Hyperactivation of mammalian sperm.

Mammalian sperm commonly show hyperactivated motility just before fertilization. The movement of hyperactivated sperm appears different in fluids of different viscosity and elasticity and in different species, but basically it involves an increase in flagellar bend amplitude and, usually, beat asymmetry. Hyperactivation may be critical to the success of fertilization, because it enhances the ability of sperm to detach from the wall of the oviduct, to move around in the labyrinthine lumen of the oviduct, to penetrate mucous substances and, finally, to penetrate the zona pellucida of the oocyte. Presumably, a signal or signals exist in the oviduct to initiate hyperactivation at the appropriate time; however, none have yet been identified with certainty. While the signal transduction cascade regulating hyperactivation remains to be completely described, it is clear that calcium ions interact with the axoneme of the flagellum to switch on hyperactivation. Although hyperactivation often occurs during the process of capacitation, divergent pathways regulate the two events.     

哺乳动物显微注精的研究

显微注精过程主要包括固定针和注射针的制备,精子的分离,卵母细胞的采集,卵母细胞的激活和培养,胚胎移植,显微注精不需要精子结构上的完整性,精子核的去凝聚与二硫键有关,卵母细胞的激活对受精成功是必须的。     

Profile of a mammalian sperm receptor

Complementary molecules on the surface of eggs and sperm are responsible for species-specific interactions between gametes during fertilization in both plants and animals. In this essay, several aspects of current research on the mouse egg receptor for sperm, a zona pellucida glycoprotein called ZP3, are addressed. These include the structure, synthesis, and functions of the sperm receptor during oogenesis and fertilization in mice. Several conclusions are drawn from available information. These include (I) ZP3 is a member of a unique class of glycoproteins found exclusively in the extracellular coat (zona pellucida) of mammalian eggs. (II) ZP3 gene expression is an example of oocyte-specific and, therefore, sex-specific gene expression during mammalian development. (III) ZP3 is a structural glycoprotein involved in assembly of the egg extracellular coat during mammalian oogenesis. (IV) ZP3 is a sperm receptor involved in carbohydrate-mediated gamete recognition and adhesion during mammalian fertilization. (V) ZP3 is an inducer of sperm exocytosis (acrosome reaction) during mammalian fertilization. (VI) ZP3 participates in the secondary block to polyspermy following fertilization in mammals. (VII) The extracellular coat of other mammalian eggs contains a glycoprotein that is functionally analogous to mouse ZP3. The unique nature, highly restricted expression, and multiple roles of ZP3 during mammalian development make this glycoprotein a particularly attractive subject for investigation at both the cellular and molecular levels.     

Flow cytometric sexing of mammalian sperm

This review reexamines parameters needed for optimization of flow cytometric sexing mammalian sperm and updates the current status of sperm sexing for various species where this technology is currently being applied. Differences in DNA content have provided both a method to differentiate between these sex-determining gametes and a method to sort them that can be used for predetermining sex in mammals. Although the DNA content of all cells for each mammalian species is highly conserved, slight but measurable DNA content differences of sperm occur within species even among cattle breeds due to different sizes of Y-chromosomes. Most mammals produce flattened, oval-headed sperm that can be oriented within a sorter using hydrodynamic forces. Multiplying the percentage the difference in DNA content of the X- or Y-chromosome bearing sperm times the area of the flat profile of the sperm head gives a simple sorting index that suggests that bull and boar sperm are well suited for separation in a flow sorter. Successful sperm sexing of various species must take into account the relative susceptibilities of gametes to the stresses that occur during sexing. Sorting conditions must be optimized for each species to achieve acceptable sperm sexing efficiency, usually at 90% accuracy. In the commercial application of sperm sexing to cattle, fertility of sex-sorted bull sperm at 2 x 10(6)/dose remains at 70-80% of unsexed sperm at normal doses of 10 to 20 x 10(6) sperm. DNA content measurements have been used to identify the sex-chromosome bearing sperm populations with good accuracy in semen from at least 23 mammalian species, and normal-appearing offspring have been produced from sexed sperm of at least seven species.     

Actin in mammalian sperm heads.

An actin-like substance has been detected in the postacrosomal region of mammalian spermatozoa by indirect immunofluorescence. The antigen was localized using an anti-actin antiserum from a patient with active chronic hepatitis. The actin-like contractile proteins may be important in sperm function and spermegg interactions during fertilization.     

Calmodulin-mediated adenylate cyclase from mammalian sperm

Calmodulin (CaM), the calcium binding protein that modulates the activity of a number of key regulatory enzymes, is present at high levels in sperm. To determine whether CaM regulates adenylate cyclase in mammalian sperm, the actions of EGTA and selected CaM antagonists on a solubilized adenylate cyclase from mature equine sperm were examined. The activity of equine sperm adenylate cyclase was inhibited by EGTA in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 2 mM. Equine sperm adenylate cyclase was also inhibited in a concentration-dependent manner by the CaM antagonists chlorpromazine and calmidazolium (IC50 = 400 and 50 microM, respectively). The inhibition of enzyme activity by these agents correlated with their known potency and specificity as anti-CaM agents. The activity of the enzyme in the presence of 200 microM calmidazolium was restored by the addition of authentic CaM (EC50 = 15 microM); full activity was restored by the addition of 50 microM CaM. La3+, an ion that dissociates CaM from tightly bound CaM-enzyme systems, inhibited equine sperm adenylate cyclase (IC50 = 1 mM). Incubation of equine sperm adenylate cyclase with La3+ dissociated endogenous CaM from the enzyme so that most of the enzyme bound to a CaM-Sepharose column equilibrated with Ca2+. Specific elution of CaM-binding proteins from the CaM-Sepharose column with EGTA yielded a CaM-depleted adenylate cyclase fraction that was stimulated 2-fold by the addition of exogenous CaM.     

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.     

Lack of species-specificity in mammalian sperm chemotaxis

Attraction of spermatozoa by way of chemotaxis to substances secreted from the egg or its surrounding cells has been demonstrated in marine species, amphibians, and mammals. This process is species- or family-specific in marine invertebrates: a chemoattractant for one marine species is usually not recognized by another species or by a member of another family. It is not known whether this selectivity is also the rule in other phyla. Furthermore, it is not at all obvious that such selectivity would be advantageous to species with internal fertilization. Here, using a directionality-based assay for chemotaxis, we studied in vitro the chemotactic response of human and rabbit spermatozoa to human, rabbit, and bovine egg-related factors. We found that spermatozoa from each of the two sources responded similarly well to egg-related factors obtained from any of the three species examined. These results indicate lack of chemotaxis-related, species specificity between these species, suggesting that their sperm chemoattractants are common or very similar. The findings further suggest that mammals do not rely on species specificity of sperm chemotaxis for avoidance of interspecies fertilization.     

Modulation of mammalian sperm motility by quercetin

The flavonoid quercetin inhibits collective motility of ejaculated ram spermatozoa in the first 2 hr of incubation; during the next 3-4 hr motility is stimulated. To explain this interesting effect, we followed the influence of quercetin on sperm glycolysis, extracellular pH, ATP content, mitochondrial respiration, and lipid peroxidation. The collective motility of untreated cells is decreased to about 40% of the original motility during two hours of incubation. During this time, the rate of glycolysis is constant, respiration rate is increasing, there is no change in ATP content, the rate of lipid peroxidation is very slow, and the extracellular pH became very acidic (pH 5.5). It is concluded that motility is decreased due to this acidification. This acidification is prevented to some extent by quercetin, which indirectly inhibits glycolysis. Quercetin inhibits motility due to the inhibition of the plasma membrane calcium pump, as we showed previously (Breitbart et al., J Biol Chem 260:11548-11553, 1985). The motility of untreated cells is arrested after 3.5 hr of incubation, whereas quercetin-treated cells show high motility, which continues for additional 2-3 hr. After 3.5 hr, the control cells show no glycolytic activity, ATP content and respiration rates are decreased, and rate of lipid peroxidation is highly increased. At this time, quercetin-treated cells show no glycolytic activity, only a small decrease in ATP content and respiratory rate, and a very low rate of lipid peroxidation. Based on these data it is concluded that sperm motility after 3.5 hr of incubation is dependent mainly on mitochondrial respiration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of damage in mammalian sperm cells

Ejaculated semen is washed for in vitro fertilization or diluted and processed to allow optimal and long-term low temperature liquid- and cryo-preservation. However, sperm are vulnerable to the washing, dilution, temperature and osmotic changes involved in sperm storage. In this review, a number of techniques are considered for detecting damaged spermatozoa. Staining protocols have been developed to detect the membrane and organelle integrity of mammalian sperm cells. Plasma membrane integrity is usually assessed after staining cells with membrane-impermeable dyes or alternatively with acetylated membrane (AM) permeable probes that are selectively de-esterified and become membrane impermeable and thus entrapped into viable cells only (AM ester loading). Organelle-specific dyes are commonly used to detect functionality of mitochondria or the acrosome. A distortion in the lateral and bilayer organization of lipids as well as the peroxidation of fatty acid moieties can be quantified and localized in living sperm. The relation of a disordering in the sperm membrane's lipid architecture and sperm deterioration versus capacitation is discussed. Finally, the integrity of sperm DNA can be measured at three different levels by assessing the degree of DNA-protamine condensation, the incidence of breaks and nicks in the DNA and the frequency of fragmentation of the nuclei into sub-haploid apoptotic bodies. The relevance of detecting DNA aberrations and especially the putative link to the incidence of apoptosis is critically considered.     

Atomic force microscopy of mammalian sperm chromatin

We have used the atomic force microscope (AFM) to image the surfaces of intact bull, mouse and rat sperm chromatin and partially decondensed mouse sperm chromatin attached to coverglass. High resolution AFM imaging was performed in air and saline using uncoated, unfixed and unstained chromatin. Images of the surfaces of intact chromatin from all three species and of an AFM-dissected bull sperm nucleus have revealed that the DNA is organized into large nodular subunits, which vary in diameter between 50 and 100 nm. Other images of partially decondensed mouse sperm chromatin show that the nodules are arranged along thick fibers that loop out away from the nucleus upon decondensation. These fibers appear to stretch or unravel, generating narrow smooth fibers with thicknesses equivalent to a single DNA-protamine complex. High resolution AFM images of the nodular subunits suggest that they are discrete, clipsoid-shaped DNA packaging units possibly only one level of packaging above the protamine-DNA complex.     

哺乳动物及人精子膜离子通道的研究进展

离子的跨膜转动对精子的生理活动起重要的作用。近年,应用膜片钳及人工膜重组等研究通道有关的电生理技术,人们直接观察到哺乳动物及人精子膜上Ｋ、Ｎａ＾＋、Ｃａ＾２＋、Ｃｌ通道的存在。这些结果为揭示精子成熟、获能精卵结合反应等生理过程的某些细节提供了有有的资料,特别是对人精子膜的研究,还为临床应用提供了可能。     

Dense fibers protect mammalian sperm against damage

The relative tensile strengths of the sperm of seven mammalian species and sea urchins have been measured by determining the minimum shear necessary to kill them (assayed by lack of motility) when they are suspended in a viscous fluid. In general, long sperm are killed by smaller shears than short sperm. However, the longer sperm are not as fragile as would be expected from theoretical predictions. Their additional tensile strength correlates well with the size of their dense fibers; a theory that includes the dense fiber contributions accurately predicts the sperm tensile strength for most of the species in which this has been measured. This added strength may be necessary to protect sperm from shear forces encountered during epididymal transport and especially during ejaculation, as these forces are strong enough to kill long sperm if they are not strengthened.     

Function of the mammalian oviductal sperm reservoir.

Sperm are stored in the isthmic region of the oviduct under conditions that maintain sperm viability and suppress motility. This region is also the site in which essential steps of the capacitation process are coordinated with the appearance of the ovulated egg. The influx of Ca(2+) and phosphorylation of sperm proteins are features of the ongoing capacitation process. Using a cell-culture system of oviductal epithelial cells, it was found that sperm bound to the epithelial cells showed a reduced Ca(2+) uptake and almost no tyrosine phosphorylation as shown by indirect immunofluorescence. Furthermore, sperm viability, measured as membrane integrity with propidium iodide, is significantly prolonged as compared to sperm in suspension. The formation of the sperm reservoir appears to be mediated by carbohydrate-protein interaction. In the pig, it has been found that mannosyl-oligosaccharides exposed by the epithelial cells are high-affinity ligands for sperm-associated lectins. Ovalbumin and mannopentaose are effective inhibitors of sperm binding to explants of oviductal epithelium. Spermadhesins, a new class of animal lectins and the major secretory products of the porcine seminal vesicle, associate with the sperm surface at ejaculation and are candidate molecules for the receptors of the epithelial carbohydrates.     

Effects of gossypol on the motility of mammalian sperm

The effects of the male contraceptive gossypol on the motility of mammalian spermatozoa are reviewed. The role of sperm motility in the processes of fertilization and the effect of the drug on these processes determine its effectiveness as a contraceptive. The promising male contraceptive potential of gossypol is discussed in the context of the serious adverse effects of the agent.