Spermatid translocation in the rat seminiferous epithelium: coupling membrane trafficking machinery to a junction plaque

In this study, we demonstrate that specialized junction plaques that occur between Sertoli cells and spermatids in the rat testis support microtubule translocation in vitro. During spermatogenesis, Sertoli cells are attached to spermatids by specialized adhesion junctions termed ectoplasmic specializations (ESs). These structures consist of regions of the plasma membrane adherent to the spermatid head, a submembrane layer of tightly packed actin filaments, and an attached cistern of endoplasmic reticulum. It has been proposed that motor proteins on the endoplasmic reticulum interact with adjacent microtubules to translocate the junction plaques, and hence the attached spermatids, within the epithelium. If this hypothesis is true, then isolated junctions should support microtubule transport. To verify this prediction, we have mechanically isolated rat spermatids, together with their attached ESs, and tested them for their ability to transport microtubules in vitro. Most assays were done in the presence of 2 mg/ml testicular cytosol and at room temperature. ESs attached to spermatids supported microtubule translocation. In some cases in which motility events were detected, microtubules moved smoothly over the junction site. In others, the movement was slow but progressive, saltatory and "inch-worm-like." No motility was detected in the absence of exogenous ATP or in the presence of apyrase (an enzyme that catalyses the breakdown of ATP). Our results are consistent with the microtubule-based motility hypothesis of spermatid translocation.     

Binding between mammalian spermatid-ectoplasmic specialization complexes and microtubules.

Ectoplasmic specializations (ESs) are submembrane specializations that consist of Sertoli cell plasma membrane linked by an ordered array of actin filaments to a cisterna of endoplasmic recticulum (ESER). They are thought to function in the spermatid-Sertoli cell adhesion junction. Microtubules occur adjacent to the cytoplasmic face of the ESER and are oriented parallel to the long axis of the Sertoli cell, the direction of spermatid translocation during spermatogenesis. Our hypothesis that spermatid orientation and translocation in the seminiferous epithelium is microtubule dependent predicts that microtubules bind to ESs. To test for binding between microtubules and ESs, we have developed an in vitro assay in which spermatid-ES complexes were isolated from the seminiferous epithelium and incubated with bovine brain microtubules that were labeled with [3H]GTP and stabilized with taxol. Binding was determined by scintillation counts from gradient fractions enriched for spermatid-ES complexes and depleted of unbound microtubules by differential centrifugation. Our data indicate that microtubules bind to spermatid-ES complexes in a substrate concentration-dependent manner and can be released with 5 mM GTP or 10 mM MgATP. Binding is competitively reduced with excess unlabeled microtubules and is inhibited by 100 microM vanadate and 2 mM N-ethylmaleimide (NEM). The amount of binding is unchanged by 10 microM vanadate, 2 mM erythro-(2-hydroxy-3-nonyl)adenine (EHNA) or 1 mM 5'-adenylylimidodiphosphate (AMP-PNP). Immunofluorescence and autoradiographic data confirm that labeled microtubules bind to ES locations on spermatid-ES complexes. These data are consistent with the hypothesis that spermatid translocation is a microtubule-based transport event.     

Tubulin hooks as probes for microtubule polarity: an analysis of the method and an evaluation of data on microtubule polarity in the mitotic spindle

The structural polarity of cellular microtubules can be visualized in situ by lysing cells in special buffers containing tubulin. Under these conditions, the tubulin polymerizes to form curved sheets which attach to the walls of the endogenous microtubules. When such decorated microtubules are cut in cross section and viewed in the electron microscope, they appear to bear hooks curving clockwise or counter- clockwise. The direction of hook curvature is defined by the orientation of the decorated microtubule and thus serves as a probe for microtubule polarity. In this paper we describe a way to analyze the relative frequencies of hooks of different curvatures so as to measure the fidelity of the relation between hook curvature and microtubule polarity. The assumptions of the method are tested and found to be valid to a reasonable accuracy. The correlation between hook curvature and microtubule orientation is shown to be at least 0.98 for the spindles of PtK cells and Haemanthus endosperm at all stages of division and at all places in the spindle. The correlation is shown to be valid for each hook that forms, so the polarity of those microtubules that bear multiple hooks is specified with even better certainty than 0.98. This property of hook decoration is used to reinvestigate the possibility that some of the microtubules of the kinetochore fiber might be oriented with their plus ends distal to the kinetochore (opposite to the direction previously shown to predominate). Close analysis fails to identify such oppositely oriented microtubules. The scoring of tubules bearing multiple hooks also shows that individual interzone fibers at anaphase are constructed from clusters of antiparallel microtubules. The method for estimating the correlation between hook decoration and microtubule polarity is shown to be applicable to many structures and circumstances, but we find that the hook decoration assay for microtubule polarity is not uniformly accurate. We suggest that future studies using hook decorations should employ the method of data analysis presented here to assess the accuracy of the results obtained.     

gamma-Tubulin overexpression in Sertoli cells in vivo: I. Localization to sites of spermatid head attachment and alterations in Sertoli cell microtubule distribution

Sertoli cells play a number of roles in supporting spermatogenesis, including structural organization, physical and paracrine support of germ cells, and secretion of factors necessary for germ cell development. Studies with microtubule disrupting compounds indicate that intact microtubule networks are crucial for normal spermatogenesis. However, treatment with toxicants and pharmacologic agents that target microtubules lack cell-type selectivity and may therefore elicit direct effects on germ cells, which also require microtubule-mediated activities for division and morphological transformation. To evaluate the importance of Sertoli cell microtubule-based activities for spermatogenesis, an adenoviral vector that overexpresses the microtubule nucleating protein, gamma-tubulin, was used to selectively disrupt microtubule networks in Sertoli cells in vivo. gamma-Tubulin overexpression was observed to cause redistribution of Sertoli cell microtubule networks, and overexpression of a gamma-tubulin-enhanced green fluorescent protein fusion protein was observed to localize to the site of elongate spermatid head attachment to the seminiferous epithelium.     

Distribution of the microtubule-dependent motors cytoplasmic dynein and kinesin in rat testis.

To examine the possible role of microtubule-based transport in testicular function, we used immunofluorescent techniques to study the presence and localization of the microtubule mechanoenzymes cytoplasmic dynein (a slow-growing end-directed motor) and kinesin (a fast-growing end-directed motor) within rat testis. Cytoplasmic dynein immunofluorescence was observed in Sertoli cells during all stages of spermatogenesis, with a peak in apical cytoplasm during stages IX-XIV. Cytoplasmic dynein immunofluorescence was also localized within Sertoli cells to steps 9-14 (stages IX-XIV) germ cell-associated ectoplasmic specializations. In germ cells, cytoplasmic dynein immunofluorescence was observed in manchettes of steps 15-17 (stages I-IV) spermatids, and small, hollow circular structures were seen in the cytoplasm of step 17 and step 18 spermatids during stages V and VI. Kinesin immunofluorescence was observed in manchettes of steps 10-18 spermatids (stages X-VI). The stage-dependent apical Sertoli cell cytoplasmic dynein immunofluorescence, in conjunction with the previously reported orientation of Sertoli cell microtubules (slow-growing ends toward the lumen) and peak secretion of androgen-binding protein and transferrin, is consistent with the hypothesis that cytoplasmic dynein is involved in Sertoli cell protein transport and secretion. Further, the localization of cytoplasmic dynein and kinesin to manchettes is consistent with current hypotheses concerning manchette function.     

An ultrastructural study of the effect of a diabetogenic dose of alloxan on the secretory ameloblasts of the rat incisor

Sertoli cells of the ground squirrel (Spermophilus lateralis), a seasonal breeder, were examined by light and electron microscopy and their structure, particularly the organization of the cytoskeleton, was related to events that occur in the seminiferous epithelium during spermatogenesis. Among the events considered and described are the apical movement of elongate spermatids, withdrawal of residual cytoplasm from germ cells, transport of smooth endoplasmic reticulum (SER) between the base and apex of the Sertoli cells, and sperm release. These events are dramatically evident in this species because the seminiferous epithelium is thin, i.e., there are few germ cells, and both the germ cells and Sertoli cells are large. Sertoli cells of the ground squirrel have a remarkably well developed cytoskeleton. Microfilaments occur throughout the cell but are most evident in ectoplasmic specializations associated with junctions. Intermediate filaments occur around the nucleus, as a layer at the base of the cell, and adjacent to desmosome-like junctions with germ cells. Intermediate filaments, together with microtubules, are also abundant in regions of the cell involved with the transport of SER, in cytoplasm associated with elongate spermatids, and in processes that extend into the residual cytoplasm of germ cells. Our observations of ultrastructure are consistent with the hypothesis that Sertoli cell microtubules are involved with the movement of germ cells within the seminiferous epithelium, and further implicate these structures as possibly playing a role in the retraction of residual cytoplasm from germ cells and the intracellular transport of SER. The abundance and organization of intermediate filaments suggest that these cytoskeletal elements may also be involved with events that occur during spermatogenesis.     

gamma-Tubulin overexpression in Sertoli cells in vivo. II: Retention of spermatids,residual bodies,and germ cell apoptosis

The degree of germ cell dependence on Sertoli cell-mediated activities has been a subject of considerable attention. Sertoli cell secretory pathways have been extensively studied both in an effort to understand their normal physiologic roles and as targets for pharmacologic and toxicant activity. To determine the degree to which normal spermatogenesis depends on key functions of the Sertoli cell microtubule network, adenoviral vectors that overexpress the microtubule nucleating protein, gamma-tubulin, were delivered to Sertoli cells in vivo. gamma-Tubulin overexpression disrupts the Sertoli cell microtubule network (as described in the companion article); leads to gross disorganization of the seminiferous epithelium, inducing retention of spermatids and residual bodies; and causes germ cell apoptosis. These data are consistent with earlier studies in which toxicants and pharmacologic agents were used to disrupt microtubule networks. These data confirm that Sertoli cell microtubule networks play an important role in maintaining the organization of the seminiferous epithelium and that in the absence of an intact Sertoli cell microtubule network, germ cell viability is impaired.     

Morphological changes in the rat sertoli cell induced by the microtubule poison carbendazim

Early morphological changes in the rat Sertoli cell induced by the fungicide carbendazim (methyl-2-benzimidazole carbamate; MBC), a metabolite of benomyl, were examined. Adult rats were treated with single doses of MBC (400mg/kg) or vehicle and examined by light and electron microscopy at 3 hr post-treatment. Sloughing of elongating spermatid clusters was observed in all stages of spermatogenesis, except for Stages III–V. Cleavage occurred near the apical region of the seminiferous epithelium where cytoplasmic processes of the Sertoli cell surround the heads of elongating spermatids. The cleaved cytoplasm remained attached to the sloughed spermatids and ectoplasmic specializations remained undamaged. Intact microtubules were observed in the apical Sertoli cell cytoplasm (including sloughed tissues) but were decreased in the body region, where aggregates of mitochondria were found. Cytoplasm near the cleavage site exhibited rarefaction, which was associated with swollen cisternae of endoplasmic reticulum. It appears that the mechanism of germ cell sloughing induced by MBC treatment involves the disruption of microtubules in the body region of the Sertoli cell, the retraction of cytoplasmic organelles and the swelling of endoplasmic reticulum.     

Short-time effects of taxol on the seminiferous epithelium of the rat

Summary The effect of taxol, an inhibitor of microtubule degradation, on the seminiferous epithelium was studied. Taxol arrested spermatogenesis at metaphase in both mitotic and meiotic germ cell division. Microtubules were seen to accumulate, especially in the cytoplasm of the spermatogonia, and also in the early spermatids and Sertoli cells. No microtubule accumulation was observed in germ cells during meiotic prophase. Formation of the flagellum was affected in developing spermatids. Peculiar lamellar structures, probably derived from degenerating mitochondria, were seen in the cytoplasm of late spermatids and Sertoli cells.The results are compared with the effects of other mitotic inhibitors such as colchicine and vinca alcaloids.     

Polarity of microtubular structures in manchette-like formations: possible role of the "11th filament".

Bundles of microtubular structures appear in the cytoplasm of spermatids of the African frog Dicroglossus occipitalis. They are observed in the vicinity of axonemes. Natural tubulin polymerization leads to the formation of hooks on microtubular structures. They can be related to experimentally induced tubulin hooks. The direction of curvature of the hooks allows us to define the polarity of the bundles. This is opposite to the polarity of axonemal microtubules: Bundles and axonemes are antiparallel. Under colchicine action, arch-like microtubular structures are shown to open in the same direction as they lock. This enables us to characterize their opening and locking site: It corresponds to the place of the "11th filament" described in microtubular structures such axonemes. The "11th filament" is thus demonstrated to be the most susceptible to natural opening and to the action of colchicine in microtubular structures.     

A role of microtubules during the formation of cell processes in neuronal and non-neuronal cells

This review discusses the role of microtubules in the formation of processes from neuronal and non-neuronal cells. In elongating axons of the neuron, tubulin molecules are transported toward the end of pre-existing microtubules, which may be nucleated at the centrosome, via a mechanism called slow axonal flow. Two different hypotheses are presented to explain this mechanism; the transport of soluble monomers and/or oligomers versus the transport of polymerized microtubules. The majority of tubulin seems to be transported as small oligomers as shown by the data presented so far. Alternatively, an active transport of polymerized microtubules driven by microtubule-based motor proteins is postulated as being responsible for the non-uniform polarity of microtubule bundles in dendrites of the neuron. Microtubule-associated proteins (MAPs) play a crucial role in stabilizing the microtubular arrays, whereas the non-uniform polarity of microtubules may be established with the aid of microtubule-based motor proteins. The signals activating centrosomal proteins and MAPs, resulting in process formation, include phosphorylation and dephosphorylation of these proteins. Not only neuronal cells, but also renal glomerular podocytes develop prominent cell processes equipped with well-organized microtubular cytoskeletons, and intermediate and actin filaments. A novel cell culture system for podocytes, in which process formation can be induced, should provide further evidence that microtubules play a pivotal role in process formation of non-neuronal cells.     

Cytoskeletal involvement in spermiation and sperm transport

The process of spermiation and sperm transport was studied using specific inhibitors of cytoskeletal elements. Within 12-24 hr after the intratesticular injection of taxol, a compound that acts to stabilize microtubules and inhibit microtubule-related processes, an unusually large number of microtubules was seen within the body of the Sertoli cell. At the same time, transport of elements within the seminiferous epithelium was affected. At the end of stage VI of the cycle, step 19 spermatids were maintained in the deep recesses of the Sertoli cell and not transported to the rim of the seminiferous tubule lumen. At stage VIII, residual bodies remained at, or near, the rim of the tubule and were not transported to the base of the tubule. They underwent only partial degradation at this site, indicating that there may have been two phases involved in their dissolution--one autophagic and one phagocytic, but the latter did not occur since the residual bodies were not transported to Sertoli lysosomes at the base of the tubule. The observations suggest that microtubules are involved in transport processes within the seminiferous epithelium. Within 1-12 hr after the intratesticular injection of 500 microM cytochalasin D, a compound which interferes with actin-related processes, normal appearing tubulobulbar complexes were not present. The tubular portion (distal tube) of the complex did not initiate development. It was assumed that filaments (which were identified as such using NBD-phallacidin and the S-1 fragment of myosin) played an important role in the development of this portion of the complex. Cells did not eliminate cytoplasm normally, as evidenced by an enlarged cytoplasmic droplet, further emphasizing the published role for tubulobulbar complexes in cytoplasmic elimination. Although sperm were released normally from stage VIII tubules, many remained within the tubular lumen and did not traverse the duct system. Cytochalasin did not inhibit fluid secretion by the Sertoli cell, as demonstrated by efferent duct ligation, but did alter myoid cell actin cytoskeletal organization, suggesting that myoid cell contractility is primarily responsible for transport of sperm. Overall, the observations suggest that cytoskeletal activity of the Sertoli cell is important for several aspects of the spermiation process as well as sperm transport.     

Polarity of microtubular structures in manchette-like formations: Possible role of the “11th filament”

Bundles of microtubular structures appear in the cytoplasm of spermatids of the African frog Dicroglossus occipitalis. They are observed in the vicinity of axonemes. Natural tubulin polymerization leads to the formation of hooks on microtubular structures. They can be related to experimentally induced tubulin hooks. The direction of curvature of the hooks allows us to define the polarity of the bundles. This is opposite to the polarity of axonemal microtubules: Bundles and axonemes are antiparallel. Under colchicine action, arch-like microtubular structures are shown to open in the same direction as they lock. This enables us to characterize their opening and locking site: It corresponds to the place of the “11th filament” described in microtubular structures such axonemes. The “11th filament” is thus demonstrated to be the most susceptible to natural opening and to the action of colchicine in microtubular structures.     

Fine structural changes in Sertoli and Leydig cells during the reproductive cycle of the ground squirrel, Citellus lateralis

During spermatogenesis in sexually mature ground squirrels Leydig and Sertoli cells were morphologically well differentiated. For Leydig cells the most prominent organelles were lipid droplets, mitochondria with tubulo-vesicular cristae and abundant agranular reticulum organized as a mass of anastomosing tubules. These morphological criteria suggest that the Leydig cells were steroidogenically active. Sertoli cells exhibited a topographical distribution of certain organelles with basal regions containing stacks of granular reticulum, and large areas of agranular reticulum. The cytoplasm surrounding maturing germ cells contained numerous microtubules, and an adluminal layer of spermatids at a certain stage of spermiogenesis became enveloped by Sertoli cytoplasm containing an enormous proliferation of agranular reticulum. The presence of these organelles in Sertoli cells suggests that during spermatogenesis they are active in the synthesis of proteins and steroids. In particular the mass of agranular reticulum surrounding late stage spermatids indicates that steroids may be required for spermatid maturation and/or spermiation. By contrast Leydig and Sertoli cells observed during testicular regression, when only spermatogonia remain in the seminiferous tubules, had undergone structural changes. Leydig cells were still numerous and large with abundant agranular reticulum that was now organized as a loose assemblage of single unbranched tubules. Sertoli cells were drastically reduced in both cytoplasmic volume and content of organelles.     

Polarity orientation of axonal microtubules

The polarity orientation of cellular microtubules is widely regarded to be important in understanding the control of microtubule assembly and microtubule-based motility in vivo. We have used a modification of the method of Heidemann and McIntosh (Nature (Lond.). 286:517-519) to determine the polarity orientation of axonal microtubules in postganglionic sympathetic fibers of the cat. In fibers from three cats we were able to visualize the polarity of 68% of the axonal microtubules; of these, 96% showed the same polarity orientation. Our interpretation is that the rapidly growing end of all axonal microtubules is distal to the cell body. We support Kirschner's hypothesis on microtubule organizing centers. (J. Cell Biol. 86:330- 334), although this interpretation raises questions about the continuity of axonal microtubules. Our results are inconsistent with a number of models for axonal transport based on force production on the surface of microtubules in which the direction of force is determined by the polarity of microtubules.     

The microtubular system and posttranslationally modified tubulin during spermatogenesis in a parasitic nematode with amoeboid and aflagellate spermatozoa

Using transmission electron microscopy and immunologic approaches with various antibodies against general tubulin and posttranslationally modified tubulin, we investigated microtubule organization during spermatogenesis in Heligmosomoides polygyrus, a species in which a conspicuous but transient microtubular system exists in several forms: a cytoplasmic network in the spermatocyte, the meiotic spindle, a perinuclear network and a longitudinal bundle of microtubules in the spermatid. This pattern differs from most nematodes including Caenorhabditis elegans, in which spermatids have not microtubules. In the spermatozoon of H. polygyrus, immunocytochemistry does not detect tubulin, but electron microscopy reveals two centrioles with a unique structure of 10 singlets. In male germ cells, microtubules are probably involved in cell shaping and positioning of organelles but not in cell motility. In all transient tubulin structures described in spermatocytes and spermatids of H. polygyrus, detyrosination, tyrosination, and polyglutamylation were detected, but acetylation and polyglycylation were not. The presence/absence of these posttranslational modifications is apparently not stage dependent. This is the first study of posttranslationally modified tubulin in nematode spermatogenesis. Mol. Reprod. Dev. 49:150–167, 1998. © 1998 Wiley-Liss, Inc.     

Sertoli-germ cell adherens junction dynamics in the testis are regulated by RhoB GTPase via the ROCK/LIMK signaling pathway

During spermatogenesis, cell-cell actin-based adherens junctions (AJs), such as ectoplasmic specializations (ESs), between Sertoli and germ cells undergo extensive restructuring in the seminiferous epithelium to facilitate germ cell movement across the epithelium. Although the mechanism(s) that regulates AJ dynamics in the testis is virtually unknown, Rho GTPases have been implicated in the regulation of these events in other epithelia. Studies have shown that the in vitro assembly of the Sertoli-germ cell AJs but not of the Sertoli cell tight junctions (TJs) is associated with a transient but significant induction of RhoB. Immunohistochemistry has shown that the localization of RhoB in the seminiferous epithelium is stage specific, being lowest in stages VII-VIII prior to spermiation, and displays cell-specific association during the epithelial cycle. Throughout the cycle, RhoB was localized near the site of basal and apical ESs but was restricted to the periphery of the nuclei in elongating (but not elongated) spermatids, spermatocytes, and Sertoli cells. However, RhoB was not detected near the site of apical ESs at stages VII-VIII. Furthermore, disruption of AJs in Sertoli-germ cell cocultures either by hypotonic treatment or by treatment with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) also induced RhoB expression. When adult rats were treated with AF-2364 to perturb Sertoli-germ cell AJs in vivo, a approximately 4-fold induction in RhoB in the testis, but not in kidney and brain, was detected within 1 h, at least approximately 1-4 days before germ cell loss from the epithelium could be detected by histological analysis. The signaling pathway(s) by which AF-2364 perturbed the Sertoli-germ cell AJs apparently began with an initial activation of integrin, which in turn activated RhoB, ROCK1, (Rho-associated protein kinase 1, also called ROKbeta), LIMK1 (LIM kinase 1, also called lin-11 isl-1 mec3 kinase 1), and cofilin but not p140mDia and profilin via phosphorylation. Immunoprecipitation and immunoblots revealed that the induction of LIMK1 was mediated via an increase in its phospho-Ser but not phospho-Tyr content. Furthermore, Y-27632 ([(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane-carboxamide, 2HCl]), a specific ROCK inhibitor, could effectively delay the AF-2364-induced germ cell loss from the seminiferous epithelium in vivo, illustrating that the integrin/RhoB/ROCK/LIMK pathway indeed plays a crucial role in the regulation of Sertoli-germ cell AJ dynamics. The fact that the RhoB pathway in the kidney and brain was not activated suggests that AF-2364 exerts its effects primarily at the testis-specific ES multiprotein complex structures between Sertoli cells and spermatids. In summary, this report illustrates that Sertoli germ cell AJ dynamics are regulated, at least in part, via the integrin/ROCK/LIMK/cofilin signaling pathway.     

KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility

Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.     

Spermiogenesis in Xenopus laevis: from late spermatids to spermatozoa

Spermatogenesis is a complex morphogenetic process in which microfilaments and microtubules have been shown to play an important role. The last steps of Xenopus spermatogenesis, i.e., the corkscrew shaping of the sperm head, have been followed to study actin and microtubule distribution by conventional and immunoelectron microscopy. During sperm head morphogenesis, actin is absent in the elongating spermatids, but it is present in the Sertoli cells where results localized at the periphery of their cytoplasm that surrounds the developing germ cells. Sertoli cell actin and microtubules may assist the elongation and the shaping of the spermatids and function in maintaining the Sertoli-spermatid association.