Identification of the 58-kDa phosphoprotein associated with motility initiation of hamster spermatozoa

In our previous paper [M. Fujinoki et al. (2001) BIOMED: Res. 22, 45-58], we reported that the 58-kDa protein obtained from hamster sperm flagella was phosphorylated at serine residues in association with the start of motility. In the present experiments, we identified and localized the 58-kDa protein. The 58-kDa protein was assumed to exist in the acrosomal region domain of the sperm head and the whole sperm flagellum. In particular, a large amount of 58-kDa protein was localized in the equatorial segment of the acrosomal region domain of the sperm head and the middle piece of the sperm flagellum. In the next step, the 58-kDa protein was identified by peptide mass finger printing and LC-MS/MS analysis. The results suggested that the 58-kDa protein was ATP synthase H(+) transporting F1 beta, which is one of the mitochondrial components. Therefore, it is likely that the 58-kDa protein is associated with ATP production in the mitochondrial sheath in the middle piece of the sperm flagellum, and H(+) transport in the sperm head and the sperm flagellum except for the middle piece, since ATP synthase also acts as an H(+) pump.     

Septins at the annulus of mammalian sperm

The annulus is an electron-dense ring structure connecting the midpiece and the principal piece of the mammalian sperm flagellum. Proteins from the septin family have been shown to localize to the annulus. A septin complex is assembled early in spermiogenesis with the cochaperone DNAJB13 and, in mature sperm, associates with Testis Anion Transporter 1; SLC26A8 (Tat1), a transmembrane protein of the SLC26 family. Studies in mice have shown that the annulus acts as a barrier to protein diffusion and controls correct organization of the midpiece. Consistent with these findings, absence of the annulus is associated with flagellum differentiation defects and asthenozoospermia in humans.     

Calcium regulation of flagellar curvature and swimming pattern in triton X-100--extracted rat sperm

Free Ca2+ changes the curvature of epididymal rat sperm flagella in demembranated sperm models. The radius of curvature of the flagellar midpiece region was measured and found to be a continuous function of the free Ca2+ concentration. Below 10(-7) M free Ca2+, the sperm flagella assumed a pronounced curvature in the same direction as the sperm head. The curvature reversed direction at 2.5 x 10(-6) M Ca2+ to assume a tight, hook-like bend at concentrations of 10(-5) to 10(-4) M free Ca2+. Sodium vanadate at 2 x 10(-6) M blocked flagellar motility, but did not inhibit the Ca2+-mediated change in curvature. Nickel ion at 0.2 mM and cadmium ion at 1 microM interfered with the transition and induced the low Ca2+ configuration of the flagellum. The forces that maintain the Ca2+-dependent curvature are locally produced, as dissection of the flagella into segments did not significantly alter the curvature of the excised portions. Irrespective of the induced pattern of curvature, the sperm exhibited coordinated, repetitive flagellar beating in the presence of ATP and cAMP. At 0.3 mM ATP the flagellar waves propagated along the principal piece while the level of free Ca2+ controlled the overall curvature. When Ca2+-treated sperm models with hooked midpieces were subjected to higher concentrations of ATP (1-5 mM), some cells exhibited a pattern of movement similar to hyperactivated motility in capacitated live sperm. This type of motility involved repetitive reversals of the Ca2+-induced bend in the midpiece, as well as waves propagated along the principal piece. The free Ca2+ available to the flagellum therefore appeared to modify both the pattern of motility and the flagellar curvature.     

Flagellar gyration and midpiece rotation during extension of the acrosomal process of Thyone sperm: how and why this occurs

The midpiece of Thyone sperm contains a large mitochondrion and a centriolar pair. Associated with one of the pair, i.e., the basal body of the flagellum, are satellite structures which apparently anchor the flagellar axoneme to the mitochondrion and to the plasma membrane covering the midpiece. Immediately before and as the acrosomal process elongates, the flagellum and the midpiece begin to rotate at 1-2 rotations per second even though the head of the sperm, by being firmly attached on its lateral surfaces to the coverslip, does not rotate at all. This rotation is not observed in the absence of flagellar beating whose frequency is much greater than that of its gyration. To understand how the midpiece rotates relative to the sperm head, it is first necessary to realize that in Thyone the flagellar axoneme projects at an acute angle to the principal axis of the sperm and is bent towards one side of this axis. Thus movement of the flagellum induces the sperm to tumble or yaw in solution. If the head is stuck, the midpiece will rotate because all that connects the sperm head to the midpiece is the plasma membrane, a liquid-like layer. A finger-like projection extends from the proximal centriole into an indentation in the basal end of the nucleus. In contrast to the asymmetry of the flagellum, this indentation is situated exactly on the principal axis of the sperm and, along with the finger-like projection, acts as a biological bearing to maintain the orderly rotation of the midpiece. The biological purpose of flagellar gyration during fertilization is discussed.     

Sperm motility defects and infertility in male mice with a mutation in Nsun7, a member of the Sun domain-containing family of putative RNA methyltransferases

Poor sperm quality is the major cause of infertility in humans. Other than sex-linked factors, the genetic basis for male infertility is poorly defined, largely due to practical difficulties in studying the inheritance of this trait in humans. As an alternative, we have conducted forward genetic screens in mice to generate relevant models. We report on the identification and characterization of a chemically-induced mutation, Ste5Jcs1, which causes affected male mice to be sterile or subfertile. Mutant sperm exhibited depressed progressive motility associated with a rigid flagellar midpiece (but not principal piece) segment, which could not be rescued by treatment with agents that stimulate cAMP or calcium signaling pathways. Overall mutant sperm ultrastructure appeared normal, including the axoneme, although the midpiece mitochondrial sheath showed abnormal electron density patterns. Positional cloning of Ste5Jcs1 led to the identification of a mutation in a novel gene called Nsun7, which encodes a protein with a Sun domain that is homologous to tRNA and rRNA cytosine methyltransferases. Therefore, Ste5Jcs1 mutation uncovers a previously unrecognized biological process in sperm that underscores the functional compartmentalization of the midpiece and principal piece of the flagellum.     

Fine structure of scorpion spermatozoa (Buthus occitanus; buthidae,scorpiones)

The mature spermatozoa of Buthus occitanus are threadlike in shape and divided into sperm head, middle piece, and end piece. The sperm head is corkscrew shaped anteriorly and in this region bears an unusual acrosomal complex consisting of a ring-shaped acrosomal vacuole associated with a subacrosomal filament and a perinuclear amorphous component. The subacrosomal filament extends posteriorly into a tube-like invagination of the elongated nucleus. The middle piece is characterized by elongated mitochondria which spiral around the anterior part of the flagellum in an extended collar separated from the flagellum by an extracellular cleft, termed the central flagellar tunnel. In addition to the usual 9 × 2 + 2 axonemal pattern in flagella, 9 × 2 + 1 and 9 × 2 + 3 patterns also were observed. The end piece is represented by the free flagellum. Similarities and diversities of scorpionid spermatozoa are discussed with respect to systematic relationships.     

蓝尾石龙子精子的超微结构

蓝尾石龙子(Eumeces elegans)附睾以2．5％戊二醛和1％锇酸双重固定,按常规制作超薄切片,用H-600透射电镜研究观察精子的超微结构。精子由头部和尾组成,头部由顶体复合体和核组成,尾由颈段、中段、主段和末段组成。头部的顶体囊前部扁平,分为皮质和髓质,顶体下锥由类结晶状的顶体下物质组成,穿孔器顶端尖,、穿孔器基板塞子状,细胞核延长,核内小管缺,核伸展部前端具一电子透明区,核肩圆,核陷窝锥形。颈段具片层结构,近端中心粒和远端中心粒的长轴呈直角,9束外周致密纤维与远端中心粒相应的9束三联微管相联,向后与轴丝相应的9束双联微管相联,中央纤维与2个中央单微管相联。中段短,含有线状嵴的柱状线粒体,由连续的规则小卵状或小梯形致密体组成线粒体间的环状结构,纤维鞘伸入中段,终环紧贴于细胞膜的内表面。线粒体与环状结构的模式为：rs1／mi1,rs2／mi2,rs3／mi3,rs4／mi4,横切面上每圈线粒体数目为10个。主段前面部分具薄的细胞质颗粒区。纤维3和8至主段前端消失。轴丝复合体呈“9 2”型。蓝尾石龙子精子超微结构与已描述的石龙子科种类比较发现,与蜓蜥群和胎生群的石龙子相似;但没有发现石龙子科精子的独征。     

Spatiotemporal association of DNAJB13 with the annulus during mouse sperm flagellum development

Background  

The sperm annulus is a septin-based fibrous ring structure connecting the midpiece and the principal piece of the mammalian sperm flagellum. Although ultrastructural abnormalities and functional importance of the annulus have been addressed in Sept4-null mutant mice and a subset of human patients with asthenospermia syndrome, little is known about how the structure is assembled and positioned to the midpiece-principal piece junction during mammalian sperm flagellum development.     

Changes in distribution and molecular weight of the acrosomal protein acrin2 (MC41) during guinea pig spermiogenesis and epididymal maturation

In this study, we examined the localization and characteristics of an intra-acrosomal protein, acrin2 (MC41), during guinea pig spermiogenesis and post-testicular sperm maturation in the epididymis, using the monoclonal antibody MC41. Immunoelectron microscopy demonstrated not only a specific domain localization of acrin2 in the apical segment of the guinea pig sperm acrosome, but also its dynamic behavior according to the spermatid differentiation and passage through the epididymis, as follows: acrin2 was exclusively localized in the membrane of the endoplasmic reticulum of early-stage spermatids but was not detectable in the developing acrosome until spermatids reached the maturation phase. In the final stage of spermiogenesis, acrin2 became localized in the outer acrosomal membrane (OAM)/matrix-associated materials both in the small region posterior to the dorsal matrix and along the ventral margin of the acrosomal apical segment. The acrosomal location of acrin2 in caput epididymidal sperm was almost identical to that observed in the final step spermatids, but during maturation it became progressively more restricted in area until on distal cauda epididymidal sperm it remained only in the dorsal region. In Western blot analysis, the MC41 antibody recognized a 165-kDa protein in the mature sperm extract. Furthermore, it was demonstrated that molecular weight reduction of the protein occurred during sperm passage through the epididymis. These findings indicate that acrin2 changes progressively in both distribution and size during development and maturation of the acrosome.     

Ultrastructural analysis of spermiogenesis in Iguana iguana (Reptilia: Sauria: Iguanidae)

Spermiogenesis in the lizard, Iguana iguana, was studied by transmission and scanning electron microscopy. During this process, structures such as the acrosomal complex in the spermatid head and the axonemal complex in the mid and principal pieces of the flagellum are formed. The nuclear content is initially compacted into thick, longitudinal chromatin filaments. Nuclear shape is determined by further compaction and by the manchette, a layer of microtubules surrounding the head. The acrosomal complex originates from Golgi vesicles and the interaction between the proacrosomal vesicle and the nucleus. The midpiece consists of a pair of centrioles, surrounded by a fibrous sheath and rings of simple and modified mitochondria. The centrioles sustain the axoneme that appears at the end of the midpiece. The axoneme extends throughout the principal piece of the flagellum with the 9 + 2 pattern, still surrounded by the fibrous sheath. In the endpiece, the axoneme continues, surrounded only by the plasma membrane. In the lumen of seminiferous tubules, immature spermatozoa retain abundant residual cytoplasm.     

ACROSOMAL DISRUPTION IN SPERM : Freeze-Fracture of Altered Membranes

"Capacitation" is a physiological event which alters sperm to permit rapid penetration through oocyte investments and fusion between gametes. Acrosomal "reaction," the physiological release of acrosomal contents, occurs after this facilitating process. In this study, acrosomal "disruption" of guinea pig and rat sperm was achieved in vitro by incubating sperm together with the follicular contents of superovulated mice. The samples contained both "reacted" and "disrupted" sperm. Thin sections of affected sperm revealed rupture and vesiculation of the plasma membrane overlying the acrosome, as well as loss of both the outer acrosomal membrane and the acrosomal content. Freeze-fracture revealed disintegration of the characteristic geometric patterns in regions of the acrosomal and plasma membranes thus disrupted and major modifications in particle distribution in the sperm tail. In the guinea pig, strands of 6–8-nm particles, usually confined to the plasma membrane of the midpiece, which overlies mitochondria, also appeared in the principal piece. Likewise, in rat sperm, bands of similarly small particles formed acute angles throughout the membrane of the principal piece. Compared with the membranes of control preparations, these membrane alterations are apparently a direct consequence of incubation with ovarian follicular contents.     

Molecular cloning and characterization of phospholipase C zeta in equine sperm and testis reveals species-specific differences in expression of catalytically active protein

Oocyte activation at fertilization is brought about by the testis-specific phospholipase C zeta (PLCZ), owing to its ability to induce oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). Whereas this is a highly conserved mechanism among mammals, important species-specific differences in PLCZ sequence, activity, and expression have been reported. Thus, the objectives of this research were to clone and characterize the intracellular Ca(2+)-releasing activity and expression of equine PLCZ in sperm and testis. Molecular cloning of equine PLCZ yielded a 1914-bp sequence that translated into a protein of the appropriate size (~73 kDa), as detected with an anti-PLCZ-specific antibody. Microinjection of 1 μg/μl of equine PLCZ cRNA supported [Ca(2+)](i) oscillations in murine oocytes that were of a higher relative frequency than those generated by an equivalent concentration of murine Plcz cRNA. Immunofluorescence revealed expression of PLCZ over the acrosome, equatorial segment, and head-midpiece junction; unexpectedly, PLCZ also localized to the principal piece of the flagellum in all epididymal, uncapacitated, and capacitated sperm. Immunostaining over the acrosome was abrogated after induction of acrosomal exocytosis. Moreover, injection of either sperm heads or tails into mouse oocytes showed that PLCZ in both fractions is catalytically active. Immunohistochemistry on equine testis revealed expression as early as the round spermatid stage, and injection of these cells supported [Ca(2+)](i) oscillations in oocytes. In summary, we report that equine PLCZ displays higher intrinsic intracellular Ca(2+)-releasing activity than murine PLCZ and that catalytically active protein is expressed in round spermatids as well as the sperm flagellum, emphasizing important species-specific differences. Moreover, some of these results may suggest potential novel roles for PLCZ in sperm physiology.     

CATSPER channel-mediated Ca2+ entry into mouse sperm triggers a tail-to-head propagation

Many Ca(2+) channel proteins have been detected in mammalian sperm, but only the four CATSPER channels have been clearly shown to be required for male fertility. Ca(2+) entry through the principal piece-localized CATSPER channels has been implicated in the activation of hyperactivated motility. In the present study, we show that the Ca(2+) entry also triggers a tail-to-head Ca(2+) propagation in the mouse sperm. When activated with 8-Br-cAMP, 8-Br-cGMP, or alkaline depolarization, a CATSPER-dependent increase in intracellular Ca(2+) concentration starts in the principal piece, propagates through the midpiece, and reaches the head in a few seconds. The Ca(2+) propagation through the midpiece leads to a Ca(2+)-dependent increase in NADH fluorescence. In addition, CatSper1-mutant sperm have lower intracellular ATP levels than wild-type sperm. Thus, a Ca(2+) influx in the principal piece through CATSPER channels can not only initiate hyperactivated motility, but can also trigger a tail-to-head Ca(2+) propagation that leads to an increase in [NADH] and may regulate ATP homeostasis.     

Protocadherin alpha3 acts at sites distinct from classic cadherins in rat testis and sperm

The testis expresses a variety of cadherin superfamily members including classic cadherins and protocadherins. This report describes the first localization of a protocadherin protein in testis and sperm. After cloning rat cDNAs for protocadherin alpha3 and alpha4, isoform-specific polyclonal antibodies were generated against protocadherin alpha3. Western blotting of rat testis showed that protocadherin alpha3 was solubilized completely by Triton X-100, in contrast to the adhesion junction components N-cadherin, beta-catenin, and p120 catenin. Corroborating this data, protocadherin alpha3 was immunolocalized to the spermatid acrosomal area, intercellular bridge, and flagellum, but not classic cadherin-based adhesion junctions. Acrosome-associated protocadherin alpha3 was first detected at step 8 of spermiogenesis, and this association remained on cauda epididymal sperm. Acrosome immunostaining was reduced, but present, in acrosome-reacted sperm. Spermatid intercellular bridges became positive for protocadherin alpha3 coincident with the appearance of plectin, occurring at spermiogenic steps 8 to 9, and elongate spermatid bridges remained positive throughout spermatogenesis. The developing flagellum was uniformly immunostained for protocadherin alpha3 up to approximately spermiogenic step 17. Subsequently, flagellar immunostaining was confined to the principal piece, and this pattern continued in cauda epididymal sperm. These data show that protocadherin alpha3 performs functions unique from classic cadherins in spermatogenesis and suggest a role for protocadherin alpha3 in organizing germ cell-specific structures including the intercellular bridge, flagellum, and acrosome.     

Asymmetry of spermiation and sperm surface charge patterns over the giant acrosome in the musk shrew Suncus murinus

Spermatozoa of the shrew Suncus murinus, a mammal with abdominal testes, exhibit four unusual features: a giant acrosome; a dorsoventral asymmetry of their spermiation; a dorsoventral asymmetry of their head surface character; and also apparent surface maturity as they enter the epididymis. A Sertoli cell-periacrosomal cisternal complex envelops the giant acrosome during spermatid maturation. Spermiation is heraled by asymmetrical disorganization of the subplasmalemmal components of this complex and is completed by retraction of the Sertoli cell from the ventral and then the dorsal face of the spermatid head. This sequence or release is correlated with an asynchronous acquisition of negative surface charges on the spermatid head-demonstrable on glutaraldehyde-stabilized cells by the binding at pH 1.8 of positively charged colloidal particles of ferric oxide. Mature epididymal spermatozoa exhibit an asymmetry in the patterns of distribution of bound colloid over the dorsal vs. ventral surfaces of the sperm head, as well as regional differences between the tail midpiece and principal piece. Surface distributions of anionic residues and lectin (Con A)-binding sites characteristic of mature Suncus spermatozoa are demonstrable within the testis, unlike the situation in most nannals where distinct modifications of the sperm surface occur during epididymal passage.     

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.     

Immunocytochemical alterations in the intra-acrosomal antigen MN7 during epididymal maturation of guinea pig spermatozoa

We have previously shown that a 90-kDa intra-acrosomal antigen, MN7, is restricted to the anterior acrosomal region of mouse, rat, and hamster spermatozoa. The present study has examined the localization and the behavior of MN7 during sperm maturation in the epididymis of the guinea pig by immunoelectron microscopy. MN7 showed not only a specific localization in the apical segment of the guinea pig sperm acrosome, but also a distinct alteration during maturation, as follows. MN7 was exclusively found both at the dorsal matrix and on the outer acrosome membrane (OAM)/matrix-associated materials in the apical segment. MN7 was initially distributed throughout the electron-lucent dorsal matrix in immature sperm but, during maturation, became more restricted to the spherical bodies within the electron-lucent area. MN7 on OAM/matrix-associated materials was first distributed along the ventral margin and the small area posterior to the dorsal matrix but, during maturation, disappeared from the ventral margin and became restricted to the dorsal region. These results indicate that MN7 is a good tool for studying the stepwise maturation of epididymal spermatozoa.     

Morphology and ultrastructure of Siberian sturgeon (Acipenser baerii) spermatozoa using scanning and transmission electron microscopy

BACKGROUND INFORMATION: Available data concerning the sperm morphology of teleost fishes demonstrate wide variation. In the present study, the spermatozoa of Siberian sturgeon (Acipenser baerii Brandt, 1869), a chondrostean fish, was investigated. In contrast with teleost fish, chondrostean spermatozoa have a head with a distinct acrosome, whereas other structures, such as a midpiece and a single flagellum, are present in spermatozoa of most species. RESULTS: The average length of the head including the acrosome and the midpiece was 7.01+/-0.83 microm. Ten posterolateral projections derived from the acrosome were present on a subacrosomal region, with mean lengths of 0.94+/-0.15 microm and widths of 0.93+/-0.11 microm. The nucleus consisted of electrodense homogeneous nuclear chromatin. Three intertwining endonuclear canals, bound by membranes, traversed the nucleus longitudinally from the acrosomal end to the basal nuclear fossa region. There were between three and six mitochondria, two types of centrioles (proximal and distal) in the midpiece and two vacuoles composed of lipid droplets. The flagellum (44.75+/-4.93 microm in length), originating from the centriolar apparatus, had a typical 9+2 eukaryotic flagellar organization. In addition, there was an extracellular cytoplasm canal between the cytoplasmic sheath and the flagellum. CONCLUSIONS: A principal components analysis explained the individual morphological variation fairly well. Of the total accumulated variance, 41.45% was accounted for by parameters related to the head and midpiece of the sperm and the length of the flagellum. Comparing the present study with previous studies of morphology of sturgeon spermatozoa, there were large inter- or intra-specific differences that could be valuable taxonomically.     

中国石龙子成熟精子的超微结构

利用透射电镜观察中国石龙子附睾成熟精子的超微结构。顶体囊前部扁平、由皮质和髓质组成 ,穿孔器中度倾斜、顶端尖 ,穿孔器基板塞子状 ,细胞核长形 ,核内小管缺 ,核前电子透亮区小 ,核肩圆 ,核陷窝锥形。颈段具片层结构 ,近端中心粒和远端中心粒的长轴呈直角 ,9束外周致密纤维与远端中心粒相应的 9束三联微管相联 ,向后与轴丝相应的 9束双联微管相联 ,中央纤维与 2个中央单微管相联。中段短 ,多层膜结构缺 ,含有线状嵴的柱状线粒体 ,不规则卵状致密体组成不连续的环状结构 ,纤维鞘伸入中段 ,具终环。线粒体与环状结构的模式为 :rs1 /mi1 ,rs2 /mi2 ,rs3/mi3,rs4 /mi4。主段前面部分具薄的细胞质颗粒区。纤维 3和 8至主段前端消失。轴丝呈“9 2”型。中国石龙子精子超微结构具有塞子状的穿孔器基板、致密体形成不连续的环状结构和纤维鞘始于ms2等特征与巨石龙子群和蜓蜥 -胎生群不同。没有发现石龙子科精子的独征