One of the two testis-specific actin-based structures is the ectoplasmic specialization (ES), an actin-based atypical adherens junction between adjacent Sertoli cells at the blood–testis barrier (BTB) termed as basal ES) and between Sertoli cells and spermatids near the luminal surface of the tubule termed as apical ES [
3]. The ES is composed of hexagonal bundles of actin filaments sandwiched between the endoplasmic reticulum and Sertoli cell plasma membrane. In the rat testis, the apical ES first appears between Sertoli cells and round spermatids in late stage VIII and early stage IX. The basal ES is part of the junctional complex forming the BTB between adjacent Sertoli cells throughout the seminiferous epithelium cycle. The primary function of the ES is to facilitate germ cell movement, with an additional anchoring function via adhesion molecules such as integrin–laminin and nectinafadin complexes that helps retain germ cells (mainly spermatids) in the seminiferous epithelium until spermiation [
3].
Another testis-specific structure is the tubulobulbar complex (TBC), which replaces the apical ES. Similar to the rat, TBCs in other mammals appear at a particular developmental stage of the seminiferous epithelium cycle [
4]. Because of the difficulties in observing and identifying basal TBCs, very little information is available about their structure and importance, whereas apical TBC formation, being an inevitable event during sperm release, has been studied to a larger extent. In this review, we present a detailed discussion of apical TBCs during spermatogenesis.
Structural details of TBCs
TBCs are cytoplasmic evaginations of mature elongated spermatids. They penetrate into the surrounding Sertoli cell cytoplasm and are composed of both tubular and bulbous portions. The tube is surrounded by fine filaments of actin and the bulb is flanked by smooth endoplasmic reticulum. Numerous double membrane vesicles are present in the vicinity of these TBCs. TBCs are very interesting specialized structures located between Sertoli cells and spermatids, appearing in the last few days prior to sperm release. They form in large numbers at the beginning of stage VII of the rat seminiferous epithelium cycle [
5] as the spermatids move towards the lumen of the seminiferous epithelium from the deep recesses of the Sertoli cell. TBCs have been studied in the testes of ten mammalian species, namely opossum, vole, guinea pig, mouse, hamster, rabbit, dog, ram, monkey, and human and up to 24 TBCs are formed per spermatid [
4,
6]. However, TBCs have been most extensively studied and characterized in rats. In rats, the TBCs that are located near the BTB are termed basal TBCs, while those at the ventral (or concave) side of the spermatid heads of step 18 and 19 spermatids attached to the Sertoli cell are termed as apical TBCs [
7].
Russell [
5] analyzed the ultrastructure of TBCs and performed a time-course study of the appearance and degradation of TBCs. In rats, the length of the entire complex is 3–5 μm. In stage VI, the spermatid heads are embedded within the deep recesses of Sertoli cells, and in early stage VII, the spermatids move towards the lumen and the TBCs start to form. The complexes at this stage are short tubular projections (0.1–0.2 μm in length) of the spermatid projecting towards the Sertoli cells. The bulbous region develops as a dilated component in the mid-region of the tube, facing the smooth endoplasmic reticulum of the Sertoli cell. By mid-stage VII, large vacuoles containing double membrane-bound vesicles accumulate around the clustered bulbs, which fuse with the lysosomes and are degraded or selectively recycled to the plasma membrane [
5,
8]. In late stage VII and early stage VIII, the ESs get displaced and the tubular portions of old TBCs start getting resorbed, while new TBCs start forming as evidenced by bristle-coated pits in Sertoli cell invaginations and short opposing evaginations of the spermatid. This suggests that several generations of TBCs are formed and resorbed prior to sperm release [
5,
8].
Resemblance of TBCs to podosomes
TBCs resemble podosomes present in other systems. Podosomes are dot-like contacts that a cell makes with the extracellular matrix. They are known to occur specifically in monocyte-derived haematopoetic cells including macrophages, osteoclasts, and dendritic cells [
9], while TBCs occur only in the seminiferous tubules of the testis. Podosomes comprise a core of F-actin and actin-associated proteins embedded in a ring structure of integrins and integrin-associated proteins. Actin-associated proteins such as cortactin, talin, and vinculin link integrins to the actin cytoskeleton [
9]. Similarly, TBC formation is shown to be generated by the assembly of the actin network involving the action of N-WASP (Neural Wiskott-Aldrich syndrome protein), the Arp2/3 (actin-related protein) complex, cortactin, and dynamin [
10]. Podosomes are dynamic in nature: they are repeatedly constructed and destroyed and hence are thought to be suitable for transient adhesions during cell motility. In addition to podosomes, a similar actin-based structure exists in yeast during budding events, highlighting the involvement of the actin cytoskeleton in clathrin-mediated endocytosis, which is likely to be conserved across eukaryotes [
11]. Similar observations seen in TBCs are supported by the fact that when adherent ESs between the Sertoli cell and spermatid start to disappear at stage VII, TBCs concomitantly appear in the areas where the ESs disappear [
12].
Proteins associated with TBC formation
Various proteins involved in actin bundling, polymerization, and stability are found in podosomes, which resemble TBCs. Hence, there have been studies to see if these proteins are present on TBCs and if they play a role in their formation. The Arp2/3 complex is a key regulator of actin polymerization and branching that promotes filament assembly through enhanced nucleation of actin subunits [
21]. Arp3 (actin-related protein 3) is localized to the concave side of the spermatid head, the area in which apical TBCs are located [
22]. Actin-binding proteins such as espin, for actin bundling, and vinculin, which shows actin crosslinking activity, are present on TBCs as shown by immunofluorescence staining [
23]. High paxillin expression was detected in TBCs with paxillin and vinculin colocalization seen in close association with the actin network suggesting interactions between the two proteins. Paxillin is generally involved in turnover of actin networks [
21]. Cofilin-1 (also known as non-muscle cofilin), which is involved in actin depolymerization, is found in apical TBCs and is thought to increase actin turnover in TBCs [
12]. Young
et al.[
24] have shown the presence of N-WASP (Wiskott–Aldrich syndrome protein) and cortactin, key components present in podosomes, are also present on TBCs. N-WASP is a key regulator of the Arp2/3 complex, which in turn generates new actin filament branches from pre-existing filaments, thereby forming a three-dimensional actin network in which filaments elongate from their barbed ends positioned at the plasma membrane [
24]. Blockage of N-WASP by Wiskostatin, a chemical inhibitor, results in mis-orientation of step 19 spermatids [
22]. CR16, a member of WASP-interacting proteins, is highly expressed in the testis, especially in Sertoli cells. In CR16 male knockout mice, sperm head morphology was affected along with diminished fertility [
25]. All these data suggest that actin and its regulatory proteins in TBCs are also involved in sperm head morphology.
Recent studies have shown actin and actin-regulatory proteins to be involved in endocytosis [
26]. Since TBCs are also endocytic devices, various endocytic proteins are known to be localized at the site of their formation [
10]. Amphiphysin 1 is concentrated at the luminal surface of seminiferous tubules at stage VIII during rat spermatogenesis. It is involved in clathrin-mediated endocytosis and also in regulation of actin cytoskeleton [
27]. Amphiphysin 1 is important for actin polymerization during phagocytosis as demonstrated by colocalization studies with actin, vinculin, and cortactin around TBCs [
28]. Dynamin family proteins are known to be involved in the process of endocytosis. Dynamin 2 and 3 are implicated in vesicle formation and clathrin-mediated endocytosis [
29] and are shown to be involved in tubular morphogenesis in TBCs [
30,
31]. Clathrin-lattice machinery is known to be triggered by assembly proteins (APs), namely AP1 and AP2 [
32,
33]. Similarly, the non-neuronal homolog of AP180 protein, PICALM, plays a significant role in clathrin internalization machinery [
34]. However, there are no reports describing their localization and function in TBCs. In addition, over 50 proteins are associated with clathrin-mediated endocytosis [
35]. Some of these are likely to be involved in TBC formation and function [
10,
28,
30‐
32]
Recently, the actin regulator Eps8 (epidermal growth factor receptor pathway substrate 8), which controls actin-based motility by capping barbed ends of actin filaments [
36] and has been detected in both Sertoli and germ cells and localized in apical TBCs, has been reported to play an important role in maintaining germ cell adhesion and BTB integrity in rat testes [
36]. Eps 15 homology domain containing protein 1 (EHD1) regulates endocytic trafficking and recycling of membrane components during spermatogenesis. Knockout models of EHD1 male mice have shown acrosomal defects, mis-orientation of spermatids and phagocytosis of failed elongated spermatids [
37]. Detailed description and function of proteins found to be localized in TBCs are presented in Table
1.
Table 1
Proteins localized at the sperm release boundary
Arp3 | Formation of branched actin network in TBCs | |
Actin-binding proteins
|
Espin | Actin bundling | |
Vinculin | Actin cross-linking |
Cortactin | Actin depolymerization: increases actin turnover in TBCs | |
Cofilin | Arp2/3 complex activation | |
Eps8 | Actin capping; regulation of bundling and Rac-GTPase: formation of actin bundles and network | |
Paxillin | Turnover of actin network | |
N-WASP | Arp2/3 complex activation | |
Profilin IV | Assembly–disassembly of F-actin, presumably present at tubulobulbar extensions of Sertoli cells | |
Adhesion molecules
|
α6 β1-integrin | Complex associated with disengagement during spermiation | |
Nectin 2 | Expressed only in Sertoli cells and stabilizes association of TBC–Sertoli cell adherens complex | Kierzenbaun et al.[ 41], Mueller et al.[ 42] |
Nectin 3 | Associated with actin and espin, forms a hetero-trans dimer with nectin 2 | Lee et al.[ 3], Inagaki et al.[ 43] |
Afadin | Nectin–actin linker protein | |
Endocytosis
|
Clathrin | Forms a layer around endocytic vesicles forming clathrin-coated pits | |
Amphiphysin 1 | Co-operates with dynamin 1 in clathrin-mediated endocytosis; also involved in actin dynamics | |
Dynamin 2 | Pinching off vesicles from the parent membrane and formation of tubular protrusions | |
Dynamin 3 | |
Early endosomal antigen (EEA) | Marker for early endosomes, linked to internalization–degradation pathway of TBCs | |
Adhesion molecules such as nectin 2, nectin 3, and α6 integrin have been reported in apical TBCs and appear to be concentrated at their ends [
24]. Guttman
et al.[
37] have shown that TBCs develop in regions previously occupied by ESs by studying the localization of molecular markers for ES, namely espin, myosin VII, and Keap I, all of which also localize to the TBCs. Afadin, which binds nectin to actin filaments, is found in ESs and TBCs; when a step-wise progression of afadin staining was investigated during maturation of step 18–19 spermatids, staining of ESs along the dorsal curvature of spermatids gradually appeared to decrease in intensity, whereas an increase in staining intensity was observed around TBCs where it formed a fingerlike staining pattern [
44].
Estrogen regulation in TBCs
Current knowledge of hormonal regulation during spermatogenesis supports the proposition that both testosterone and FSH have similar and independent actions that are essential for quantitatively normal spermatogenesis in adulthood [
45]. Testosterone is also known to maintain the binding competency of Sertoli cells and Sertoli cell–spermatid junctional interaction [
46]. The process of spermiation, involving formation of TBCs and subsequent release of sperm into the tubular lumen, is known to be responsive to hormones. The major hormonal regulators of spermiation are FSH and androgens [
38]. It has been shown that in rats, suppression of either FSH, androgen, or both causes spermiation failure. The combined suppression of FSH and testosterone had a significantly greater effect on spermiation than the two alone. This failure appears to be only due to the inability of the spermatid to disengage from the Sertoli cell cytoplasm, the formation of ES and TBCs seems unaffected. In this model, an adhesion molecule, β1-integrin, is likely to mediate spermatid disengagement as indicated by its localization around retained spermatids by Beardsley
et al.[
40,
47]. Also, testosterone capsules implanted in hypophysectomized rats, which led to decrease in intratesticular testosterone concentration, prevented TBCs from forming in many late spermatids during their release. These spermatids displayed an accumulation of cytoplasm (swelling) in the perinuclear region of the head in turn suggesting a role of testosterone or its metabolites in TBC formation [
48]. Our studies using a model that involved administration of exogenous estradiol to rats leading to suppression of FSH and intratesticular testosterone, and a concomitant increase in intratesticular estrogen, showed marked spermiation failure. The failure was likely due to the absence of TBCs, shown in electron micrographs, and lack of localization of F-actin (which is a marker for TBCs) [
49,
50]. Looking at the molecular aspects, Arp2/3 complex transcript levels were unaltered in the FSH and T suppression model, whereas, transcripts of one of the subunits of Arp2/3 complex, namely Arpc1b, involved in complex stabilization, was found to be reduced in the exogenous estradiol model [
51,
52]. These observations could indicate that formation of TBCs is most likely mediated by estrogen rather than by FSH and androgens; however, the molecular mechanism of estrogen action in TBC formation is yet to be investigated.