Protein kinase C is present in human sperm: possible role in flagellar motility.

We report the presence of protein kinase C (PKC) in ejaculated human sperm as revealed by enzymatic activity assay and indirect immunohistochemistry. PKC is localized in the equatorial segment and in the principal piece of the tail. Addition of phorbol 12-myristate 13-acetate resulted in increased flagellar motility that was blocked by known PKC inhibitors such as sphingosine, staurosporine, and 1-(5-isoquinoylinylsulfonyl)-2-methylpiperazine. A very good correlation (r = 0.9, P less than 0.001) was found between the percentage of PKC-stained sperm cells and motility. We propose that PKC is involved in the regulation of flagellar motility in human sperm.     

Human sperm express cannabinoid receptor Cb1, the activation of which inhibits motility, acrosome reaction, and mitochondrial function

Cannabinoids and endocannabinoids negatively influence sperm functions. These substances have been demonstrated in many mammalian tissues, including male and female reproductive tracts, and previous studies have shown the presence of functional receptors for cannabinoids in human sperm. The present study, by means of RT-PCR and Western blot techniques, demonstrates that human sperm express the CB(1), but not CB(2), cannabinoid receptor (CB-R) subtype located in the head and middle piece of the sperm. The activation of this receptor by anandamide reduces sperm motility and inhibits capacitation-induced acrosome reaction. Activation of the CB(1)-R did not induce any variation in sperm intracellular calcium concentrations, but produced a rapid plasma membrane hyperpolarization that was reduced by the K(+) channel blocker tetraethylammonium. The effects of anandamide on human sperm motility were dependent on the reduction of sperm mitochondrial activity as determined by rhodamine 123 fluorescence. The specificity of anandamide effects in human sperm were confirmed by the effects of the CB(1)-R antagonist SR141716. These findings provide additional evidence that human sperm express functional CB(1)-R, the activation of which negatively influences important sperm functions, and suggest a possible role for the cannabinoid system in the pathogenesis of some forms of male infertility.     

Biochemical and structural characterization of apolipoprotein A-I binding protein, a novel phosphoprotein with a potential role in sperm capacitation

The physiological changes that sperm undergo in the female reproductive tract rendering them fertilization-competent constitute the phenomenon of capacitation. Cholesterol efflux from the sperm surface and protein kinase A (PKA)-dependent phosphorylation play major regulatory roles in capacitation, but the link between these two phenomena is unknown. We report that apolipoprotein A-I binding protein (AI-BP) is phosphorylated downstream to PKA activation, localizes to both sperm head and tail domains, and is released from the sperm into the media during in vitro capacitation. AI-BP interacts with apolipoprotein A-I, the component of high-density lipoprotein involved in cholesterol transport. The crystal structure demonstrates that the subunit of the AI-BP homodimer has a Rossmann-like fold. The protein surface has a large two compartment cavity lined with conserved residues. This cavity is likely to constitute an active site, suggesting that AI-BP functions as an enzyme. The presence of AI-BP in sperm, its phosphorylation by PKA, and its release during capacitation suggest that AI-BP plays an important role in capacitation possibly providing a link between protein phosphorylation and cholesterol efflux.     

Coevolution of head, neck, and tail domains of myosin heavy chains

Myosins, a large family of actin-based motors, have one or two heavy chains with one or more light chains associated with each heavy chain. The heavy chains have a (generally) N-terminal head domain with an ATPase and actin-binding site, followed by a neck domain to which the light chains bind, and a C-terminal tail domain through which the heavy chains self-associate and/or bind the myosin to its cargo. Approximately 140 members of the myosin superfamily have been grouped into 17 classes based on the sequences of their head domains. I now show that a phylogenetic tree based on the sequences of the combined neck and tail domains groups 144 myosins, with a few exceptions, into the same 17 classes. For the nine myosin classes that have multiple members, phylogenetic trees based on the head domain or the combined neck/tail domains are either identical or very similar. For class II myosins, very similar phylogenetic trees are obtained for the head, neck, and tail domains of 47 heavy chains and for 29 essential light chains and 19 regulatory light chains. These data strongly suggest that the head, neck, and tail domains of all myosin heavy chains, and light chains at least of class II myosins, have coevolved and are likely to be functionally interdependent, consistent with biochemical evidence showing that regulated actin-dependent MgATPase activity of Dictyostelium myosin II requires isoform specific interactions between the heavy chain head and tail and light chains.     

日本血吸虫精子的超微结构

目的：了解日本血吸虫正常精子的超微结构。方法：半超薄切片定位雄虫睾丸和含有成熟精子的雌虫输卵管，常规透射电镜制样并观察。结果：日本血吸虫精子由头、尾两部分组成，头部呈长卵圆形，平均长6．2μm，宽1．4μm，无顶体构造，前端钝圆，后端尖细，质膜下有1圈纵行的微管，核1个，前端有少量线粒体，尾部鞭毛1根，在中、后段主体部分鞭毛轴丝外周为9组二联管，中央为一团弥散的电子致密物质，呈9×2＋《1》型；但在过渡区鞭毛轴丝中央无电子致密物质，呈9×2＋0型。结论：日本血吸虫精子超微结构与其他血吸虫相似，具有同源性，但明显区别于复殖目大多数吸虫的构造。     

Structure and genome release of Twort-like Myoviridae phage with a double-layered baseplate

Bacteriophages from the family Myoviridae use double-layered contractile tails to infect bacteria. Contraction of the tail sheath enables the tail tube to penetrate through the bacterial cell wall and serve as a channel for the transport of the phage genome into the cytoplasm. However, the mechanisms controlling the tail contraction and genome release of phages with “double-layered” baseplates were unknown. We used cryo-electron microscopy to show that the binding of the Twort-like phage phi812 to the Staphylococcus aureus cell wall requires a 210° rotation of the heterohexameric receptor-binding and tripod protein complexes within its baseplate about an axis perpendicular to the sixfold axis of the tail. This rotation reorients the receptor-binding proteins to point away from the phage head, and also results in disruption of the interaction of the tripod proteins with the tail sheath, hence triggering its contraction. However, the tail sheath contraction of Myoviridae phages is not sufficient to induce genome ejection. We show that the end of the phi812 double-stranded DNA genome is bound to one protein subunit from a connector complex that also forms an interface between the phage head and tail. The tail sheath contraction induces conformational changes of the neck and connector that result in disruption of the DNA binding. The genome penetrates into the neck, but is stopped at a bottleneck before the tail tube. A subsequent structural change of the tail tube induced by its interaction with the S. aureus cell is required for the genome’s release.The worldwide emergence of antibiotic-resistant pathogenic bacteria creates a need for new antibacterial treatments, including phage-based therapy (1). Bacteriophage phi812 from the genus Twort-like virus, subfamily Spounavirinae, can infect at least 95% of Staphylococcus aureus strains, including those strains resistant to antibiotics (2). Thus, phi812 has the potential to be used as antibacterial phage-therapy agent (3, 4).     

Bypassing natural sperm selection during fertilization: the azh mutant offspring experience and the alternative of spermiogenesis in vitro

Molecular aspects of spermiogenesis can be studied using mouse mutants and spermatids developed in vitro. The azh/azh mutant is an attractive model system because structural abnormalities in the sperm head and the ectopic position of the manchette are associated with tail bending and looping. Spermatids, developing an axoneme in vitro and capable of cell motility, offer the possibility of the dynamic analysis of tail development. Offspring generated by intracytoplasmic injection of azh/azh sperm heads into normal mouse oocytes complement the mouse mutant approach. A central question of sperm tail development is the role of the manchette, a transient microtubular structure assembled soon after the organization of the axoneme. The fractionation of intact manchettes by gradient centrifugation has enabled a biochemical analysis of constitutive tubulin isotypes and transiently associated proteins. For example, keratins Sak57, Odf1, and Odf2 are initially stored in the manchette before being sorted to the outer dense fibers and fibrous sheath of the developing spermatid tail. Additional proteins associated with the manchette include two proteases, the 26S proteasome and N-arginine convertase (both sorted to the developing spermatid tail), a spermatid perinuclear RNA binding protein, Spag4, an Odf1-binding protein, and type 4 cAMP-specific phosphodiesterase D. Keratin 9 and delta-tubulin are two proteins found in the perinuclear ring of the manchette, the insertion site of the microtubular mantle. Available data indicate that the manchette is a highly dynamic structure providing microtubular tracks to structural proteins participating in the sperm tail development.     

Molecular diffusion in sperm plasma membranes during epididymal maturation

Fluorescence recovery after photobleaching (FRAP) analysis has been used to measure lipid diffusion in different regions of the sperm plasma membrane. Our goal has been to understand how some membrane components are confined to specific surface domains, whilst others are freely diffusing and in some cases are able to migrate against large concentration gradients. Results with a variety of fluorescent lipid reporter probes (ODAF, NBD-PC, NBD-cholesterol) show that diffusion coefficients (D) are generally three to four times higher on the sperm acrosome than on the principal piece of the tail and increase significantly during epididymal maturation (ram, mouse, goat, dog and monkey sperm). Cholesterol diffusion is approximately 10 times faster on the sperm head than the tail and has a heterogenous distribution when detected with filipin. Lipid diffusion is very temperature sensitive but remarkably insensitive to changes in external pH and osmotic pressure. There was no evidence that the posterior ring or annulus functioned as diffusion barriers to lipids. On this basis it was possible to construct models of increasing complexity to describe the behaviour of a lipid molecule on the sperm surface, beginning with simple linear diffusion progressing to random diffusion and eventually to constrained diffusion.     

The tail binds to the head–neck domain,inhibiting ATPase activity of myosin VIIA

Myosin VIIA is an unconventional myosin, responsible for human Usher syndrome type 1B, which causes hearing and visual loss. Here, we studied the molecular mechanism of regulation of myosin VIIA, which is currently unknown. Although it was originally thought that myosin VIIA is a dimeric myosin, our electron microscopic (EM) observations revealed that full-length Drosophila myosin VIIA (DM7A) is a monomer. Interestingly, the tail domain markedly inhibits the actin-activated ATPase activity of tailless DM7A at low Ca²⁺ but not high Ca²⁺. By examining various deletion constructs, we found that deletion of the distal IQ domain, the C-terminal region of the tail, and the N-terminal region of the tail abolishes the tail-induced inhibition of ATPase activity. Single-particle EM analysis of full-length DM7A at low Ca²⁺ suggests that the tail folds back on to the head, where it contacts both the motor core domain and the neck domain, forming an inhibited conformation. We concluded that unconventional myosin that may be present a monomer in the cell can be regulated by intramolecular interaction of the tail with the head.     

RANBP17 is localized to the XY body of spermatocytes and interacts with SPEM1 on the manchette of elongating spermatids

We identified Ran-binding protein 17 (RANBP17) as one of the interacting partners of sperm maturation 1 (SPEM1) using yeast 2-hybrid screening and immunoprecipitation assays. Expression profiling analyses suggested that RANBP17 was preferentially expressed in the testis. Immunofluorescent confocal microscopy revealed a dynamic localization pattern of RANBP17 during spermatogenesis. In primary spermatocytes RANBP17 was mainly localized to the XY body. In the subsequent spermiogenesis, RANBP17 was first observed in the nuclei of round spermatids (steps 1-7) and then confined to the manchette of elongating spermatids (steps 8-14) together with its interacting partner SPEM1. In the Spem1-null testes, levels of RANBP17 were significantly elevated. As a member of a large protein family involved in the nucleocytoplasmic transport, RANBP17 may have a role in sex chromosome inactivation during the meiotic phase of spermatogenesis, and also in the intramanchette transport during spermiogenesis. Interactions between RANBP17 and SPEM1, for the first time, point to a potential function of SPEM1 in the RANBP17-mediated nucleocytoplasmic transport.     

Expression and localization of delta-, kappa-, and mu-opioid receptors in human spermatozoa and implications for sperm motility

CONTEXT: Endogenous opioid peptides signal through delta-, kappa-, and mu-opioid receptors. Some of these peptides such as endorphins and enkephalins are present in the male reproductive tract, but the presence of the corresponding receptors in human sperm cells has not yet been reported. OBJECTIVE: Our objective was to study the expression and localization of delta-, kappa-, and mu-opioid receptors on human spermatozoa and the implication in sperm motility. METHODS: The expression of receptors was studied by RT-PCR, Western blot, and immunofluorescence techniques. We evaluated the effects of activation of each opioid receptor by specific agonist and antagonist. RESULTS: Human spermatozoa express delta-, kappa-, and mu-opioid receptors. These receptors were located in different parts of the head, in the middle region, and in the tail of the sperm. Progressive motility of spermatozoa, an important parameter to evaluate male fertility, was found to be significantly reduced after incubation with the mu-receptor agonist morphine, whereas this effect was antagonized in the presence of the corresponding antagonist naloxone. The delta-receptor antagonist naltrindole significantly reduced progressive motility immediately after its addition. However, the delta-receptor agonist DPDPE had no significant effect. Finally, neither the kappa-receptor agonist U50488 nor its antagonist nor-binaltorphimine significantly affected the progressive motility of human spermatozoa. CONCLUSION: We report for first time the presence of functional delta-, kappa-, and mu-opioid receptors in human sperm membranes. These findings are indicative of a role for the opioid system in the regulation of sperm physiology.     

Further studies on the involvement of protein kinase C in human sperm flagellar motility

Addition of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) or the membrane-permeable diacylglycerol analog, 1-oleoyl-2-acetylglycerol to human sperm resulted in increased motility. The biologically inactive 4 alpha-phorbol 12,13 didecanoate had no effect on flagellar motility. Basal motility was markedly reduced in the absence of Ca2+ in the incubation medium, but TPA-induced sperm motility persisted even in the absence of Ca2+. Sperm motility was also enhanced by the Ca2+ ionophore ionomycin in a Ca2(+)-dependent, protein kinase c (PKC)-independent fashion. Although all stimulants examined here reached maximal response at about 15 min of incubation, nevertheless whereas the effect of TPA and 1-oleoyl-2-acetylglycerol declined at 60 min of incubation, that of ionomycin still persisted. Human sperm PKC activity is extremely low and represents only about 20% and 25% of the specific activity recovered from PC-12 and rat pituitary cells, respectively. Immunohistochemical analysis using various type-specific PKC antibodies revealed staining only in the equatorial segment and the principal piece of the tail. Thus, PKC is present in human ejaculated sperm and is involved in flagellar motility.     

Ultrastructural observation of spermatozoa and fertilization in Schistosoma japonicum

The ultrastructure of the sperm and the process of fertilization are described in Schistosoma japonicum. The sperm of S. japonicum has an elongated head and a single tail. The head measures 6.2 x 1.4 microm in average size. No acrosome is present. A mass of mitochondria locates in front of the nucleus. A layer of about 100-120 peripheral microtubules is parallel with the long axis of the head under plasma membrane. The nucleus is dense with some electron-lucent patches. The tail is a single flagellum with unique axoneme, which originates from a centriole. The structure of axoneme includes two types: 9 x 2 + in the main part of the flagellum, and 9 x 2 + 0 near the end of the flagellum. The sperm ultrastructure of S. japonicum is similar to that of other schistosomes, apart from the fact that two types of configuration coexisted in the same axoneme, and there is no striated root found in S. japonicum. The sperm differs distinctly from other Digenea. The aberrant ultrastructure of S. japonicum reflects that its evolution is far away from other genera in Digenea. Fertilization occurs at the posterior part of oviduct, in which region the oviduct wall lacks lamellae. Some cortical granules (CG) fuse with plasma membrane, and discharge their content on the surface of the fertilized ovum. The other CGs break down or degenerate in the cytoplasm. By the secondary mature division, the secondary oocyte finally divides to form a female pronucleus. During this period a male pronucleus also forms. The female and male pronucleus approach each other, come into contact in the central region and finally fuse to form a zygote. The function of CGs is discussed.     

Ca(2+)-independent induction of acrosome reaction by protein kinase C in human sperm.

We report that activated protein kinase C (PKC) can induce acrosome reaction independently of elevated Ca2+. Addition of 12-O-tetradecanoyl phorbol-13-acetate or the membrane-permeable diacylglycerol analog 1-oleoyl-2-acetylglycerol to ejaculated human sperm resulted in stimulation of acrosomal reaction (2- to 3-fold), provided the sperm underwent capacitation. Induction of acrosome reaction by 12-O-tetradecanoyl phorbol-13-acetate was blocked by the PKC inhibitor staurosporine or by down-regulation of endogenous PKC, but not by removal of extracellular Ca2+. Acrosome reaction was also enhanced by the Ca2+ ionophore ionomycin in a Ca(2+)-dependent, PKC-independent fashion. Immunohistochemical analysis with type-specific PKC antibodies revealed the presence of PKC alpha and PKC beta II in the equatorial segment, whereas PKC beta I and PKC epsilon staining was found in the principal piece of the tail. Acrosome reaction, thus far believed to be induced only by elevated Ca2+, can therefore be triggered by activated PKC in a Ca(2+)-independent fashion. The PKC subtypes potentially involved in acrosome reaction are most likely alpha and beta II, whereas the beta I- and epsilon-subspecies might be involved in regulation of flagellar motility of human sperm.     

斯氏狸殖吸虫精子形成的透射电镜观察

用透射电镜观察了斯氏狸殖吸虫精细胞的变化和精子的主要形态结构特征。从而揭示了该虫精子形成的过程。证实了斯氏狸殖吸虫的精细胞和精子有顶体结构。成熟的精子可分为头部和尾部。头部由核质充满,核质可延伸至精子尾部中段的前端。头尾之间为连接段。尾部又由中段和末段组成。尾部的中段起始部的两侧各有一条轴丝。每条轴丝各由9对外周微管和2个中央微管组成,在每2条中央微管的外围,均由一个纤维鞘包绕,其间形成一个车轮样的9 2微管系统结构,其腹侧排列着线粒体。尾部的末段,主要为2条紧靠的轴丝。     

Histone H1 and the origin of protamines

We present evidence that chordate protamines have evolved from histone H1. During the final stages of spermatogenesis, the compaction of DNA in many organisms is accomplished by the replacement of histones with a class of arginine-rich proteins called protamines. In other organisms, however, condensation of sperm DNA can occur with comparable efficiency in the presence of somatic-type histones or, alternatively, an intermediate class of proteins called protamine-like proteins. The idea that the highly specialized sperm chromosomal proteins (protamines) and somatic chromosomal proteins (histones) could be related dates back almost to the discovery of these proteins. Although this notion has frequently been revisited since that time, there has been a complete lack of supporting experimental evidence. Here we show that the emergence of protamines in chordates occurred very quickly, as a result of the conversion of a lysine-rich histone H1 to an arginine-rich protamine. We have characterized the sperm nuclear basic proteins of the tunicate Styela montereyensis, which we show consists of both a protamine and a sperm-specific histone H1 with a protamine tail. Comparison of the genes encoding these proteins to that of a sister protochordate, Ciona intestinalis, has indicated this rapid and dramatic change is most likely the result of frameshift mutations in the tail of the sperm-specific histone H1. By establishing an evolutionary link between the chromatin-condensing histone H1s of somatic tissues and the chromatin-condensing proteins of the sperm, these results provide unequivocal support to the notion that vertebrate protamines evolved from histones.     

Structural organization of yeast and mammalian mediator complexes

Structures of yeast Mediator complex, of a related complex from mouse cells and of thyroid hormone receptor-associated protein complex from human cells have been determined by three-dimensional reconstruction from electron micrographs of single particles. All three complexes show a division in two parts, a "head" domain and a combined "middle-tail" domain. The head domains of the three complexes appear most similar and interact most closely with RNA polymerase II. The middle-tail domains show the greatest structural divergence and, in the case of the tail domain, may not interact with polymerase at all. Consistent with this structural divergence, analysis of a yeast Mediator mutant localizes subunits that are not conserved between yeast and mammalian cells to the tail domain. Biochemically defined Rgr1 and Srb4 modules of yeast Mediator are then assigned to the middle and head domains.     

Identification of a hyaluronidase, Hyal5, involved in penetration of mouse sperm through cumulus mass

A glycosylphosphatidylinositol (GPI)-anchored hyaluronidase, PH-20, on the sperm surface has long been believed to assist sperm penetration through the cumulus mass surrounding the eggs. However, mouse sperm lacking PH-20 were still capable of penetrating the cumulus mass despite a delayed dispersal of cumulus cells. Intriguingly, a 55-kDa hyaluronan-hydrolyzing protein was abundantly present in wild-type and PH-20-deficient mouse sperm. In this study, we purified the 55-kDa mouse protein from soluble protein extracts released from epididymal sperm by acrosome reaction and identified as a hyaluronidase, Hyal5. Hyal5 was exclusively expressed in the testis and formed a 160-kbp gene cluster together with Hyalp1, Hyal4, and Ph-20 on mouse chromosome 6. Hyal5 was a single-chain hyaluronidase present on the plasma and acrosomal membranes of sperm presumably as a GPI-anchored protein. Moreover, hyaluronan zymography revealed that Hyal5 is enzymatically active in the pH range 5-7 and inactive at pH 3 and 4. Both Hyal5-enriched PH-20-free soluble protein extracts and PH-20-deficient mouse sperm were capable of dispersing cumulus cells from the cumulus mass. Cumulus cell dispersal was strongly inhibited by the presence of a hyaluronidase inhibitor, apigenin. These results suggest that in the mouse, Hyal5 may function principally as a "cumulus matrix depolymerase" in the sperm penetration through the cumulus mass and in the local hyaluronan hydrolysis near or on the surface of the egg zona pellucida to enable the proximal region of sperm tail to move freely. PH-20 may compensate in part for the functional roles of Hyal5.     

Protein kinase C and Mammalian spermatozoa acrosome reaction.

The presence of protein kinase C (PKC) in mammalian sperm was demonstrated by enzymatic activity assay and immunohistochemistry at the light and electron microscopy levels. The sperm head PKC is localized in the acrosome, equatorial segment, and postacrosomal region. In the flagellum, PKC is associated with the segmented column of the neck and is distributed along the mid, principal, and end pieces. Immunoreactive sites are observed in patches along the axoneme and outer dense fibers and are evenly distributed between these regions. Functional studies suggest the involvement of PKC in flagellar motility and acrosome reaction. The cross-talk between the signaling cascades that operate during sperm activation is discussed. (Trends Endocrinol Metab 1997;8:337-341). (c) 1997, Elsevier Science Inc.