are formed in association with intercellular junctions in the seminiferous epithelium. They consist of a cylindrical double-membrane core composed of the plasma membranes of the two attached cells, cuffed by a dendritic network of actin filaments. TBCs are proposed to be subcellular machines that internalize intercellular junctions during the extensive junction remodeling that occurs during spermatogenesis. At the apical sites of attachment between Sertoli cells and spermatids, junction disassembly is part of the sperm release mechanism. In this study, we used immunological probes to explore junction internalization and recycling at apical TBCs in the rat seminiferous epithelium. We demonstrate that β1-integrin and nectin 2 were concentrated at the ends of TBCs and for the first time show that the early endosome marker RAB5A was also distinctly localized at the ends of TBCs that appear to be the 'bulbar' regions of the complexes. Significantly, we also demonstrate that the 'long-loop' recycling endosome marker RAB11A was co-distributed with nectin 2 at junctions with early spermatids deeper in the epithelium. Our results are consistent with the hypothesis that TBCs associated with late spermatids internalize adhesion junctions and also indicate that some of the internalized junction proteins may be recycled to form junctions with the next generation of spermatids.

Altered phosphorylation and distribution status of vimentin in rat seminiferous epithelium following 17β-estradiol treatment

Vimentin, type III intermediate filament, has stage-specific localization in the Sertoli cell. In the rat, during stages I-V and XI-XIV of the seminiferous epithelium, vimentin is localized in the perinuclear area with filaments projecting into the apical region toward the developing germ cells. These filaments decrease in length at stages VI-VII with perinuclear staining in stages VIII-IX, when spermiation occurs. Our earlier studies following 17β-estradiol treatment to adult male rats demonstrated an increase in germ cell apoptosis, spermiation failure and disruption of Sertoli cell microfilaments and microtubules. The present study was undertaken to determine the stage-specific distribution of vimentin and its involvement in spermiation failure and germ cell apoptosis. Immunofluorescence studies revealed that in contrast to the perinuclear localization with small extensions in control stages VII-IX, long extensions radiating apically to the spermatids in deep recess were observed in the treated group. Immunoprecipitation studies showed marked absence of phosphorylated vimentin in stages VII-VIII in the treated group. Further, localization of plectin, cytoskeletal linker protein, showed decrease in all the stages of spermatogenesis following estradiol treatment. Interestingly, for the first time the localization of plectin in the tubulobulbar complex was observed. In conclusion, the study suggests that estradiol treatment leads to an effect on vimentin phosphorylation, which could have inhibited the disassembly of vimentin leading to retention of apical projection in stages VII-VIII. These effects could be presumably due to a decrease in plectin, affecting the reorganization of vimentin and therefore the apical movement of spermatids, leading to spermiation failure.

Hormonal Regulation of the Tubulobulbar Complex in the Testis.

Spermatogenesis is absolutely dependent on follicle stimulating hormone (FSH) and androgens; acute suppression of these hormones inhibits germ cell development and thus sperm production. The removal of intercellular junctions and release of spermatids by the Sertoli cell, a process known as spermiation, is particularly sensitive to acute hormone suppression. Retention of spermatids following hormone suppression is characterised by the persistence of intercellular junctions between the Sertoli cell and the adjacent elongated spermatid. Recent reports have suggested that the tubulobulbar complex (TBC) and the related endocytotic pathways may be important mediators of intercellular junction internalisation by the Sertoli cell, and hence sperm release. We hypothesised that the TBC is regulated by FSH and androgens in the testis. The aim of this study was to characterise the hormonal dependence of TBC formation and function by the Sertoli cell. Using a previously characterised in vivo model of acute hormone suppression in rats, we assessed the expression of known and predicted TBC components by array analysis and immunohistochemistry. Following hormone suppression, known components including clathrin, Wasl and the Arp2/3 complex, and previously uncharacterised components including Eps15 and Picalm appear mislocalised or absent, suggesting that the TBC may fail to form appropriately in the absence of hormone action in vivo. Eps15 and Picalm were also suppressed following acute hormone suppression in primary Sertoli cells in vitro by Western blot analysis. In order to assess the impact of FSH and androgens upon clathrin-mediated endocytosis, we measured the kinetics of fluorescently labelled transferrin uptake by Sertoli cells using flow cytometry as a marker of endocytotic function. Using this approach, we confirmed that endocytosis is hormonally stimulated in the Sertoli cell.We have previously demonstrated that FSH and androgen regulate a subset of Sertoli cell miRNAs. It was of particular interest that miR-23b, which is stimulated following hormone suppression, was predicted to repress several TBC associated proteins. To assess this, we cloned the 3′untranslated regions of each gene into a dual luciferase vector, and validated novel inhibitory actions of miR-23b on Eps15, Picalm, Wasl and Dynamin 3. This inhibition provides a facile mechanism by which hormones can act to regulate TBC formation in the testis.In conclusion, we have demonstrated that hormonal suppression in the testis disrupts the localisation of known and predicted components of the TBC. Disrupted removal of intercellular junctions may contribute to the failure of sperm release following hormone suppression, and identifies a novel mechanism by which hormones control male fertility. Research supported by the National Health and Medical Research Council of Australia, Program Grant #494802 and CDF fellowship to CAH #1013533. (platform)

Multi‐tasking focal adhesion protein zyxin is present at tubulobulbar complexes

Tubulobulbar complexes (TBCs) are actin‐related double‐membrane projections that form at intercellular junctions in the seminiferous epithelium of the mammalian testis. They form at basal junction complexes between neighboring Sertoli cells and apical junctions between Sertoli cells and germ cells. They are proposed to internalize intact junctions during sperm release and during the translocation of spermatocytes through basal junction complexes between neighboring Sertoli cells. Although TBCs are testis‐specific, they morphologically resemble podosomes in osteoclasts. Previously, we have reported that a complex of N‐WASp, Arp2/3, cortactin and dynamin that is found at podosomes also is present at TBCs. Here we demonstrate 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) [generous gift from M.C. Beckerle, Huntsman Cancer Institute] was used to label fixed fragments and frozen sections of testis. Zyxin staining was intense in Sertoli cell regions known to contain tubular regions of TBCs. Zyxin is comprised of a proline rich N‐terminal domain and three LIM domains in its C‐terminal half. Although known to interact with the cytoplasmic domain of integrins, zyxin may be part of the mechanism of tubulobulbar complex formation. Supported by an NSERC Discovery Grant to AWV.Grant Funding Source: NSERC

Spectrin and plectin are present at apical tubulobulbar complexes in the seminiferous epithelium

Tubulobulbar complexes (TBCs) are actin‐related double membrane structures that are suspected to internalize intercellular junctions, a process that is crucial for junction turnover and sperm release. At apical sites of adhesion between spermatids and Sertoli cells, each TBC consists of a tubulobulbar projection of the spermatid plasma membrane, the surrounding attached plasma membrane of the adjacent Sertoli cell, and an actin network cuff. In other systems, spectrin is part of the membrane skeleton that links actin filaments to the plasma membrane. Others previously have reported that the protein is present at differentiating germ cells and epididymal spermatozoa. Plectin links cytoskeletal elements to each other and to the plasma membrane. We probed the seminiferous epithelium for spectrin, plectin and actin on fixed frozen sections of rat testis and fixed epithelial fragments. Both in sections and in epithelial fragments, beta‐spectrin II and plectin are localized to apical actin‐related tubulobulbar. Neither protein is concentrated at actin‐related intercellular adhesion junctions (ectoplasmic specializations). Our results demonstrate that both spectrin and plectin are present in Sertoli cells at tubulobulbar complexes. Supported by an NSERC Discovery Grant to AWV.Grant Funding Source: NSERC

Tubulobulbar complexes in Sertoli cell primary cultures

In the seminiferous epithelium, unique double membrane structures termed tubulobulbar complexes (TBCs) develop in association with basal junction complexes between Sertoli cells. These actin‐related structures are thought to internalize intercellular junctions during the movement of spermatocytes from basal to adluminal compartments of the epithelium. We are establishing a primary Sertoli cell culture system to study the role of basal TBCs in junction turnover. We confirm that Sertoli cells isolated from 20 day‐old rat pups morphologically differentiate when grown on Matrigel in Transwells. The cells are high cuboidal to columnar in shape with basal nuclei, and with basal junction complexes similar to the appearance of junction complexes between Sertoli cells in vivo. Significantly, TBCs and TBC‐like structures develop in association with intercellular junctions. Double‐membrane coated‐pits, together with double‐membrane tubular structures capped by coated‐pits are observed in regions related to ectoplasmic specializations. When immunostained for integral junction proteins, “rod‐like” structures are observed to extend into the cell from the periphery. These observations indicate that TBCs are able to develop in differentiated Sertoli cells in culture, making further functional analyses of TBCs possible in vitro. Supported by an NSERC Discovery Grant to AWV.Grant Funding Source: NSERC

The actin filament network associated to Sertoli cell ectoplasmic specializations

Junctional devices in Sertoli cells conform the blood-testis barrier and play a key role in maturation and differentiation of germ cells. The spacial distribution of ectoplasmic specializations of Sertoli cells was studied by beta-actin immunolabelling, using laser confocal and transmission electron microscopy. For confocal microscopy, beta-actin immunolabelling of ectoplasmic specializations was studied over the background of either prosaposin or glutaredoxin immunolabelling of the Sertoli cytoplasm. Labelling was found near the basal lamina, surrounding early spermatocytes (presumably in leptotene-zygotene) or at one of two levels in the seminiferous epithelium: (1) around deep infoldings of the Sertoli cell cytoplasm, in tubular stages before spermiation, and (2) in the superficial part of the seminiferous epithelium, in tubular stages after or during spermiation. For transmission electron microscopy, beta-actin immunolabelling of ectoplasmic specializations was also used. Ectoplasmic specializations were found at two different levels of the seminiferous epithelium. We also used freeze fracture to analyze the characteristics of tubulo-bulbar complexes, a known component of apical ectoplasmic specializations. Also, these different approaches allowed us to study the complex arrangement of the actin cytoskeleton of Sertoli cells branches, which surround germ cells in different stages of the spermatogenic cycle. Our results show a consistent labelling for beta-actin before, during and after the release of spermatozoa in the tubular lumen (spermiation) suggesting a significant role of the actin network in spermatic cell differentiation. In conclusion, significant interrelations among the beta-actin network, the junctional complexes of the blood-testis barrier and the ectoplasmic specializations were detected at different stages of the seminiferous cycle.

The myotubularin family of lipid phosphatases in disease and in spermatogenesis

The MTM (myotubularin)/MTMR (myotubularin-related) protein family is comprised of 15 lipid phosphatases, of which nine members are catalytically active. MTMs are known to play a fundamental role in human physiology as gene mutations can give rise to X-linked myotubular myopathy or Charcot-Marie-Tooth disease, which manifest in skeletal muscle or in peripheral neurons respectively. Interestingly, studies have shown MTMR2 and MTMR5, two MTM family members, to be highly expressed in the testis, particularly in Sertoli and germ cells, and knockout of either gene resulted in spermatogenic defects. Other studies have shown that MTMR2 functions in endocytosis and membrane trafficking. In the testis, MTMR2 interacts and co-localizes with c-Src/phospho-Src-(Tyr⁴¹⁶), a non-receptor protein tyrosine kinase that regulates the phosphorylation state of proteins at the apical ES (ectoplasmic specialization), a unique type of cell junction found between Sertoli cells and elongating/elongated spermatids. In the present review, we highlight recent findings that have made a significant impact on our understanding of this protein family in normal cell function and in disease, with the emphasis on the role of MTMs and MTMRs in spermatogenesis. We also describe a working model to explain how MTMR2 interacts with other proteins such as c-Src, dynamin 2, EPS8 (growth factor receptor pathway substrate 8) and ARP2/3 (actin-related protein 2/3) at the apical ES and the apical TBC (tubulobulbar complex; tubular-like invaginations that function in the disassembly of the apical ES and in the recycling of its components) to regulate spermiation at late stage VIII of the seminiferous epithelial cycle.

Molecular dynamics of the blood–testis barrier components during murine spermatogenesis

The blood-testis barrier (BTB) separates the seminiferous epithelium into the adluminal and basal compartments. During murine spermatogenesis, preleptotene/leptotene spermatocytes migrate from the basal to the adluminal compartment through the BTB during stages VIII-IX. In the present study, we focused on the tight junction (TJ) molecules and analyzed their spatiotemporal expression during the murine seminiferous epithelial cycle. Structural analysis revealed that the principal components of the BTB, for example, claudin-3, claudin-11, occludin, and zonula occludens-1 (ZO-1), were localized at the basal and luminal sides of the preleptotene/leptotene spermatocytes during the migration stages (VIII-IX). Although we detected claudin-11, occludin, and ZO-1 throughout spermatogenesis, claudin-3 was only detected during stages VI-IX. Quantitative PCR using dissected seminiferous tubules from three stages (Early: II-VI, Middle: VII-VIII, Late: IX-I) clarified that the mRNA levels of TJ molecules were not correlated with the histoplanimetrical protein levels during spermatogenesis. Additionally, tubulobulbar complexes, considered to be involved in the internalization of TJ, were observed at the BTB site. Furthermore, a significant reduction in the mRNA levels of genes for the degradation of occludin (Itch) and endocytic recycling (Rab13) were observed during the Late and Middle stages, respectively. Therefore, we hypothesized that the lag between mRNA and protein expression of TJ molecules may be due to posttranslational modulation, for example, tubulobulbar complexes and endocytic recycling processes. In conclusion, these findings indicate that the integrity of the BTB is maintained throughout spermatogenesis, and the stage-specific localization of claudin-3 protein plays an important role in regulating BTB permeability.

Rab 11 is associated with Nectin 2 positive vesicles in Sertoli cells at newly forming adhesion junctions with early elongating spermatids in rat testis

Tubulobulbar complexes are podosome‐like structures that internalize intercellular junctions in the seminiferous epithelium. Near the ends of the complexes, enlarged swollen areas develop and separate away from the complexes forming double membrane vesicles. Although these vesicles are degraded by Sertoli cells, it also is possible that some of the junction molecules in the vesicles are sorted and are recycled to junctions assembling at other locations in the cell. In this study, we double label perfusion‐fixed and mechanically fragmented seminiferous epithelium of rat testis for Nectin 2 and Rab 11. The rabbit anti‐Nectin 2 antibody that reacts with rat protein was provided by Dr. Yoshimi Takai, Kobe University, Japan. As expected on the basis of data from studies in mice, Nectin 2 is found at adhesion junctions associated with late spermatids, and in vesicles at the ends of tubulobulbar complexes. Although Rab 11 is not associated with any of the Nectin 2 positive vesicles at tubulobulbar complexes, Rab 11 is associated with Nectin 2 positive vesicles at newly forming adhesion junctions attached to early elongating spermatids. Our data support the conclusion that some of the Nectin 2 being incorporated into newly forming adhesion junctions is from recycling pathways, possibly from junctions being internalized by tubulobulbar complexes. Supported by a NSERC discovery grant to AWV.Grant Funding Source: NSERC

148. HORMONALLY REGULATED miRNAS TARGET THE TUBULOBULBAR COMPLEX IN THE TESTIS

Spermatogenesis is absolutely dependent on follicle stimulating hormone (FSH) and androgens; acute suppression of these hormones inhibits germ cell development and thus sperm production. The removal of intercellular junctions and release of spermatids by the Sertoli cell, a process known as spermiation, is particularly sensitive to acute hormone suppression(1). To define the molecular mechanisms that mediate FSH and androgen effects in the testis, we investigated the expression and hormonal regulation of micro-RNAs (miRNA), small non-coding RNAs that regulate protein translation and modify cellular responses. By array analysis, we identified 23 miRNAs that were upregulated >2-fold in stage VIII seminiferous tubules following hormone suppression, and in vitro in primary Sertoli cells. We subsequently validated the expression and hormonal regulation of several miRNAs, including miR-23b, -30d and -690 by quantitative PCR in primary Sertoli cells. Bioinformatic analysis of potential targets of hormonally-suppressed miRNAs identified genes associated with Focal adhesions (54 genes, P = –ln(17.97)) and the Regulation of the actin cytoskeleton (52 genes, P = –ln(10.16)), processes known to be intimately associated with adhesion of spermatids to Sertoli cells(2, 3). Furthermore, this analysis identified numerous components of the testicular tubulobulbar complex (TBC) as being targets of hormonally sensitive miRNAs. The TBC is a podosome-like structure between Sertoli and adjacent spermatids in the testis, which internalises intact inter-cellular junctions by endocytotic mechanisms prior to spermiation(4). We then demonstrate the hormonal regulation of predicted miRNA target proteins, and validate novel inhibitory miRNA interactions with Pten, nWASP, Eps15 and Picalm by luciferase knockdown in vitro. We hypothesise that hormonally suppressed miRNAs inhibit TBC function, and subsequently, endocytosis of intercellular junctions. In conclusion, we have demonstrated that hormonal suppression in the testis stimulates the expression of a subset of Sertoli cell miRNAs that are likely regulators of cell adhesion protein networks involved in spermiation. (1) Saito K, O’Donnell L, McLachlan RI, Robertson DM 2000 Spermiation failure is a major contributor to early spermatogenic suppression caused by hormone withdrawal in adult rats. Endocrinology 141: 2779–2.(2) O’Donnell L, Stanton PG, Bartles JR, Robertson DM 2000 Sertoli cell ectoplasmic specializations in the seminiferous epithelium of the testosterone-suppressed adult rat. Biol Reprod 63: 99–108.(3) Beardsley A, Robertson DM, O’Donnell L 2006 A complex containing alpha6beta1-integrin and phosphorylated focal adhesion kinase between Sertoli cells and elongated spermatids during spermatid release from the seminiferous epithelium. J Endocrinol 190(3): 759–70.(4) Young JS, Guttman JA, Vaid KS, Vogl AW 2009 Tubulobulbar complexes are intercellular podosome-like structures that internalize intact intercellular junctions during epithelial remodeling events in the rat testis. Biol Reprod 80: 162–74.

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 5: Intercellular junctions and contacts between germs cells and Sertoli cells and their regulatory interactions, testicular cholesterol, and genes/proteins associated with more than one germ cell generation

In the testis, cell adhesion and junctional molecules permit specific interactions and intracellular communication between germ and Sertoli cells and apposed Sertoli cells. Among the many adhesion family of proteins, NCAM, nectin and nectin-like, catenins, and cadherens will be discussed, along with gap junctions between germ and Sertoli cells and the many members of the connexin family. The blood-testis barrier separates the haploid spermatids from blood borne elements. In the barrier, the intercellular junctions consist of many proteins such as occludin, tricellulin, and claudins. Changes in the expression of cell adhesion molecules are also an essential part of the mechanism that allows germ cells to move from the basal compartment of the seminiferous tubule to the adluminal compartment thus crossing the blood-testis barrier and well-defined proteins have been shown to assist in this process. Several structural components show interactions between germ cells to Sertoli cells such as the ectoplasmic specialization which are more closely related to Sertoli cells and tubulobulbar complexes that are processes of elongating spermatids embedded into Sertoli cells. Germ cells also modify several Sertoli functions and this also appears to be the case for residual bodies. Cholesterol plays a significant role during spermatogenesis and is essential for germ cell development. Lastly, we list genes/proteins that are expressed not only in any one specific generation of germ cells but across more than one generation.

Podosome‐like tubulobulbar complexes internalize intact junctions in the seminiferous epithelium

Tubulobulbar complexes are actin‐rich double‐plasma membrane protrusions with associated vesicles that form at intercellular junctions in the seminiferous epithelium of the mammalian testis. One role that has been proposed for these 'podosome‐like' structures is that they internalize 'intact' intercellular junctions during the translocation of spermatocytes through basal junction complexes between Sertoli cells, and during sperm release from Sertoli cells at the apex of the epithelium. If tubulobulbar complexes internalize junctions, then components of tubulobulbar complexes that contain junction proteins also should be associated with endosomal markers. In this study, we probed apical tubulobular complexes in fixed epithelial fragments of rat and mouse testes for adhesion molecules (alpha‐6 integrin in rat, and nectin 2&3 in mouse) and early endosomal markers (Rab 5 in rat and EEA1 in mouse). The antibody to Rab 5 labeled the distal ends of tubulobulbar complexes that also labeled strongly with the antibody to alpha‐6 integrin. EEA1 surrounded vesicles at the ends of tubulobulbar complexes that also were positive for nectin 2 and nectin 3. These results are consistent with the proposal that tubulobulbar complexes are subcellular machines that internalize intact intercellular junctions in the testes.Grant Funding SourceNSERC

148. HORMONAL REGULATION OF miRNA IN THE TESTIS

Acute suppression of circulating reproductive hormones (FSH and testosterone) inhibits sperm release (spermiation) (1), although the molecular mechanisms of spermiation failure are poorly understood. Micro-RNAs (miRNAs) are small non-coding RNAs that regulate protein expression, and are essential for normal spermatogenesis. Recent studies suggest that miRNAs are exquisitely sensitive to hormonal control by FSH, LH and testosterone (2–4). This suggests that hormonal regulation of miRNAs in the testis following acute hormonal suppression may contribute to spermiation failure. Therefore, we hypothesised that gonadotrophin regulated miRNAs control spermiation outcome. We used array analysis to show that miRNA expression is hormonally regulated by FSH and testosterone in our rat in vivo model of spermiation failure and also in primary rat Sertoli cells by. qPCR validation revealed that miR-7b, -23a, -30c, -125b, -148b, -197, -483, -592, and -690 are all hormonally sensitive testicular miRNAs. Bioinformatic analyses of potential gene targets of these miRNAs predicted numerous protein components localised in the testicular tubulobulbar complex (TBC). The TBC is a podosome-like structure found between Sertoli cells and adjacent germ cells in the testis, and is thought to internalise intact inter-cellular structures and regulate spermatid head shape prior to spermiation. WASP, a TBC protein that regulates actin filament dynamics, contained a conserved binding site for miR-690 within its 3'UTR. Increased miR-690 expression following hormone suppression corresponded to a decrease in WASP protein expression in vivo and in vitro. In addition, transfection of miR-690 into HEK293T cells down-regulated WASP protein. Our results suggest that following hormone suppression, miR-690 is stimulated in the Sertoli cell, thereby inhibiting WASP protein expression. We conclude that miRNA-mediated disruption of TBC integrity potentially regulates spermatid disengagement. This study describes new molecular mechanisms in the testis that may control spermiation outcome of potential significance in male hormonal contraception.

Disruption of Tubulobulbar Complex by High Intratesticular Estrogens Leading to Failed Spermiation

Spermiation is the final phase of spermatogenesis leading to release of mature spermatids into the lumen of the seminiferous tubules. Morphologically, it involves a series of events, namely removal of excess spermatid cytoplasm, removal of ectoplasmic specialization, formation of tubulobulbar complex, and final disengagement of the spermatid from the Sertoli cell. Previous studies in our laboratory have shown that administration of 17beta-estradiol at a dose of 100 microg/kg body weight for 10 d resulted in failure of spermiation. This was accompanied by a suppression of FSH and intratesticular testosterone with a concomitant rise in intratesticular 17beta-estradiol. The present study was undertaken to determine the cause of failure and subsequently the molecular events in spermiation. Electron microscopic and confocal studies revealed an absence of tubulobulbar complex in step 19 spermatids after estradiol treatment, highlighting the significance of these structures in spermiation. It was further observed that treatment affected the Sertoli cell cytoskeleton and Arp2/3 complex that is critical for de novo polymerization of actin during tubulobulbar complex formation. In conclusion, the present study reports the role of 17beta-estradiol in inhibiting the formation of tubulobulbar complex, which could be one of the mechanism by which environmental estrogens influence male fertility.

Cortactin (CTTN), N-WASP (WASL), and Clathrin (CLTC) Are Present at Podosome-Like Tubulobulbar Complexes in the Rat Testis1

Tubulobulbar complexes are actin filament-rich plasma membrane protrusions that 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. Tubulobulbar complexes morphologically resemble podosomes found at cell/substrate attachments in other systems. In this study we probe apical tubulobulbar complexes in fixed epithelial fragments and fixed frozen sections of rat testis for two key actin-related components found at podosomes, and for the endocytosis-related protein clathrin. N-WASP and cortactin, two regulators of actin network assembly known to be components of podosomes, are concentrated at tubulobulbar complexes. Clathrin-positive structures occur in Sertoli cell regions containing tubulobulbar complexes when analyzed by immunofluorescence microscopy and occur at the ends of the complexes when evaluated by immunoelectron microscopy. Our results are consistent with the conclusion that tubulobulbar complexes are podosome-like structures. We propose that the formation of tubulobulbar complexes may be clathrin initiated and that their growth is due to the dendritic assembly of a membrane-related actin network.

Tubulobulbar Complexes Are Intercellular Podosome-Like Structures That Internalize Intact Intercellular Junctions During Epithelial Remodeling Events in the Rat Testis1

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.

Clathrin is present in regions containing tubulobulbar complexes in the testis

Tubuobulbar complexes (TCs) are elongate 2–3μm membrane‐based and actin‐related structures that form at intercellular junctions in the seminiferous epithelium. They are proposed to be responsible for the internalization of “intact” junctions during spermatogenesis. Large swollen regions develop and bud from the end of each TC that is internalized by the related Sertoli cell. A “coated pit” caps the end of each TC and is suspected to induce TC formation. To determine if clathrin is associated with the coated pits, we immunolocalized fixed seminiferous epithelial fragments, of rat testis with an antibody raised against clathrin. Small foci of staining were evident in Sertoli cell regions associated with late spermatids, known to contain tubulobulbar complexes. The antibody reacted specifically with a band at the appropriate molecular weight for clathrin heavy chain on Western blots of rat testis and seminiferous epithelium that was not present on control replicate blots. We confirmed the staining by immunoelectron microscopy where gold particles in antibody treated sections were concentrated in small patches in regions containing tubulobulbar complexes. We conclude that clathrin is associated with TCs.

Dynamins, Spermatogenesis and Contraceptive Development

Dynamins are large GTPases of ∼100 kDa known to participate in endocytosis and interact with the actin-based cytoskeletal network in multiple tissues. Recent studies have shown that dynamins play a critical role in the internalization of integral membrane proteins via either clathrin-mediated or clathrin-independent endocytosis. Furthermore, recent studies have shown that dynamin II interacts with junctional complex adaptors, namely ZO-1 and β-catenin, at the blood-testis barrier in the seminiferous epithelium of adult rat testes. This interaction may be responsible for pulling away tight junction- and adherens junction-based protein complexes, thereby facilitating blood-testis barrier opening to permit preleptotene and leptotene spermatocyte migration, which is a critical event in spermatogenesis occurring at stage VIII of the seminiferous epithelial cycle. In this short review, we highlight some of the latest findings on dynamins in the field, and discuss how this information can be used to further expand the functional studies to tackle the role of dynamins in spermatogenesis. It is likely that dynamins per se or their interacting protein partners can become a target for male contraceptive research to compromise spermatogenesis, leading to transient male infertility without perturbing the hypothalamic-pituitary-testicular axis. Keywords: Testis, GTPases, dynamins, blood-testis barrier, ectoplasmic specialization, tubulobulbar complex, adherens junction, anchoring junction, Sertoli cells

Cortactin is a component of tubulobulbar complexes in Sertoli cells

Tubulobulbar complexes (TCs) are double membrane tubular structures that project into Sertoli cells from sites of intercellular attachment. Large double membrane vesicles bud from the ends of the complexes and are degraded by Sertoli cells. TCs have been proposed to be part of the mechanism by which intercellular junctions are disassembled during sperm release and during the movement of spermatocytes from basal to adluminal compartments of the seminiferous epithelium. TCs resemble podosomes that develop at sites of cell attachment to substrate in other systems. The morphogenesis of podosomes is thought to be regulated by the interaction of the Arp2/3 complex, dynamin and cortactin. Although we previously have determined that the Arp2/3 complex and dynamin 3 are present at TCs, it is not known if cortactin also occurs at the sites. To determine whether or not cortactin is present at TCs we immunolabelled fixed frozen sections and fixed fragments of seminiferous epithelium with an antibody to cortactin. Intense labeling of TCs was present in sections and in epithelial fragments. Weak staining of ectoplasmic specializations also was detected. Controls were negative and immunoblots of testis and seminferous epithelium indicated that the antibody was specific. We conclude that cortactin is a component of TCs and therefore is a candidate regulator of TC morphogenesis.