Characterization and immunolocalization of beta-D-glucuronidase in mouse testicular germ cells and spermatozoa.

Spermatozoa released from the seminiferous tubules are terminally differentiated cells with no known synthetic activity. Their components are synthesized in the spermatogenic cells during spermatogenesis. In this study, we report the characterization and immunolocalization of beta-glucuronidase in mouse testicular germ cells and spermatozoa. The enzyme is an exoglycohydrolase with dual localization, being present in lysosomes and endoplasmic reticulum of several mouse and rat tissues. The purified germ cell preparations (spermatocytes, round spermatids, and condensed/elongated spermatids) when assayed for beta-glucuronidase activity showed that the spermatocytes contained five times more enzyme activity per cell than the spermatids. Polyacrylamide gel electrophoresis, carried out under native and denaturing conditions, demonstrated that the germ cells express only the lysosomal form of the enzyme (pI 5.5-6.0) with a subunit molecular mass of 74 kDa. Immunocytochemical studies revealed a positive reaction in the Golgi membranes, Golgi-associated vesicles, and lysosomes of late spermatocytes (pachytene spermatocytes) and a stage-specific localization during spermiogenesis. The forming or formed acrosome of the elongated spermatids (stages 9-16) and epididymal spermatozoa was highly immunopositive. Comparison of immunoprecipitation curves and kinetic properties of the enzyme present in spermatocytes and spermatozoa revealed no major differences. Taken together, our results demonstrate that beta-glucuronidase activities present in the lysosomes of spermatocytes and the sperm acrosome are kinetically and immunologically similar.

Composition and organization of tubulin isoforms reveals a variety of axonemal models.

In the flagellum of mammalian spermatozoa, glutamylated and glycylated tubulin isoforms are detected according to longitudinal gradients and preferentially in axonemal doublets 1-5-6 and 3-8, respectively. This suggested a role for these tubulin isoforms in the regulation of flagellar beating. In the present work, using antibodies directed against various tubulin isoforms and quantitative immunogold analysis, we aimed at investigating whether the particular accessibility of tubulin isoforms in the mammalian sperm flagellum is restricted to this model of axoneme surrounded with periaxonemal structures or is also displayed in naked axonemes. In rodent lung ciliated cells, all studied tubulin isoforms are uniformly distributed in all axonemal microtubules with a unique deficiency of glutamylated tubulin in the transitional region. A similar distribution of tubulin isoforms is observed in cilia of Paramecium, except for a decreasing gradient of glutamylated tubulin labeling in the proximal part of axonemal microtubules. In the sea urchin sperm flagellum, predominant labeling of tyrosinated and detyrosinated tubulin in 1-5-6 and 3-8 doublets, respectively, were observed together with decreasing proximo-distal gradients of glutamylated and polyglycylated tubulin labeling and an increasing gradient of monoglycylated tubulin labeling. In flagella of Chlamydomonas, the glutamylated and glycylated tubulin isoforms are detected at low levels. Our results show a specific composition and organization of tubulin isoforms in different models of cilia and flagella, suggesting various models of functional organization and beating regulation of the axoneme.

Expression of glycylated tubulin during the differentiation of spermatozoa in mammals.

Using quantitative immunogold analyses of tubulin isoforms we previously demonstrated a unique differential expression of glutamylated tubulin in the flagellum of mouse and man spermatozoa [Fouquet et al., 1997: Tissue Cell 29:573-583]. We have performed similar analyses for glycylated tubulin using two monoclonal antibodies, TAP 952 and AXO 49, directed to mono- and polyglycylated tubulin respectively. Glycylated tubulin was not found in centrioles and cytoplasmic microtubules (manchette) of germ cells. In mouse and man, axonemal tubulin was first monoglycylated and uniformly distributed in all doublets at all levels of the flagellum in elongating spermatids. In human mature spermatozoa axonemal microtubules were enriched in monoglycylated tubulin from the base to the tip of the flagellum. In mouse sperm flagellum a similar gradient of monoglycylated tubulin was also observed in addition to an opposite gradient of polyglycylated tubulin. In both species, monoglycylated tubulin labeling predominated in doublets 3-8 whereas glutamylated tubulin labeling [Fouquet et al., 1997] predominated in doublets 1-5-6. These differential labelings were suppressed after motility inhibition of mouse spermatozoa by sodium azide treatment and in non-motile human spermatozoa lacking dynein arms. The unique distribution of these tubulin isoforms and the known inhibition of motility induced by their specific antibodies are consistent with a complementary role of tubulin glycylation and glutamylation in the regulation of flagellar beating in mammalian spermatozoa.

Glutamylation of centriole and cytoplasmic tubulin in proliferating non-neuronal cells.

We have examined the distribution of glutamylated tubulin in non-neuronal cell lines. A major part of centriole tubulin is highly modified on both the alpha- and beta-tubulin subunits, whereas a minor part of the cytoplasmic tubulin is slightly modified, on the beta-tubulin only. Furthermore, we observed that tubulin glutamylation varies during the cell cycle: an increase occurs during mitosis on both centriole and spindle microtubules. In the spindle, this increase appears more obvious on the pole-to-pole and kinetochore microtubules than on the astral microtubules. The cellular pattern and the temporal variation of this post-translational modification contrast with other previously described tubulin modifications. The functional significance of this distribution is discussed.

Gamma-tubulin during the differentiation of spermatozoa in various mammals and man.

The distribution of gamma-tubulin as a marker of microtubule organizing centres (MTOC) was studied during spermiogenesis in rodents and in rabbit, monkey and man. A polyclonal antibody directed against human gamma-tubulin was used both for indirect immunofluorescence (IIF) and post-embedding immunogold procedures. In all species, gamma-tubulin was detected in the proximal and distal centrioles of round spermatids. In elongating spermatids, gamma-tubulin was predominantly found in the pericentriolar material (PCM) of both centrioles and particularly around the adjunct of the proximal centriole. At this level, some labelling was also associated with manchette microtubules, but other parts of the manchette and the nuclear ring were never labelled. We propose a role for distal centriole gamma-tubulin in axoneme nucleation and centriolar adjunct gamma-tubulin in manchette nucleation. The disappearance of gamma-tubulin in mature spermatozoa indicates that sperm aster nucleation should be dependent on oocyte gamma-tubulin. Remnants of gamma-tubulin in some human spermatozoa suggest that paternal gamma-tubulin also could contribute to sperm aster formation.

Expression of tubulin isoforms during the differentiation of mammalian spermatozoa.

Using the GT 335 mAb we have previously demonstrated a differential expression of glutamylated tubulin isoforms during spermatogenesis and in spermatooza of the mouse and human. Moreover, the proximodistal decrease of the immunolabeling and its predominance in doublets 1-5-6, corresponding to the plane of the flagellar wave, suggested that the glutamylated tubulin could be involved in a functional heterogeneity of microtubules in peripheral doublets of the sperm flagellum. In order to characterize further the importance of glutamylated tubulin in the sperm model, we analyzed tubulin isoforms by immunoblotting and quantitative immunogold, using antibodies to the C-terminal domain of both subunits including non-glutamylated and glutamylated epitopes. The unique differential immunolabeling of the glutamylated tubulin was confirmed with three mAbs 406-3, 392-2 and B3, in addition to GT 335. This differential labeling was interpreted as a differential accessibility of tubulin epitopes since it was greatly reduced in human spermatozoa lacking dynein arms and after motility inhibition of normal spermatozoa by azide pretreatment. We suggest that the glutamylated tubulin interacts with other axonemal and/or periaxonemal proteins which could be involved in flagellar beating and its regulation.

Glutamylated tubulin as a marker of microtubule heterogeneity in the human sperm flagellum.

Four site-directed monoclonal antibodies (mAbs) to tubulin: DM1A and DM1B general anti-alpha and anti-beta tubulin, 6-11B-1 anti-acetylated alpha tubulin and GT335 anti-glutamylated alpha and beta tubulin were used to study the distribution of tubulin isoforms in the human sperm flagellum. Since indirect immunofluorescence (IIF) did not give reliable results, a quantitative study of the immunogold labelling of the flagellum was performed at five levels: the mid-piece, three successive regions of the principal piece and the terminal piece. A uniform labelling was observed with DM1A, DM1B and 6-11B-1 mAbs. In contrast, the labelling of glutamylated tubulin detected with GT335 mAb decreased from the middle piece to the terminal piece both for peripheral doublets and the central pair. The changes in labelling of peripheral doublets were related to the pattern of outer dense fibre (ODF) changes. Thus doublets 1-5-6, associated with the largest number of ODF, were the most heavily labelled. This predominant labelling corresponded to the plane of flagellar beating suggesting a functional heterogeneity of peripheral doublets.

Comparative immunogold analysis of tubulin isoforms in the mouse sperm flagellum: unique distribution of glutamylated tubulin.

The distribution of different tubulin isoforms in the mouse sperm flagellum was studied using four site-directed antibodies to tubulin: DM1A and DM1B general anti alpha and beta-tubulin, 6-11B-1 anti-acetylated alpha-tubulin, and GT335 anti-glutamylated alpha and beta-tubulin. Quantitative immunogold analyses were performed on five regions of the flagellum: the middle piece, three successive regions of the principal piece, and the terminal piece. A uniform labeling was observed with DM1A and DM1B along the entire flagellum both for peripheral doublets and the central pair. Similar results were obtained with 6-11B-1 directed to acetylated alpha-tubulin, an N-terminal-modified tubulin isoform. In contrast, the labeling for glutamylated alpha and beta-tubulin, C-terminal modified isoforms, was not uniform. The highest intensity was found in the middle piece and the terminal piece. The labeling which decreased significantly both for peripheral doublets and central pair along the principal piece was considered as a loss of glutamylated tubulin accessibility. From the middle piece to the end of the principal piece, this labeling was predominant in doublets 1-5-6, corresponding to the plane of the flagellar wave. However, the labeling for doublets 2-3-4-7-8-9 was heterogeneous, showing an increasing asymmetry. These results suggest that in the mammalian sperm cell model, the glutamylated tubulin might be involved in a functional heterogeneity among peripheral doublets of the flagellum.

Differential distribution of glutamylated tubulin in the flagellum of mouse spermatozoa.

Using a monoclonal antibody (GT 335) we previously demonstrated that glutamylation is a predominant posttranslational modification of alpha and beta tubulin isoforms in the axoneme of mouse spermatozoa (Fouquet et al., Cell Motil. Cytoskel. 27, 49, 1994). However, we noted that the staining intensity and/or distribution of glutamylated tubulin were not identical using either indirect immunofluorescence (IIF) or immunoelectron microscopy. To test this discrepancy various permeabilization procedures were performed for IIF: methanol or acetone alone or in combination, including freezing pretreatment and with or without paraformaldehyde fixation. Each procedure gave a particular labeling of sperm axoneme. The diversity of axoneme labeling in mouse spermatids and spermatozoa appeared dependent both on the absence or presence of periaxonemal sheaths and permeabilization procedures. For comparison with IIF and to avoid problematic premeabilization treatments a quantitative postembedding immunogold approach was preferred. In these conditions the labeling predominated in the middle piece of the sperm flagellum and decreased progressively in the principal piece. However, the labeling of the terminal piece was similar to that of the middle piece. These results suggested a differential glutamylated tubulin distribution along the axoneme of the mouse sperm flagellum.

The cytoskeleton of mammalian spermatozoa.

The mammalian spermatozoa are endowed with a unique cytoskeleton which consists both of ubiquitous and specific proteins, some of them arising from gene haploid transcription. In the head, a dense perinuclear layer is made of basic proteins (calicin, cylicin, etc) associated with calmodulin and actin remnants. In the flagellum, the axonemal microtubules are mainly composed of glutamylated tubulin isoforms; the periaxonemal outer dense fibers and fibrous sheath are considered as related cytoskeletal structures on the basis of some common polypeptides.

Differential distribution of glutamylated tubulin during spermatogenesis in mammalian testis.

The distribution of glutamylated tubulin has been analyzed in mammalian testis using the specific mAb GT335 by immunoelectron microscopy and immunoblotting. In spermatozoa of various species, immunogold labeling showed the presence of glutamylated tubulin in all of the microtubules of axoneme and centrioles, whereas the microtubule network of the spermatid manchette was unlabeled. In earlier germ cells, centriole was the only microtubule structure to be labeled. A similar distribution was observed using the anti-acetylated tubulin antibody (6-11B-1), confirming previous results of Hermo et al. [Anat. Rec. 229:31-50, 1991]. However, among testicular somatic cells, microtubules of some Sertoli cell branches were not acetylated but glutamylated. 2-D PAGE of mouse and hamster sperm extracts showed a high level of alpha and beta-tubulin heterogeneity, comparable to that found in brain. Immunoblotting with GT335 revealed a large amount of glutamylated tubulin resolved into numerous alpha as well as beta-tubulin isoforms. This suggests that the major testis-specific tubulin isotypes (m alpha 3/7 and m beta 3) are also glutamylatable. These results show a subcellular sorting of posttranslationally modified tubulin isoforms in spermatids, glutamylation being associated with the most stable microtubule structures.

Spectrin and ankyrin like proteins in spermatids and spermatozoa of the hamster and some other mammals.

The presence of spectrin and ankyrin-like proteins was investigated during the differentiation and maturation of spermatozoa in mammalian species which have previously been studied for actin and calmodulin. These actin-binding proteins were characterized by immunoblotting and localized by immunoelectron microscopy. Neither spectrin nor ankyrin could be detected in the F-actin rich subacrosomal layer of spermatids in any species. In hamster and mouse maturing spermatids and spermatozoa, spectrin was mainly evidenced around the fibrous sheath of the flagellum whereas ankyrin was detected only in the neck. In rabbit spermatozoa, spectrin was evidenced in the outermost cytoplasmic layer of the post-acrosomal region and a light ankyrin labeling appeared in the neck. In rat, monkey and human sperm cells, these 2 proteins were not demonstrated. These results showed that as for actin there was no uniform pattern of distribution of spectrin and ankyrin among the 6 species studied.

Association of spectrin with manchette microtubules in mammalian spermatids.

In hamster and mouse spermatozoa a spectrin immunogold labeling was found under the plasma membrane in the principal piece of the flagellum. During spermatid differentiation, the spectrin labeling was associated with the manchette, a transient microtubular network involved in nuclear shaping and organelle translocation.

Species-specific localization of actin in mammalian spermatozoa: fact or artifact?

Actin has been characterized and localized in sperm cells of many mammals. Nevertheless, the reported localizations obtained by different methods and/or antibodies varied from species to species and even for the same species. To clarify the question, sperm actin distribution was reinvestigated under uniform technical conditions. Immunogold post-embedding procedures were performed using a polyclonal and two monoclonal antibodies of known specificity to localize actin in spermatids and spermatozoa of rabbit, mouse, rat, monkey, and human. In these species, actin (F-actin) was detected with the three antibodies between the nucleus and the acrosome of round and elongating spermatids. Species-specific changes occurred in maturing spermatids. In the rabbit, actin labeling decreased and disappeared from the tip to the base of the subacrosomal layer. In testicular and epididymal spermatozoa actin was detected only with a monoclonal antibody (Amersham) successively in the neck, postacrosomal area, and subacrosomal bulges. In mouse late spermatids a transitory labeling of the neck was detected only with the polyclonal antiactin. In testicular and epididymal spermatozoa an actin labeling was observed in the principal piece of the tail. In rat, monkey, and human sperm cells actin remained undetected. These results suggest that there is a redistribution of actin in late spermatids and spermatozoa which is a species-specific process but not an artifact of methodological origin. Thus, a function for actin in sperm, if any, remains to be demonstrated.

Perinuclear cytoskeleton of acrosome-less spermatids in the blind sterile mutant mouse.

The perinuclear cytoskeleton of mammalian spermatids is thought to play a major role in nucleus-acrosome association and in shape changes of the head during spermiogenesis. To test these hypotheses acrosome-less spermatids in blind-sterile mutant mice were investigated for the development of the subacrosomal layer. Immunogold procedures were used for the detection of actin and calmodulin. In addition to various other abnormalities many acrosome-less round and elongating spermatids developed a subacrosomal layer with an actin and calmodulin distribution similar to that observed in normal spermatids. However, in mutant elongating spermatids the apical part of the nucleus was truncated and/or folded. The expected elongation and shaping of the nucleus only occurred in its caudal part associated with an hypertrophied and somewhat ectopic manchette. These abnormalities and those previously observed in mutant and experimental models indicated that the subacrosomal layer may form independently of the acrosome. It is suggested that the subacrosomal filamentous actin is a transitory scaffolding which might be involved in the assemblage of other proteins of the perinuclear cytoskeleton. However, by itself, this layer is not sufficient to ensure a normal shaping of the nucleus. Acrosome-nucleus interactions mediated by the subacrosomal layer seem necessary to shape the cranial spermatid head. The manchette appears to be involved only in the caudal nuclear shaping.

Sperm actin and calmodulin during fertilization in the hamster: an immune electron microscopic study.

The distribution of actin and CaM in hamster spermatozoa was examined during the early events of fertilization using postembedding immunogold procedures. Actin was immunolocalized with a polyclonal antibody and two monoclonal antibodies. CaM was immunodetected with a polyclonal antibody. In epididymal sperm, actin labeling was found solely in the principal piece of the flagellum. CaM labeling was observed in the postacrosomal lamina, subacrosomal ring, and tip of the perforatorium. These distributions were not modified after capacitation and acrosome reaction. During the successive steps of sperm-egg fusion actin remained undetected in the sperm head whereas its location did not change in the flagellum. CaM distribution remained unmodified until the sperm head begins to decondense. At later stages of sperm head decondensation the postacrosomal lamina and its CaM labeling disappeared, whereas gold particles were still detected in the subacrosomal layer. The predominant location of actin into the egg cortex, particularly the microvillus-free area was confirmed. Except for the CaM labeling of the meiotic spindle, no special CaM location could be found throughout the egg. Thus, in hamster, a role for sperm actin in sperm-egg fusion appears unlikely. In contrast the CaM present in the Ca(2+)-rich postacrosomal lamina could be involved in the regulation of egg activation.

Localization of calmodulin in perinuclear structures of spermatids and spermatozoa: a comparison of six mammalian species.

The distribution of Calmodulin was examined during spermiogenesis and sperm epididymal maturation in rabbit, hamster, mouse, rat, monkey, and human. An affinity-purified antibody to Calmodulin was used to characterize this protein in sperm extracts by immunoblot analysis. Post-embedding immunogold procedures were used to localize Calmodulin at the ultrastructural level. The pattern of Calmodulin distribution was similar in the six species studied. A diffuse labeling was observed in round spermatids. Gold particles accumulated first in the subacrosomal layer of elongating spermatids. The perinuclear ring was also labeled. During the maturation phase of spermatids, Calmodulin labeling extended to the postacrosomal sheath. Dramatic changes occurred at spermiation so that in testicular sperm Calmodulin immunostaining was predominant in the postacrosomal sheath. Some labeling was still detected in restricted areas of the subacrosomal layer. This feature varied from species to species. Calmodulin location did not change during sperm epididymal maturation. A role for Calmodulin in the control of manchette development and regulation of subacrosomal actin aggregation state during spermiogenesis is proposed. The unique location of Calmodulin in the postacrosomal sheath of all species that have been studied in this work, together with the known presence of calcium in this area suggest a pivotal role for Calmodulin in sperm-egg fusion process.

Immunoelectron microscopic distribution of actin in hamster spermatids and epididymal, capacitated and acrosome-reacted spermatozoa.

The distribution of actin in hamster sperm cells was studied during spermiogenesis, epididymal transit, in vitro capacitation and acrosome reaction by immunogold procedures using a polyclonal and two monoclonal antiactin antibodies. A predominant actin labeling (F-actin) was detected in the subacrosomal space of spermatids. Actin labeling was also observed under the plasma membrane of intercellular bridges and along the outer acrosomal membrane. In late spermatids there was both F-actin depolymerization and a loss of actin immunolabeling, thus suggesting a dispersion of G-actin monomers. No obvious labeling was evidenced in residual bodies. This pattern was observed with the three antiactin probes. In contrast, an actin labeling reappeared over the fibrous sheath of the flagellum in epididymal spermatozoa but only when the polyclonal antibody was used. Only one single actin reactive band was detected by immunoblotting of sperm extracts. Since the sperm tails were NBD phallacidin negative they were considered to contain either G-actin or actin oligomers rather than bundles of actin filaments. It is suggested that G-actin originating in the head of late spermatids was redistributed to the flagellum of epidymal spermatozoa. No further changes were noted after capacitation and acrosome reaction thus indicating no apparent effect on actin polymerization and distribution.

Immunogold distribution of actin during spermiogenesis in the normal rabbit and after experimental cryptorchidism.

Immunogold procedures for actin detection were used in combination with experimental cryptorchidism in the rabbit as a model to shed more light on the function of subacrosomal actin during spermiogenesis. In the normal testis, actin was localized in the perinuclear substance (PNS) from round spermatid onward but it was not detected in late spermatids. Actin labeling in each type of spermatid was essentially unmodified after 24 hr of cryptorchidism. However, among relevant immediate and delayed effects, discontinuous acrosomes overlying a continuous PNS with normal actin labeling were noted. Nuclear invaginations were seen in combination with subacrosomal dilatations; at this site actin labeling was found only in the PNS closely apposed to the nuclear envelope. In subacrosomal areas lacking PNS, actin labeling also was lacking. These results suggest that the subacrosomal actin (F-actin) is a component of the PNS that is tightly bound to the nuclear envelope rather than the overlying inner acrosomal membrane. Therefore, a function for the subacrosomal actin either in anchoring the acrosome to the nucleus or in capping the inner acrosomal membrane appears unlikely. The data rather suggest a capping function for the nuclear membrane during spermiogenesis.

Comparison of LR white resin, Lowicryl K4M and Epon postembedding procedures for immunogold staining of actin in the testis.

The efficiency of various postembedding procedures for actin immunogold detection was compared using testicular tissue as a model. Whatever the fixative, testes embedded in LR White resin or in Lowicryl K4M showed few differences as regard ultrastructural preservation and gave similar actin antigenicity preservation. A purified polyclonal antibody (IgG) and a monoclonal antibody (IgM) visualized with gold secondary antibody yielded high labeling intensity whereas the IgG-protein-A gold association was less efficient. Crude antisera gave a low specific staining/background ratio. Samples of testes, fixed in different conditions, were also embedded in Epon, omitting propylene oxide and lowering polymerization temperature to 40 degrees-50 degrees C. This slight modification improved ultrastructural preservation which was better than with hydrophilic resins, as well as made possible immunogold detection of actin though antigenicity preservation was lesser than with these resins. Thus, in Epon embedded samples actin labeling, using IgG antiactin-gold secondary antibody, was similar to that observed after hydrophilic resin-protein-A gold procedures. In addition to actin labeling of various somatic cells it was confirmed that actin is a consistent component of the subacrosomal space of spermatids during the greater part of spermiogenesis in rat.