Marie-Louise Kann

Nm23/NDP kinases in human male germ cells: role in spermiogenesis and sperm motility?

Nucleoside diphosphate (NDP) kinases, responsible for the synthesis of nucleoside triphosphates and produced by the nm23 genes, are involved in numerous regulatory processes associated with proliferation, development, and differentiation. Their possible role in providing the GTP/ATP required for sperm function is unknown. Testis biopsies and ejaculated sperm were examined by immunohistochemical and immunofluorescence microscopy using specific antibodies raised against Nm23-H5, specifically expressed in testis germinal cells and the ubiquitous NDP kinases A to D. Nm23-H5 was present in sperm extract, together with the ubiquitous A and B NDP kinases (but not the C and D isoforms) as shown by Western blotting. Nm23-H5 was located in the flagella of spermatids and spermatozoa, adjacent to the central pair and outer doublets of axonemal microtubules. High levels of NDP kinases A and B were observed at specific locations in postmeiotic germinal cells. NDP kinase A was transiently located in round spermatid nuclei and became asymmetrically distributed in the cytoplasm at the nuclear basal pole of elongating spermatids. The distribution of NDP kinase B was reminiscent of the microtubular structure of the manchette. In ejaculated spermatozoa, the proteins presented specific locations in the head and flagella. Nm23/NDP kinase isoforms may have specific functions in the phosphotransfer network involved in spermiogenesis and flagellar movement.

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 α and β‐tubulin, 6‐11B‐1 anti‐acetylated α‐tubulin, and GT335 anti‐glutamylated α and β‐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 α‐tubulin, an N‐terminal‐modified tubulin isoform. In contrast, the labeling for glutamylated α and β‐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.

The cytosolic chaperonin CCT associates to cytoplasmic microtubular structures during mammalian spermiogenesis and to heterochromatin in germline and somatic cells

Spermiogenesis, the haploid phase of spermatogenesis, is characterised by a dramatic cytodifferentiation of spermatids. The two major steps, nuclear shaping and cytoplasmic reorganisation of the organelles, rely on an extensive remodelling of the microtubule cytoskeleton. Folding of alpha- and beta-tubulin is mediated by the cytoplasmic chaperonin containing TCP-1 (CCT), highly expressed in testis. We studied CCT cellular distribution throughout spermatogenesis by immunofluorescence and immunoelectron microscopy. We unveil two main cytoplasmic localisations for CCT: at the centrosome and at the microtubules of the manchette, a structure unique to male germ cells. Both structures are essential for spermatid differentiation and may require CCT function. Although CCT is essentially cytoplasmic, a few reports suggest that a subset may have a nuclear localisation. We demonstrate that in the nucleus of germline and somatic cells, part of CCT associates to heterochromatin. In interphase cells, CCT seems generally confined to constitutive heterochromatin. Nevertheless, in condensing nucleus of future spermatozoon, it is also associated with chromatin undergoing compaction. Finally, in fully-condensed mitotic chromosomes, CCT is located all along the chromosomes. Our finding that CCT is associated with constitutive heterochromatin and to compacting chromatin raises the possibility that it may be implicated in maintenance and remodelling of heterochromatin.

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.

The cytoskeleton of mammalian spermatozoa.

Summary— The mammalian spermatozoa are endowed with a unique cytoskeleton which consists both of ubiquitous and specific proteins, some of them arising from a 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.

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.

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.

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.

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.

Association of spectrin with manchette microtubules in mammalian spermatids

Summary— 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.