Calmodulin immunoelectron microscopy: redistribution during ram spermatogenesis and epididymal maturation. II

Affinity-purified monospecific antibodies and indirect immunogold and immunoferritin labeling on ultra-thin sections of low-temperature Lowicryl K4M-embedded samples were used to study the redistribution of calmodulin in ram spermatids and epididymal spermatozoa at the electron microscopic level. Calmodulin appeared as an integral component of well-defined structures or organelles of these cells. In young spermatids, calmodulin was localized in the nucleus, cytoplasm, and developing acrosome. During spermatogenesis and epididymal maturation, calmodulin left the acrosome to reach the perinuclear substance and finally became concentrated in the post-acrosomal area of the head, although some calmodulin remained associated with the tip of the acrosome. Such a redistribution is consistent with the preferential location of Ca2+ in the post-acrosomal cytoplasm of ejaculated spermatozoa. Calmodulin was also observed in the flagellum associated with the plasma membrane and with the motility apparatus, between coarse fibers and axonemal microtubules. These changes in calmodulin distribution may account for the Ca2+-dependent regulation of spermatogenesis and sperm maturation. Calmodulin therefore appears to be a pleiotropic regulator of male gamete development and functions.     

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.     

Differential localization of two isoforms of the regulatory subunit RIIα of cAMP-dependent protein kinase in human sperm: Biochemical and cytochemical study

In the present study, immunogold labeling of ultrathin sections of ejaculated sperm was used to obtain insight into the ultrastructural localization and presumable function of type II cAMP-dependent protein kinase in sperm motion. In the flagellum, a human-specific isoform of the RIIα subunit was located on the axonemal microtubule wall, whereas a different isoform of broader specificity was present in the cytoplasm at the periphery of the coarse fibers and fibrous sheath. This isoform was also found in the mitochondria. The human-specific RIIα subunit is likely linked to microtubules by a unique binding protein of M_r 72kD. These findings are in agreement with the concept of a concerted mechanism involving phosphorylation of both the axonemal microtubules and the fibrous structures for the regulation of mammalian sperm motion. © 1994 Wiley-Liss, Inc.     

Immunocytochemical localization of tubulins in spermatids and spermatozoa of Euptoieta hegesia (Lepidoptera: Nymphalidae)

A comparative analysis of the distribution of tubulin types in apyrene and eupyrene sperm of Euptoieta hegesia butterflies was carried out, also verifying the presence of tubulin in lacinate appendages of the eupyrene sperm. Ultrathin sections of LR White embedded spermatids and spermatozoa were labeled for alpha, beta, gamma, alpha-acetylated and alpha-tyrosinated tubulins. Apyrene and eupyrene spermatids show the same antibody recognition pattern for tubulins. All tubulin types were detected in axonemal microtubules. Alpha and gamma tubulins were also detected on the cytoplasmic microtubules. However, for beta and tyrosinated tubulins only scattered labeling was detected on cytoplasmic microtubules and acetylated tubulin was not detected. In apyrene and eupyrene spermatozoa only the axoneme labeling was analyzed since cytoplasmic microtubules no longer exist in these cells. Alpha, beta and tyrosinated tubulins were easily detected on the apyrene and eupyrene axoneme; gamma tubulin was strongly marked on eupyrene axonemes but was scattered on the apyrene ones. Acetylated tubulin appeared with scattered labeling on the axoneme of both sperm types. Our results demonstrate significant differences in tubulin distribution in apyrene and eupyrene axonemal and cytoplasmic microtubules. Extracellular structures, especially the lacinate appendages, were not labeled by antibodies for any tubulin.     

Accessory tubules and axonemal microtubules of Apis mellifera sperm flagellum differ in their tubulin isoform content

In the insect sperm flagellum, an extra set of nine additional microtubules, named accessory tubules, is present surrounding the axoneme. Using a sarcosyl/urea extraction, we were able to fractionate the microtubular cytoskeleton of the sperm flagellum of the insect Apis mellifera resulting in the dissociation of the axonemal microtubule protein components and the accessory tubules. This has allowed us to compare the tubulin isoform content of axonemal microtubules and accessory tubules by immunoelectron microscopy and immunoblotting using a panel of monoclonal antibodies directed against different tubulin post-translational modifications (PTMs). All the PTMs occurring in axonemal tubulin are also present in accessory tubules, which indicates the close relativeness of accessory tubules to axonemal rather than to cytoplasmic microtubules. However, our results demonstrate the presence of significant differences in the tubulin isoform content of axonemal microtubules and accessory tubules. First, the tubulin tyrosination extent of accessory tubules is far lower than that of axonemal microtubules, thus confirming at the molecular level their morphogenetic origin as outgrowths from the B-subtubule of each microtubular doublet. Second, although polyglycylation seems to occurr at the same extent in both microtubular systems, alpha-tubulin exhibits a larger amount of monoglycylated sites in axonemal microtubules than in accessory tubules. Third, a greater amount of beta-tubulin molecules is glutamylated in axonemal microtubules than in accessory tubules. Moreover, highly acidic isoforms, likely molecules with longer polyglutamate side chains, are present only in axonemal microtubules. Taken together, our data are indicative of a higher level of tubulin heterogeneity in axonemal microtubules than in accessory tubules. They also show a segregation of post-translationally modified isoforms between accessory tubules and axonemal microtubules and suggest the implication of PTMs in the functional specialization of the two microtubular systems.     

Sperm and spermatozeugma ultrastructure in the inseminating species Tyttocharax cochui, T. tambopatensis, and Scopaeocharax rhinodus (Pisces: Teleostei: Characidae: Glandulocaudinae: Xenurobryconini)

This article presents the scanning and transmission electron microscopy of the spermatozoa and sperm packets of three inseminating species of the glandulocaudine tribe Xenurobryconini. All three species, Scopaeocharax rhinodus, Tyttocharax cochui, and T. tambopatensis produce unencapsulated sperm packets (= spermatozeugmata) of similar morphology. The external anterior surface of each spermatozeugma is comprised of elongate sperm heads arranged in parallel, and the posterior part is made up of tightly packed flagella. The interior of the anterior portion consists of alternating layers of sperm heads and flagella. The remarkable integrity of each packet appears to be maintained through an electron-dense secretion seen among all parts of the cells. Spermatozeugma formation takes place within the spermatocysts at the end of spermiogenesis and at spermiation fully formed packets are released. Morphology of the mature spermatozoa was similar in all three species. Each nucleus is elongate, flattened along most of its length, and tapers at either end. The two centrioles are nearly parallel to one another and are located just anterior to the nucleus. Elongate mitochondria are located along the nucleus. The single flagellum, which lacks axonemal fins, is initially contained within a short cytoplasmic collar. Accessory microtubules run parallel to the long axis of the nucleus just beneath the plasma membrane. During spermiogenesis, no nuclear rotation occurs and the cytoplasmic canal containing the flagellum elongates along with the nucleus. However, prior to spermiation all but the anterior portion of the collar degenerates. The sperm modifications observed in these species are discussed as adaptations to the unique reproductive habit of insemination.     

Possible conversion of axonemal microtubules to pellicular microtubules in Trypanosoma gambiense treated with vinblastine, colchicine plus concanavalin A

The effects of vinblastine alone and in combination with vinblastine, colchicine and concanavalin A on microtubules of Trypanosoma gambiense cultured in vitro were studied ultrastructurally. Trypanosomes treated with vinblastine at 20 micrograms/ml, showed fusion of the extracellular flagellum with the plasma membrane of the parasite. As a result, the axoneme with the paraxial rod in the extracellular flagellum was taken into the cytoplasm. Although the axonemal and pellicular microtubules in T. gambiense differ in function and origin, the axonemal microtubules of the extracellular flagellum that was taken into the cytoplasm could be converted to pellicular microtubules by treatment with a combination of vinblastine (20 micrograms/ml), concanavalin A (10 micrograms/ml) and colchicine (100 micrograms/ml).     

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. © 1996 Wiley-Liss, Inc.     

Distribution of microtubules and microfilaments in thyroid follicular epithelial cells of normal,TSH-treated,aged, and hypophysectomized rats

Summary We investigated the distribution of microtubules and microfilaments in rat thyroid follicular epithelial cells by applying an immunofluorescence technique with monoclonal antibodies against tubulin and by staining sections with rhodamine-phalloidin. In normal thyroid cells, microtubules run longitudinally from the apical region to the basal region intersecting with each other. In addition, intense labelling with tubulin antibodies was observed in the apical part of the cell. The ultrastructural examinations showed that microtubules often run along the apical plasma membrane. Dot-like labelling with anti-tubulin antibodies was often observed in the perinuclear space, but no microtubules were recognized in the nucleus. Microfilaments bound to rhodamine-phalloidin were distributed mainly beneath the apical plasma membrane, and the portion along the basolateral membrane was scarcely positive. The apical pole of the follicle cell was also decorated by anti-microtubule-associated protein-2 (MAP-2). After TSH stimulation, the intensity of immunocytochemical staining against tubulin was remarkably increased in the cytoplasm. Simultaneously, at the apical region, the staining intensity of rhodamine-phalloidin was increased. Microtubules and microfilaments appeared in the pseudopods after TSH injection. In hypophysectomized or aged rats, thyroid follicular epithelial cells decreased in height, and both immunofluorescent labelling against tubulin and rhodamine-phalloidin labelling were markedly decreased. These results indicate that the distribution and polymerization of microtubules and microfilaments in thyroid follicular epithelial cells vary with the functional stage.     

Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms

Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of alpha-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic alpha-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only alpha-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal alpha-tubulin isoform acetylated in vivo on the epsilon-amino group of lysine(s) (L'Hernault, S.W., and J.L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal alpha-tubulin, unassembled alpha-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic alpha-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated alpha-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of alpha-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated alpha-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo.     

Sperm ultrastructure of<Emphasis Type="Italic"> Mantophasma zephyra</Emphasis> (Insecta,Mantophasmatodea)

The ultrastructure of Mantophasma zephyra spermatozoa is described. Sperm cells have a trilayered acrosome with conspicuous extra-acrosomal material which expands along the nucleus. The nucleus is crossed anteriorly by a canal and its posterior end is embedded in the centriole adjunct material. A centriole with microtubular triplets is present. The flagellum has a 9+9+2 axonemal pattern, two partially crystallised mitochondrial derivatives, two membranous sacs and three connecting bands. The accessory microtubules are filled with dense material and have 16 protofilaments in their tubular wall. The intertubular material is not very expanded. In the seminal vesicles spermatozoa are stuck together to form spermatodesms, and their heads are also joined by adherens junctions. A cladistic analysis based on sperm features indicates a close relationship of Mantophasmatodea with Mantodea.     

Spermatogenesis in animals as revealed by electron microscopy

Summary The first indication of differentiation of the Jensen's ring has been detected in an early stage of spermiogenesis of Felis catus Linné when the pair of centrioles takes up a position immediately beneath the plasma membrane. The chromatoid bodies appear in the early spermatid cytoplasm through the nuclear pore complex. In a more advanced stage, such bodies have been found in association with the striated columns, the distal centriole or the proximal part of flagellum and the Jensen's ring. As the spermiogenesis proceeds, the bodies have decreased their size and density, and finally disappear in mature spermatozoa. The chromatoid bodies seem, therefore, to share with the centriole the capacity to form the connecting piece. As a consequence of disorganization of triplet microtubules of the centriole, a noticeable material appears in the center of lumen of the centriole to be identifiable as a distinct precursor of the central pair of axonemal complex. Microtubules are first developed as the sheath of principal piece of the sperm flagellum, originating from the plasma membrane surrounding the axonemal complex.     

Proteins associated with soluble adenylyl cyclase in sea urchin sperm flagella

Adenylyl cyclases (ACs) synthesize cAMP and are present in cells as transmembrane AC and soluble AC (sAC). In sperm, the cAMP produced regulates ion channels and it also activates protein kinase-A that in turn phosphorylates specific axonemal proteins to activate flagellar motility. In mammalian sperm, sAC localizes to the midpiece of flagella, whereas in sea urchin sperm sAC is along the entire flagellum. Here we show that in sea urchin sperm, sAC is complexed with proteins of the plasma membrane and axoneme. Immunoprecipitation shows that a minimum of 10 proteins is tightly associated with sAC. Mass spectrometry of peptides derived from these proteins shows them to be: axonemal dynein heavy chains 7 and 9, sperm specific Na+/H+ exchanger, cyclic nucleotide-gated ion channel, sperm specific creatine kinase, membrane bound guanylyl cyclase, cyclic GMP specific phosphodiesterase 5A, the receptor for the egg peptide speract, and alpha- and beta-tubulins. The sAC-associated proteins could be important in linking membrane signal transduction to energy utilisation in the regulation of flagellar motility.     

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.     

Differential localization of annexins in ram germ cells: a biochemical and immunocytochemical study.

We used antibodies that specifically bind annexins on Western blots to determine the distribution and abundance of these proteins in ram spermatids and sperm by immunogold electron microscopy. Annexins I and II were found essentially within the entire acrosome of spermatids. During epididymal maturation, they concentrated in the postacrosomal region or the acrosomal equatorial segment, respectively. They were also present in sperm flagellum, on the surface of the coarse fibers and fibrous sheath. These findings show that during ram germ cell maturation, annexins I and II are exported from the spermatid acrosome towards structurally and functionally defined parts of the sperm. Annexins III, IV, and V were not found in ram germ cells. Annexin VI was isolated from testis and sperm. In spermatids, it was found to be associated with endoplasmic reticulum and the mitochondria but was absent from the acrosome. In sperm, it was confined to the flagellum, the mitochondria, and on the coarse fibers and fibrous sheath. The presence of three annexins, in addition to calmodulin, in functional areas may indicate differential ways for sperm to control and regulate events that are known to be calcium dependent, such as flagellar motility, acrosome reaction, and fertilization.     

Cryopreservation, actin localization and thermotropic phase transitions in ram spermatozoa

The effects of controlled stress, i.e. cooling, upon the distribution of actin in ram spermatozoa were examined to investigate the hypothesis that cytoskeletal proteins are involved in the maintenance of sperm plasma membrane integrity. The normal distribution of actin on the spermatozoon was initially determined. A monoclonal antibody (IgM) interacted exclusively with the post-acrosomal region and the principal piece of the flagellum. By the use of a polyclonal antibody, actin was detected on the acrosome (excluding the equatorial segment), the post-acrosomal region and the whole of the flagellum. The actin was present in its non-filamentous form. Spermatozoa fixed at 39 degrees C and then treated for the immunofluorescent detection of actin with the monoclonal antibody were mostly unstained (proportion stained = 4.4% (+/- 1.6; s.e.m.); n = 8 ejaculates). Provided spermatozoa were permeabilized by greater than 0.025% Triton X-100 before immunofluorescence, actin was localized in the postacrosomal region of all sperm heads, and to a minor extent on the principal piece of the flagellum. Use of the polyclonal antibody confirmed that the post-acrosomal antigen was unmasked by detergent treatment. Slow cooling, over 2-h periods to various temperatures between 5 and 15 degrees C, also induced an increase in the proportion of cells showing post-acrosomal actin immunoreactivity. Cooling through the temperature range 15 to 10 degrees C markedly increased the proportion of immunoreactive cells (mean +/- s.e.m.; 12 +/- 4.5% at 15 degrees C; 27 +/- 4.5% at 10 degrees C; n = 4 ejaculates). Further cooling to 5 degrees C failed to elicit increased staining. Ultrastructural examination of cooled spermatozoa confirmed that a subpopulation of spermatozoa exhibited post-acrosomal actin immunoreactivity after cooling. These results are compatible with the suggestion that actin fulfills a stabilizing function in spermatozoa.     

An essential quality control mechanism at the eukaryotic basal body prior to intraflagellar transport

Constructing a eukaryotic cilium/flagellum is a demanding task requiring the transport of proteins from their cytoplasmic synthesis site into a spatially and environmentally distinct cellular compartment. The clear potential hazard is that import of aberrant proteins could seriously disable cilia/flagella assembly or turnover processes. Here, we reveal that tubulin protein destined for incorporation into axonemal microtubules interacts with a tubulin cofactor C (TBCC) domain-containing protein that is specifically located at the mature basal body transitional fibres. RNA interference-mediated ablation of this protein results in axonemal microtubule defects but no effect on other microtubule populations within the cell. Bioinformatics analysis indicates that this protein belongs to a clade of flagellum-specific TBCC-like proteins that includes the human protein, XRP2, mutations which lead to certain forms of the hereditary eye disease retinitis pigmentosa. Taken with other observations regarding the role of transitional fibres in cilium/flagellum assembly, we suggest that a localized protein processing capacity embedded at transitional fibres ensures the 'quality' of tubulin imported into the cilium/flagellum, and further, that loss of a ciliary/flagellar quality control capability may underpin a number of human genetic disorders.     

Flagellum retraction and axoneme depolymerisation during the transformation of flagellates to amoebae inPhysarum

Summary The transformation ofPhysarum polycephalum flagellates to myxamoebae is characterised by disappearance of the flagellum. This transition, from the flagellate to the myxamoeba was observed by phase contrast light microscopy and recorded by time lapse video photography to determine whether flagellates shed their flagella or they are absorbed within the cell. In addition, the kinetics of flagellum disappearance were also studied. Our observations indicate that the flagellum was absorbed within the cell; the process occurred within seconds. Flagellum resorbtion was preceded by typical morphological cell changes. The shape of the nucleus altered and its mobility within the cell decreased. It was not possible to observe the flagellum within the cell with phase contrast video recordings. Thin section electron microscopy was used to study this intracellular phenomenon. Several stages of flagellum dissolution could be identified within the cell. The two most important stages were: an axoneme surrounded by the flagellar membrane within a plasma membrane lined pocket or vacuole and the naked axoneme without its membrane, free within the cell cytoplasm. The existence of cytoplasmic microtubules prevented identification of any further dissolution stages of the flagellum. A group of microtubules adjacent to the flagellum but within the cytoplasm was observed in flagellates and also in those cells which possesed enveloped axonemes. The flagellum did not dissociate from the kinetosomes before resorbtion.Immunofluorescence studies with the 6-11-B-1 monoclonal antibody indicated that acetylated microtubules exist in myxamoebae after transformation from flagellates for up to 40 min. Acetylated tubulin is not limited to the centrioles in these cells.     

The fine structure of spermiogenesis in the Amblypygi and the Uropygi (Arachnida)

Summary Spermiogenesis in one species from each of the arachnid groups Amblypygi and Uropygi is described by electron microscopy: The whip spider,Tarantula marginemaculata (Amblypygi), and the whip-scorpion,Mastigoproctus giganteus, (Uropygi). In both species the earliest spermatid has a spherical nucleus and soon acquires an anterior acrosome and a posterior flagellar tail. The flagellun is peculiar in having a 9 + 3 axonemal pattern. By the mid-spermatid stage, the nucleus becomes conspicuously elongated, possibly through the agency of a manchette of microtubules. In the late spermatid, the elongated nucleus begins to coil posteriorly; simultaneously the middle piece and the tail flagellum begin to retract into the cell body to form a coiled intracellular axonema. Membranous profiles appear in the peripheral cytoplasm, possibly to accommodate a decrease in the total area of plasma membrane. The mature sperm is a spherical cell, which includes the following organelles in twisted and fully coiled configuration: an elongated nucleus, an acrosome and an acrosomal filament, a long middle piece with helically arranged mitochondria and an intracellular axonema.