Maintenance of motility in mouse sperm permeabilized with streptolysin O.
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One approach to studying the mechanisms governing sperm motility is to permeabilize sperm and examine the regulation of motility by manipulating the intracellular milieu of the cell. The most common method of sperm permeabilization, detergent treatment, has the disadvantage that the membranes and many proteins are extracted from the cell. To avoid this problem, we have developed a method that uses streptolysin O to create stable pores within the plasma membrane while leaving internal membranes intact. Sperm were permeabilized, preincubated, and then treated with 0.6 U/ml of streptolysin O. Permeabilization was assessed by fluorescent dye technologies and endogenous protein phosphorylation using exogenously added [gamma-32P]ATP. Streptolysin O-induced permeabilization rendered the sperm immotile, and the effect was Ca2+-dependent. When the cells were treated simultaneously with a medium containing ATP, streptolysin O-treated sperm maintained flagellar movement. These results demonstrate that the streptolysin O permeabilization model system is a useful experimental method for studying the mechanisms that regulate sperm motility since it allows the flagellar apparatus to be exposed to various exogenously added molecules. (+info)
Mechanical stimulation of starfish sperm flagella.
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1. The responses of starfish sperm flagella to mechanical stimulation with a microneedle were analysed. Flagellar movement was recorded by high-speed microcinematography and by stroboscopic observation. 2. The amplitude of the bending wave of a flagellum was restricted over its entire length when the microneedle was brought near to the flagellum at its proximal region. Beyond the restricted part, the amplitude of the wave, and the bend angle, became smaller than those of a normally beating flagellum, while the curvature was practically unchanged. 3. When the tip of the microneedle was in contact with the flagellum, propagation of the bending wave beyond the microneedle was inhibited. The part of the flagellum between the base and the microneedle continued beating in some cases and stopped beating in other cases. The flagellum beyond the arrested part stopped beating and remained straight. When the microneedle was removed, the bending wave which existed in the part of the flagellum proximal to the microneedle, or the wave which was passively formed de novo at the time of the removal of the microneedle, propagated over the arrested part towards the tip. 4. A flagellum amputated by a microneedle in a medium containing ATP continued beating with a small amplitude, small curvature, small bend angle and low frequency. When the amputated flagellum was passively bent by a microneedle at the region near the point of amputation, this bend propagated towards the tip with a constant bend angle. 5. The beating frequency of the flagellum could be modulated by the application of a rhythmic external force generated by vibrating a microneedle near the flagellum. The beating was completely synchronized with vibration of the microneedle in the frequency range from 23 Hz to 43 Hz. (+info)
Rhophilin, a small GTPase Rho-binding protein, is abundantly expressed in the mouse testis and localized in the principal piece of the sperm tail.
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Tissue distribution and cellular localization of rhophilin, a 71 kDa Rho-binding protein, were examined in mice. Rhophilin mRNA was highly expressed in adult testis, but was absent in the testis of W/WV mice deficient in germ cells. An anti-rhophilin antibody detected a band of an expected size in sperm extracts, which was enriched in the tail fraction. Immunofluorescence analysis revealed two lines of striated staining running in parallel in the principal piece of the sperm tail. These results suggest that rhophilin is expressed in germ cells and localized in the fibrous sheath of the sperm tail. (+info)
Rat testis motor proteins associated with spermatid translocation (dynein) and spermatid flagella (kinesin-II).
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In this study, we report sites in the seminiferous epithelium of the rat testis that are immunoreactive with antibodies to the intermediate chain of cytoplasmic dynein and kinesin II. The study was done to determine whether or not microtubule-dependent motor proteins are present in Sertoli cell regions involved with spermatid translocation. Sections and epithelial fragments of perfusion-fixed rat testis were probed with an antibody (clone 74.1) to the intermediate chain of cytoplasmic dynein (IC74) and to kinesin-II. Labeling with the antibody to cytoplasmic dynein was dramatically evident in Sertoli cell regions surrounding apical crypts containing attached spermatids and known to contain unique intercellular attachment plaques. The antibody to kinesin II reacted only with spermatid tails. The levels of cytoplasmic dynein visible on immunoblots of supernatants collected from spermatid/junction complexes treated with an actin-severing enzyme (gelsolin) were greater than those of controls, indicating that at least some of the dynein may have been associated with Sertoli cell junction plaques attached to spermatids. Results are consistent with the conclusion that an isoform of cytoplasmic dynein may be responsible for the apical translocation of elongate spermatids that occurs before sperm release. Also, this is the first report of kinesin-II in mammalian spermatid tails. (+info)
Fertility of ejaculated and testicular megalohead spermatozoa with intracytoplasmic sperm injection.
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In this study the fertility and outcome of intracytoplasmic sperm injection (ICSI) using megalohead spermatozoa from the ejaculates and testicles was evaluated. Seventeen males with megalohead and pinhead sperm forms in their ejaculate were studied in 22 cycles. A high number of sperm heads without tails and abundant round spermatid forms were commonly observed. Round-headed spermatozoa were seldom accompanied by these severely abnormal spermatozoa. The majority of megalohead spermatozoa were observed to have multiple tails, were predominant in the sample, and were used for ICSI. Ejaculated megalohead spermatozoa were used for ICSI in 15 cycles, while testicular spermatozoa were used in seven cycles where there were no vital spermatozoa or spermatozoa of low vitality in the ejaculate. The same abnormal morphology was observed in the testicles as in the ejaculated spermatozoa in the same males. Mean (+/- SD) low motility 4.7 +/- 5.6% and sperm count (3.8 +/- 4.19 x 10(6)) were common findings in these severely teratozoospermic patients. A low fertilization rate (43.2%) was achieved by using megalohead sperm forms (group I, n = 17) in comparison with the control group (60.2%) which had zero normal sperm morphology according to strict criteria (group II, n = 30) (P <0.01). Furthermore, a low pregnancy rate (9.1%) was obtained in the megalohead sperm group in comparison with the control group (40%) (P <0.05). Low fertilization and pregnancy rates may be due to a high incidence of chromosomal abnormalities from severely defective spermatozoa in the ejaculate. Couples should be counselled and warned about possible low fertilization and pregnancy rates with ICSI when only pinhead and megalohead forms with a high number of sperm heads without tails are present in the ejaculate. (+info)
Effects of the central pair apparatus on microtubule sliding velocity in sea urchin sperm flagella.
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To produce oscillatory bending movement in cilia and flagella, the activity of dynein arms must be regulated. The central-pair microtubules, located at the centre of the axoneme, are often thought to be involved in the regulation, but this has not been demonstrated definitively. In order to determine whether the central-pair apparatus are directly involved in the regulation of the dynein arm activity, we analyzed the movement of singlet microtubules that were brought into contact with dynein arms on bundles of doublets obtained by sliding disintegration of elastase-treated flagellar axonemes. An advantage of this new assay system was that we could distinguish the bundles that contained the central pair apparatus from those that did not, the former being clearly thicker than the latter. We found that microtubule sliding occurred along both the thinner and the thicker bundles, but its velocity differed between the two kinds of bundles in an ATP concentration dependent manner. At high ATP concentrations, such as 0.1 and 1 mM, the sliding velocity on the thinner bundles was significantly higher than that on the thicker bundles, while at lower ATP concentrations the sliding velocity did not change between the thinner and the thicker bundles. We observed similar bundle width-related differences in sliding velocity after removal of the outer arms. These results provide first evidence suggesting that the central pair and its associated structures may directly regulate the activity of the inner (and probably also the outer) arm dynein. (+info)
Spag4, a novel sperm protein, binds outer dense-fiber protein Odf1 and localizes to microtubules of manchette and axoneme.
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Outer dense fibers are structures unique to the sperm tail. No definite function for these fibers has been found, but they may play a role in motility and provide elastic recoil. Their composition had been described before, but only two of the fiber proteins, Odf1 and Odf2, are cloned. We cloned Odf2 by virtue of its functional and specific interaction with Odf1, which, we show, is mediated by a leucine zipper. Further work demonstrated that the 84-kDa Odf2 protein localizes to both the cortex and the medulla of the fibers, whereas the 27-kDa Odf1 protein is present only in the medulla. Here we report the cloning and characterization of a new Odf1-interacting protein, Spag4. Spag4 mRNA is spermatid specific, and the 49-kDa Spag4 protein complexes specifically with Odf1, but not Odf2, mediated by a leucine zipper. It also self-associates. In contrast to Odf1 and Odf2, Spag4 protein localizes to two microtubule-containing spermatid structures. Spag4 is detectable in the transient manchette and it is associated with the axoneme in elongating spermatids and epididymal sperm. Our data suggest a role for Spag4 in protein localization to two major sperm tail structures. (+info)
Role of tyrosine phosphorylation of flagellar proteins in hamster sperm hyperactivation.
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Despite extensive study of sperm motility, little is known of the mechanism of mammalian sperm hyperactivation. Here we describe a novel method for preparation of rodent sperm flagella and use it to show a correlation between tyrosine phosphorylation of flagellar proteins and hyperactivation of hamster sperm. When hyperactivation was produced by a 3.5-h incubation in a medium supporting capacitation, four major tyrosine-phosphorylated peptides of 90-, 80-, 62-, and 48-kDa mass were detected in flagellar extracts. Incubation with calyculin A, an inhibitor of protein phosphatases 1 and 2A, produced hyperactivation within 40 min but only a single 80-kDa phosphotyrosine-containing flagellar component. Conversely, incubation with inhibitors of either protein kinase A (H8) or protein tyrosine kinase (tyrphostin 47) prevented both hyperactivation and the production of tyrosine-phosphorylated flagellar peptides. These results indicate a strong correlation of hyperactivation with the tyrosine phosphorylation of sperm flagellar peptides, and they strongly implicate an 80-kDa component as a major mediator of the mechanism that produces hyperactivated motility of hamster sperm. (+info)