Cytogenetic analysis of sperm chromosomes and sperm nuclei in a male heterozygous for a reciprocal translocation t(5;7)(q21;q32) by in situ hybridisation. (49/9715)

We have studied the meiotic segregation of a reciprocal translocation t(5;7)(q21;q32) in a male carrier, using the human sperm-hamster oocyte fusion technique and the whole chromosome painting. A total of 296 sperm complements were analysed by dual chromosome painting. The frequencies of alternate, adjacent-1, adjacent-2 and 3:1 segregation were 49.7%, 32.4%, 16.2% and 1.7% respectively. Aneuploidy frequencies for chromosomes not involved in the translocation were determined by FISH on decondensed sperm heads using probes from chromosomes X, Y, 6, 18 and 21. A total of 20,118 spermatozoa was analysed, 10,201 by two-colour FISH (probes for chromosomes 6 and 21) and 9917 by three-colour FISH (probes for chromosomes X, Y, and 18). There was no evidence of an interchromosomal effect, since disomy frequencies were within the range of normal controls.  (+info)

Direct estimation of the recombination frequency between the RB1 gene and two closely linked microsatellites using sperm typing. (50/9715)

In this study, single sperm typing has been used for high-resolution recombination analysis between the retinoblastoma gene and two closely linked extragenic microsatellites (D13S284 and D13S1307). The analysis of 1198 single sperm from three donors allowed the determination of recombination fractions between RB1.20 and D13S284 and RB1.20 and D13S1307 of 0.022 and 0.033, respectively. These results show that RB1 gene and the two microsatellites are closely linked, which validates their potential use in indirect genetic diagnosis of retinoblastoma.  (+info)

Activation of the proteasomes of sand dollar eggs at fertilization depends on the intracellular pH rise. (51/9715)

The mechanism of the activation of intracellular proteasomes at fertilization was measured in living sand dollar eggs using the membrane-impermeant fluorogenic substrate, succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid. When the substrate was microinjected into unfertilized eggs, the initial velocity of hydrolysis of the substrate (V0) was low. V0 measured 5 to 10 min after fertilization was five to nine times the prefertilization level and remained high throughout the first cell cycle. Hydrolysis of the substrate was inhibited by clasto-lactacystin beta-lactone, a specific inhibitor of the proteasome. There has been in vitro evidence that calcium may be involved in regulation of proteasome activity to either inhibit the increase in peptidase activity associated with PA 28 binding to the 20S proteasome or stimulate activity of the PA 700-proteasome complex. Since both intracellular free Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) increase after fertilization, hydrolysis of the proteasome substrate was measured under conditions in which [Ca2+]i and pHi were varied independently during activation. When the pHi of unfertilized eggs was elevated by exposure to 15 mM ammonium chloride in pH 9 seawater, V0 increased to a level comparable to that measured after fertilization. In contrast, [Ca2+]i elevation without pHi change, induced by calcium ionophore in sodium-free seawater, had no effect on V0 in the unfertilized egg. Moreover, when unfertilized eggs were microinjected with buffers modulating pHi, V0 increased in a pH-dependent manner. These results indicate that the pHi rise at fertilization is the necessary prerequisite for activation of the proteasome, an essential component in the regulation of the cell cycle.  (+info)

Spatiotemporal dynamics of the [Ca2+]i rise induced by microinjection of sperm extract into mouse eggs: preferential induction of a Ca2+ wave from the cortex mediated by the inositol 1,4,5-trisphosphate receptor. (52/9715)

Hamster sperm extract (SE) possessing Ca2+ oscillation-inducing activity was microinjected into the peripheral or central region of mouse eggs, and the first increase in intracellular Ca2+ concentration ([Ca2+]i), together with the spread of fluorescence-labeled SE in the ooplasm, was investigated by imaging with confocal microscopy. Injection into the periphery always induced a Ca2+ wave that started from the injection site after a delay of 5 to 30 s depending on the concentration of SE. The diluted SE caused a wave of two-step [Ca2+]i rises, which was always observed at fertilization. Injection into the center could induce a radial Ca2+ wave with relatively high dose of SE, but lower dose of SE caused a [Ca2+]i rise after a longer delay which was initiated synchronously over the ooplasm or was preceded in a peripheral area. Injection of diluted SE remarkably prolonged the delay time and reduced the rate of [Ca2+]i rise. The critical concentration of SE needed to induce [Ca2+]i rise was significantly lower in the periphery. These results indicate that the sensitivity to SE is higher in the cortex. SE-induced [Ca2+]i rises were blocked by an antibody against the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R). The cortex was substantially more sensitive to injected InsP3 induction of Ca2+ release than the center. It is suggested that the cortex of mouse eggs may involve a functionally specialized organization of InsP3Rs and Ca2+ pools in which a cytosolic sperm factor(s) could act upon sperm-egg fusion to cause Ca2+ release, leading to the Ca2+ wave at fertilization.  (+info)

Calcium-dependent actin filament-severing protein scinderin levels and localization in bovine testis, epididymis, and spermatozoa. (53/9715)

We assessed the levels and localization of the actin filament-severing protein scinderin, in fetal and adult bovine testes, and in spermatozoa during and following the epididymal transit. We performed immunoblots on seminiferous tubules and interstitial cells isolated by enzymatic digestion, and on bovine chromaffin cells, spermatozoa, aorta, and vena cava. Immunoperoxidase labeling was done on Bouin's perfusion-fixed testes and epididymis tissue sections, and on spermatozoa. In addition, immunofluorescence labeling was done on spermatozoa. Immunoblots showed one 80-kDa band in chromaffin cells, fetal and adult tubules, interstitial cells, spermatozoa, aorta, and vena cava. Scinderin levels were higher in fetal than in adult seminiferous tubules but showed no difference between fetal and adult interstitial cells. Scinderin levels were higher in epididymal than in ejaculated spermatozoa. Scinderin was detected in a region corresponding with the subacrosomal space in the round spermatids and with the acrosome in the elongated spermatids. In epididymal spermatozoa, scinderin was localized to the anterior acrosome and the equatorial segment, but in ejaculated spermatozoa, the protein appeared in the acrosome and the post-equatorial segment of the head. In Sertoli cells, scinderin was detected near the cell surface and within the cytoplasm, where it accumulated near the base in a stage-specific manner. In the epididymis, scinderin was localized next to the surface of the cells; in the tail, it collected near the base of the principal cells. In Sertoli cells and epididymal cells, scinderin may contribute to the regulation of tight junctional permeability and to the release of the elongated spermatids by controlling the state of perijunctional actin. In germ cells, scinderin may assist in the shaping of the developing acrosome and influence the fertility of the spermatozoa.  (+info)

Protein tyrosine phosphorylation during hyperactivated motility of cynomolgus monkey (Macaca fascicularis) spermatozoa. (54/9715)

Capacitation and capacitation-related hyperactivated motility do not occur spontaneously in cynomolgus monkey (Macaca fascicularis) spermatozoa; instead, both have an absolute requirement for exogenous stimulation with caffeine and dibutyryl (db)cAMP. In the present study, we 1) defined sorting criteria for automated analysis of macaque sperm exhibiting hyperactivated motility (HA) and 2) investigated protein tyrosine phosphorylation involvement in dbcAMP- and caffeine-stimulated capacitation and HA. Motion characteristics were assessed by computer-assisted motion analysis. Tyrosine phosphorylation of sperm tail proteins was determined by immunocytochemistry with PY-20 antiserum. Automated sorting criteria for HA were curvilinear velocity (VCL) >/= 150 microm/sec; amplitude of lateral head displacement (ALH) >/= 8.0 microm, and linearity (LIN) +info)

Elasticity measurements show the existence of thin rigid cores inside mitotic chromosomes. (55/9715)

Chromosome condensation is one of the most critical steps during cell division. However, the structure of condensed mitotic chromosomes is poorly understood. In this paper we describe a new approach based on elasticity measurements for studying the structure of in vitro assembled mitotic chromosomes in Xenopus egg extract. The approach is based on a unique combination of measurements of both longitudinal deformability and bending rigidity of whole chromosomes. By using specially designed micropipettes, the chromosome force-extension curve was determined. Analysis of the curvature fluctuation spectrum allowed for the measurement of chromosome bending ridigity. The relationship between the values of these two parameters is very specific: the measured chromosome flexibility was found to be 2,000 times lower than the flexibility calculated from the experimentally determined Young modulus. This requires the chromosome structure to be formed of one or a few thin rigid elastic axes surrounded by a soft envelope. The properties of these axes are well-described by models developed for the elasticity of titin-like molecules. Additionally, the deformability of in vitro assembled chromosomes was found to be very similar to that of native somatic chromosomes, thus demonstrating the existence of an essentially identical structure.  (+info)

Voltage-operated Ca2+ channels and the acrosome reaction: which channels are present and what do they do? (56/9715)

Evidence from pharmacological studies suggests that induction of the acrosome reaction of mammalian spermatozoa by solubilized zona pellucida, and possibly by progesterone, is dependent upon Ca2+ influx through voltage-operated Ca2+ channels. Studies on Ca2+ accumulation and membrane potential in ligand-stimulated or artificially depolarized spermatozoa support such a conclusion. Electrophysiological studies on rodent spermatogenic cells have revealed the presence of a 'T' type voltage-operated Ca2+ current. This current has pharmacological attributes consistent with those of the putative channel responsible for Ca2+ influx mediating the acrosome reaction. However, use of molecular techniques to study human and rodent testis and spermatogenic cells has detected the presence of three different voltage-operated Ca2+ channel subunits. One of these (alpha lE) may generate T-currents, though this is currently disputed. Voltage-operated Ca2+ channel structure and the relationship between channel subunit expression and the characteristics of consequent Ca2+ currents is briefly reviewed. The nature and function of T-channel-mediated Ca2+ influx is examined in the context of the time-course of ligand- and depolarization-induced elevation of [Ca2+]i in mammalian spermatozoa. It is likely that a secondary Ca2+ response (mobilization of stored Ca2+ or activation of a second Ca(2+)-influx pathway) is required for the acrosome reaction. Evidence for the existence and participation of various candidates is discussed (including voltage-operated Ca2+ channels, which may be functionally expressed only in mature spermatozoa), the available evidence favouring a secondary Ca(2+)-influx pathway. Immediate priorities for future research in this area are proposed.  (+info)