Modulation of the asymmetry of sea urchin sperm flagellar bending by calmodulin

Sea urchin spermatozoa demembranated with Triton X-100 in the presence of EGTA, termed potentially asymmetric, generate asymmetric bending waves in reactivation solutions containing EGTA. After they are converted to the potentially symmetric condition by extraction with Triton and millimolar Ca++, they generate symmetric bending waves in reactivation solutions containing EGTA. In the presence of EGTA, their asymmetry can be restored by addition of brain calmodulin or the concentrated supernatant obtained from extraction with Triton and millimolar Ca++. These extracts contain calmodulin, as assayed by gel electrophoresis, radioimmunoassay, activation of brain phosphodiesterase, and Ca++-dependent binding of asymmetry-restoring activity to a trifluorophenothiazine-affinity resin. Conversion to the potentially symmetric condition can also be achieved with trifluoperazine substituted for Triton during the exposure to millimolar Ca++, which suggests that the calmodulin-binding activity of Triton is important for this conversion. These observations suggest that the conversion to the potentially symmetric condition is the result of removal of some of the axonemal calmodulin and provide additional evidence for axonemal calmodulin as a mediator of the effect of Ca++ on the asymmetry of flagellar bending.     

Sperm-activating peptide induces asymmetric flagellar bending in sea urchin sperm

Speract, a sperm-activating peptide (SAP) from sea urchin eggs, induces various sperm responses including a transient increase in the intracellular Ca2+ concentration. However, it has not been clarified how speract modulates sperm motility and whether it functions as a chemoattractant. To confirm the effect of speract on sperm motility, we observed the flagellar bending response to speract in sperm of Hemicentrotus pulcherrimus, in experiments using caged speract and a lighting system for a microscope newly developed with a power LED. We found that speract induces increases in curvature of swimming paths and changes flagellar bending shape to asymmetric. These facts show that speract directly regulates flagellar motility, and suggest that speract-induced increases in intracellular Ca2+ concentration play an actual role in regulation of the flagellar movement.     

Initiation of hyperactivated flagellar bending in mouse sperm within the female reproductive tract

To determine where and when hyperactivation is initiated in vivo, the flagellar curvature ratios (fcr) of mouse sperm within the female reproductive tract were measured from videotape recordings and compared with those of epididymal sperm incubated under capacitating conditions in vitro. The fcrs and linearities of trajectory were significantly lowered after 90 min of incubation in vitro, indicating that hyperactivation had been initiated by that time. The flagellar curvature ratios of sperm at the colliculus tubarius, within the uterotubal junction, and in the isthmus, measured at 1-2 h postcoitus and approximately 1 h before and 1 h after ovulation, were found to have fcrs that were not different from those of sperm incubated for 90 min in vitro. It was concluded that the tract sperm had initiated hyperactivated flagellar bending before the time of ovulation and before entering the oviduct. Only sperm in the lower isthmus 1 h before ovulation had fcrs that were significantly different from sperm incubated for 90 min in vitro, but not from sperm measured at the beginning of incubation in vitro. This could be the result of motility suppression in the lower isthmus.     

Ultrastructure of the ejaculated human sperm

Summary The ultrastructure of the ejaculated human sperm follows the general pattern known from other mammalian sperms. However, certain features are peculiar to the human sperm. The chromatin is often granular giving it an unmature appearance, the mitochondrial helix is often unorderly arranged, the longitudinal columns of the fibrous sheath and the annular ring are not very conspicuous.The nucleus is covered by the acrosome with its anterior, reactive and posterior, unchangeable equatorial segment and most posteriorly by the postnuclear cap.Generally the gross and fine morphology of the human sperm is more varying and less regular than it is known from other mammalian sperms.This investigation was supported by United States Public Health Service, National Institutes of Health (HD 02493) and the Lalor Foundation.     

Regulation of flagellar bending by cAMP and Ca2+ in hamster sperm

Hamster sperm were immotile in the medium at free Ca2+ concentrations ([Ca2+]) below 1 x 10(-4) M. The flagellum was acutely bent in the opposite direction to the curve of the hook-shaped heads. This phenomenon seemed to be caused by the decrease in the intracellular cAMP concentration, since the cAMP concentration was low at [Ca2+] below 1 x 10(-4) M and increased abruptly at 1 x 10(-3) M, at which sperm were swimming actively. In addition, sperm became motile due to treatment with 8-bromo-cAMP, a membrane permeable analogue of cAMP, in a medium without Ca2+. These results suggested that extracellular Ca2+ is involved in the regulation of flagellar movement via increasing intracellular cAMP concentration. By the treatment with W-13, a calmodulin inhibitor, sperm also became motile, although cAMP concentration remained at a low level. These results suggested that cAMP is not always required for the flagellar movement when the function of calmodulin is depressed.     

Sialyl glycoprotein distribution on the plasma membrane of ejaculated ram spermatozoa

Localization of sialyl residues on unfixed ejaculated ram sperm membrane using the direct covalent probes of either ferritin hydrazide or latex hydrazide revealed a unique regional distribution on the plasmalemma covering the sperm head only. Three different labelling zones were identified based on the intensity and the nature of the sialyl glycoconjugates: a patchy-like zone which included the plasma membrane overlaying the post-nuclear cap and the convex side of the apical body of the acrosome; highly ordered heavily labelled zones including the plasmalemma adjacent to the concave apical body of the acrosome and to the posterior part of the equatorial acrosomal segment; a paucity-labelling zone which included the plasma membrane underlying the principal acrosomal region and the anterior part of the equatorial acrosomal segment. The possible physiological role of the highly ordered labelled zones is discussed.     

Actin in ejaculated human sperm cells

Previous studies have identified an "actin-like" protein in human sperm by indirect immunofluorescence microscopy with various probes, but no real biochemical confirmation of actin was made. In this study, two-dimensional (2-D) gels of Nonidet P-40 (NP-40) extracts of purified human sperm cells revealed a protein with appropriate pI and molecular weight coordinates for actin. When excised from a 2-D gel, cleaved with N-chlorosuccinimide, and separated on a sodium dodecyl sulfate slab gel, this putative actin showed a cleavage pattern identical to those from known actin. Furthermore, when sperm were immediately purified from whole semen and extracted with 0.3% NP-40, actin was detected almost entirely in the soluble fraction, indicative of unpolymerized actin. Conclusions from these experiments support those implied by direct immunofluorescence microscopy: actin is present in human sperm and appears to be mainly unpolymerized.     

Automatic evaluation of ram sperm morphometry

This study was designed to develop a new method based on fluorescence microscopy and image analysis for the automatic assessment of sperm morphometry and to study separately the effect of drying and fixation on the parameters of head sperm morphometry in the ram. The study was divided into two experiments. In the first experiment, ejaculates from 25 adult males were collected using an artificial vagina, diluted and divided into four sample aliquots. The first was labeled directly with Hoechst 33342 (FRESH), and the others were processed as smears. Between smears, one group was directly labeled with Hoechst after air drying (DRIED), and the other were fixed either with glutaraldehyde (GLUT), or with methanol (MET), and labeled with Hoechst afterward. Digital images of the fluorescence-labeled sperm were recorded with a digital camera, and sperm heads were automatically captured and analyzed using the ImageJ program. The method used allowed a fast and automatic selection of most sperm heads for a given image with high precision. There was a general trend toward significant decrease in head length, width, area and perimeter of air-dried sperm compared with fresh sperm. On average, this decrease was of 4.1% in length, 4.3% in width, 9.1% in area, and 2.8% in perimeter. Between semen smears, fixation with glutaraldehyde significantly increased head sperm dimensions. The smears fixed with glutaraldehyde method is recommended for a more practical use than with fresh samples, providing better quality images than the other methods, and because the morphometric results obtained were more similar to the FRESH group than those of the DRIED and MET. In the second experiment, ejaculates from adult males were used to compare the sperm head morphometric results obtained with the new method developed (using the GLUT treatment as reference) with a more conventional CASMA method (semen smears stained with Hemacolor and processed with the ISAS commercial software, HEM). The GLUT method allowed the analysis of 100% of sperm, whereas only 93% of sperm could be analyzed using HEM. Spermatozoa displayed a bigger size when processed with HEM than with GLUT method in all primary sperm head morphometric parameters. A significant correlation was observed between the two methods used in this experiment for all morphometric size parameters. The new method developed allows automatic determination of sperm head morphometry in a reduced time, which facilitates its use in routine semen analysis. It was concluded that the automation of sperm morphometry is feasible using fluorescence microscopy and image analysis and that the effect of drying and fixation was less important than previously stated.     

Assessment of chromatin status (SCSA) in epididymal and ejaculated sperm in Iberian red deer, ram and domestic dog

Abnormal chromatin condensation is not detected using classical techniques for sperm analysis. SCSA has demonstrated its usefulness in sperm chromatin analysis in several species (human, bull, stallion and boar). In this work, we studied sperm samples from red deer, ram and dog to analyze the differentiation of chromatin structure applying SCSA in epididymal and ejaculated spermatozoa. Epididymal samples were obtained from the caput, corpus and cauda by means of cuts, and ejaculated ones were obtained by electroejaculation (deer), artificial vagina (ram) and digital manipulation (dog). SCSA results suggested different critical points in sperm maturation (spermatozoa with loose chromatin to more condensed chromatin) among species: from corpus to cauda in ram and from caput to corpus in deer and dog. Moreover, we also detected differences in ruminants and dog, reflected in the appearance of SCSA plots. Indeed, ram and deer samples rendered two peaks within the sperm main population (sperm with condensed chromatin), whereas only one was detected in dog. Although some differences were observed between cauda and ejaculated samples, SCSA parameters indicated good chromatin condensation, making these samples suitable for germplasm banking. Some species-dependent modifications in the analysis of the results may be necessary to take full advantage of its analytical power.     

Adenylate kinase activity in ejaculated bovine sperm flagella

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) activity was detected in the flagella of ejaculated bovine spermatozoa. This activity provided sufficient ATP to produce normal motility in cells permeabilized with digitonin and treated with 0.5 mM MgADP. In the presence of ADP, adenylate kinase activity was inhibited by P1,P5-di(adenosine 5')-pentaphosphate (Ap5A), an adenylate kinase-specific inhibitor, and motility was stopped. ATP-supported motility was not affected by Ap5A. Mitochondrial adenylate kinase activity allowed AMP to stimulate respiration in permeabilized sperm. Adenylate kinase activity in tail fragments was most active in a pH range from 7.6 to 8.4, and a similar pH sensitivity was observed for this enzyme activity in a hypotonic extract of whole sperm. The apparent km of adenylate kinase activity in permeabilized tail fragments was about 1.0 mM ADP in the direction of ATP synthesis. The fluctuation of nucleotide concentrations in normal and metabolically stimulated sperm suggested that adenylate kinase was most active when the cell was highly motile, although adenylate kinase activity did not appear to be coupled strictly with motility.     

Cryopreservation and fertility of ejaculated and epididymal stallion sperm

The cryopreservation of epididymal sperm is important to preserve genetic material from valuable deceased males. This study evaluated the viability of sperm samples from eight stallions under three conditions: (1) collected using an artificial vagina (EJ-0h), (2) recovered from the epididymal cauda immediately after orchiectomy (EP-0h), and (3) recovered from the epididymal cauda after 24h of storage at 5°C (EP-24h). To obtain EJ-0h sperm, two ejaculates were collected from each stallion. After 1 week, the stallions were submitted to bilateral orchiectomy, and one of the removed epididymides was flushed to obtain EP-0h sperm. The contralateral epididymis was stored at 5°C for 24h before being flushed to obtain EP-24h sperm. The sperm samples were analyzed at three different times: immediately after sperm recovery, after dilution in the freezing extender, and post-thawing. A fertility trial was performed using 39 estrous cycles. After ovulation induction with 1mg of deslorelin acetate (i.m.), mares were inseminated with 800×10(6) sperm. The total number of sperm recovered was 7.8±4.7×10(9) for EJ-0h sperm, 12.9±9.2×10(9) for EP-0h sperm and 12.0±8.0×10(9) for EP-24h sperm. The sperm motility, evaluated by total motility, progressive motility and the percentage of rapid cells, was similar among the samples before and after freezing (P>0.05). However, the plasma membrane integrity was different between EJ-0h and EP-0h pre-freezing and between EJ-0h and EP-24h post-thawing (P<0.05). The conception rates were similar between groups inseminated with sperm recovered from the epididymal cauda immediately after orchiectomy (EP-0h), after 24h of storage at 5°C of the epididymal cauda (EP-24h) and with ejaculated sperm (EJ-0h) (P>0.05). In conclusion, the viability and fertility of cauda epididymal sperm are similar to those of ejaculated sperm.     

Analysis of flagellar bending in hamster spermatozoa: characterization of an effective stroke

The mechanism by which flagella generate the propulsive force for movement of hamster spermatozoa was analyzed quantitatively. Tracing points positioned 30, 60, 90, and 120 microm from the head-midpiece junction on the flagellum revealed that they all had zigzag trajectories. These points departed from and returned to the line that crossed the direction of progression. They moved along the concave side (but not the convex side) of the flagellar envelope that was drawn by tracing the trajectory of the entire flagellum. To clarify this asymmetry, the bending rate was analyzed by measuring the curvatures of points 30, 60, 90, and 120 microm from the head-midpiece junction. The bending rate was not constant through the cycle of flagellar bending. The rate was higher when bending was in the direction described by the curve of the hook-shaped head (defined as a principal bend [P-bend]) to the opposite side (R-bend). We measured a lower bending rate in the principal direction (R-bend to P-bend). To identify the point at which the propulsive force is generated efficiently within the cycle of flagellar bending, we calculated the propulsive force generated at each point on the flagellum. The value of the propulsive force was positive whenever the flagellum bent from an R-bend to a P-bend (when the bending rate was lowest). By contrast, the propulsive force value was zero or negative when the flagellum bent in the other direction (when the bending rate was higher). These results indicate that flagellar bending in hamster spermatozoa produces alternate effective and ineffective strokes during propulsion.     

Acrosomal status in fresh and capacitated human ejaculated sperm

The acrosomal status of human sperm was evaluated by immunofluorescence utilizing a specific monoclonal antibody that recognizes target antigen(s) localized in the acrosomal cap region. Spontaneous acrosomal loss was first examined in sperm preparations used for successful in vitro fertilization of human eggs. In these sperm populations, less than 20% of the sperm underwent degenerative or spontaneous acrosomal loss following 24 h of incubation. The correlation of acrosomal loss with changes in motility and viability suggested that sperm senescence was not necessarily coupled to acrosomal loss. Chemical induction of acrosomal loss by calcium ionophore A23187 and lysophosphatidylcholine (LPC) was characterized. Maximal ionophore induction (10 microM A23187 in media containing calcium) was observed in cells exposed to capacitating conditions in vitro; sperm exposed to noncapacitating conditions did not readily acquire the ability to respond to ionophore. The reaction induced by ionophore was slow (60 min), and at least 30% of the cells were always resistant to induction. In contrast, LPC induced rapid, synchronous acrosomal loss in either freshly ejaculated or capacitated sperm in the presence or absence of extracellular calcium, suggesting that this loss was not a physiologic reaction. These studies may provide a basis for evaluating capacitation and ultimately fertility potential in the human male.