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PubMed2005

Exposure of actin on the surface of the human sperm head during in vitro culture relates to sperm morphology, capacitation and zona binding.

BACKGROUND: The aim of this study was to determine the relationship between the proportion of motile sperm with actin exposed on the surface of the head and sperm function. METHODS: Semen samples were obtained from normozoospermic men and sperm function tests were performed. Motile sperm selected by swim-up were incubated with actin monoclonal antibody (A-mAb, 1:100) for 2 h, then anti-mouse IgG Dynabeads were used to detect sperm-bound A-mAb. Sperm capacitation was increased by phorbol myristate acetate (PMA) and decreased by bicarbonate-free medium. RESULTS: The proportion of sperm with exposed actin increased with time for up to 2 h incubation. Bicarbonate-free medium significantly decreased the proportion of sperm with exposed actin. PMA significantly enhanced this phenomenon. Sperm bound to zona pellucida (ZP) had a significantly higher proportion with exposed actin than did sperm remaining in medium. Of the 79 samples studied, an average of 9.4% (range 1-27%) of motile sperm had exposed actin after 2 h incubation and this was significantly correlated with sperm normal morphology and ZP binding. CONCLUSION: Exposure of actin on the surface of the sperm head during in vitro culture may be related to membrane modification during sperm capacitation and hence may be a useful marker for this subpopulation of sperm.

Actins↗

PubMed1991

Use of sperm viability and acrosomal status assays in combination with immunofluorescence technique to ascertain surface expression of sperm antigens.

Sperm antigens are being sought for the development of prototype contraceptive vaccines. For vaccine-induced antisperm antibodies to inhibit the function of viable sperm, target antigens must be expressed on the sperm surface before, during or after the acrosome reaction. The purpose of this study was to develop an approach to detect accurately surface binding of antibodies to viable acrosome-intact or acrosome-reacted sperm, and to confirm sperm surface binding characteristics of a panel of antisperm monoclonal antibodies submitted to the World Health Organization Antifertility Vaccines Task Force. Monoclonal antibodies were tested using a combination of carboxyl-fluorescein diacetate (CoFDA) sperm viability, and rhodamine immunofluorescence assays to ascertain surface binding to viable membrane-intact human sperm, and, in parallel studies, with a combination of fluorescein isothiocyanate (FITC) conjugated Pisum sativum agglutinin (PSA) and rhodamine immunofluorescence assays to ascertain the acrosomal status of antibody-positive sperm. Only 14 out of 32 antibodies that were previously considered to be surface-reactive were confirmed to be positive with these methods. Five were positive on unfixed viable (CoFDA-labelled) fresh sperm in this study, and 9 different antibodies were positive on unfixed acrosome-reacted sperm. We conclude that this multifaceted approach is more accurate and informative than previous techniques used for detection of sperm surface antigens such as radioimmunoassay, standard immunofluorescence, sperm agglutination assays and enzyme-linked immunosorbent assays, which can give false positive results due to the presence of contaminating non-sperm cells, insoluble seminal plasma material, or permeabilized non-viable sperm. Furthermore, the combination of assays employed in this study does not depend on sperm motility, as do the immunobead and sperm immobilization assays, and provides information about the acrosomal status of antibody-positive cells.

Acrosome↗

PubMed1991

The method of sperm collection significantly influences sperm motion parameters following ethane dimethanesulphonate administration in the rat.

Sperm motion analysis following exposure to a reproductive toxicant is one means of evaluating the functional integrity of the testis and epididymis. In this study we sought to determine whether the method used to collect sperm from the proximal cauda epididymidis, where sperm are not completely mature, has a significant influence on sperm motion parameters. Two methods of collecting rat sperm for motion analysis were used: one based on an aspiration technique selected from the literature; the other, a new approach based on diffusion of sperm from the epididymal tubule. The two methods were tested for sensitivity to effects on sperm motility parameters 4 days after a single exposure to ethane dimethanesulphonate (EDS). Since EDS is known to decrease serum testosterone (T), an additional group of rats received T-filled implants just prior to dosing to determine if the decrease in serum T alone had an effect on sperm motility. The results of the study yielded strikingly different interpretations of the effect of a 65 mg/kg BW dose of EDS on the motility of sperm taken from the proximal cauda epididymidis. Sperm collected by "aspiration" showed no significant decrease in the percentage of motile or progressively motile sperm compared to vehicle-treated animals. On the contrary, sperm collected by "diffusion" showed large, significant decreases in the percentages of both motile and progressively motile sperm. This difference was due largely to lower percentages of motile and progressively motile sperm in control sperm samples collected by aspiration. Similarly, the motion parameters of sperm collected by the aspiration method were unaffected by EDS/T treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2006

Computer-assisted analysis of sperm motion in goats and its relationship with sperm migration in cervical mucus.

In vitro sperm migration in cervical mucus relates to sperm concentration at the utero-tubal junction and to in vivo fertilization performance in goats. The present study aimed to characterize, using Computer-Assisted Sperm Analysis (CASA), motility patterns depicted by buck sperm and their relation to the migration efficiency in homologous (goat) and heterologous (heifer) cervical mucus in vitro. Semen was collected from 23 sexually mature bucks from three breeds by artificial vagina and sperm were assessed for motility parameters with a Hobson Sperm analyzer following extension in Sperm Analysis Medium (SAM). To study the relationship between kinematics parameters and the ability of sperm to migrate in cervical mucus, in a first experiment, motility performance of buck sperm suspended in SAM was compared against seminal plasma. In a second experiment, kinematics parameters of sperm were characterized. In a third experiment, bucks with sperm that differed in specific motion parameters were compared for the ability of their sperm to migrate through goat and bovine cervical mucus collected at estrus. In a fourth experiment, ejaculates that were compared in their migration ability and were assessed simultaneously for their motility parameters. Overall, sperm suspended in SAM medium had better velocity and similar linearity and lateral head displacement than those suspended in seminal plasma; furthermore, caprine sperm swam relatively fast (relative to bovine and ovine sperm), following a very linear trajectory. Under the conditions used, velocity parameters, linearity and lateral head displacement seemed to be related to sperm migration efficiency in homologous mucus but not in bovine cervical mucus.

Animals↗

PubMed2004

Percoll gradient-centrifuged capacitated mouse sperm have increased fertilizing ability and higher contents of sulfogalactosylglycerolipid and docosahexaenoic acid-containing phosphatidylcholine compared to washed capacitated mouse sperm.

Although Percoll gradient centrifugation has been used routinely to prepare motile human sperm, its use in preparing motile mouse sperm has been limited. Here, we showed that Percoll gradient-centrifuged (PGC) capacitated mouse sperm had markedly higher fertilizing ability (sperm-zona pellucida [ZP] binding and in vitro fertilization) than washed capacitated mouse sperm. We also showed that the lipid profiles of PGC capacitated sperm and washed capacitated sperm differed significantly. The PGC sperm had much lower contents of cholesterol and phospholipids. This resulted in relative enrichment of male germ cell-specific sulfogalactosylglycerolipid (SGG), a ZP-binding ligand, in PGC capacitated sperm, and this would explain, in part, their increased ZP-binding ability compared with that of washed capacitated sperm. Analyses of phospholipid fatty acyl chains revealed that PGC capacitated sperm were enriched in phosphatidylcholine (PC) molecular species containing highly unsaturated fatty acids (HUFAs), with docosahexaenoic acid (DHA; C22: 6n-3) being the predominant HUFA (42% of total hydrocarbon chains of PC). In contrast, the level of PC-HUFAs comprising arachidonic acid (20:4n-6), docosapentaenoic acid (C22:5n-6), and DHA in washed capacitated sperm was only 27%. Having the highest unsaturation degree among all HUFAs in PC, DHA would enhance membrane fluidity to the uppermost. Therefore, membranes of PGC capacitated sperm would undergo fertilization-related fusion events at higher rates than washed capacitated sperm. These results suggested that PGC mouse sperm should be used in fertilization experiments and that SGG and DHA should be considered to be important biomarkers for sperm fertilizing ability.

Animals↗

PubMed1986

Motility characteristics of sperm from the uterus and oviducts of female mice after mating to congenic males differing in sperm transport and fertility.

Motile sperm were videotaped after removal from the uterus and isthmus of the oviducts of female mice 1 h after mating with congenic males carrying none, one, or two t complexes. Males carrying one t complex (tw32/+) are fertile, and sperm carrying the t complex have an advantage in fertilization; males carrying two complexes (tw32/t0) are sterile. For each sperm, 2 sec of movement of the head-midpiece junction were traced from the videotape. For each tracing, five motility parameters were used: curvilinear velocity (Vc), and index of the sperm's mean swimming speed; coefficient of variation of move length (CVML), an index of speed constancy; progressiveness ratio (PR), an index of all deviation of the sperm's movement from a straight line; linear index (LI), an index of the straightness of the sperm's trajectory; and curvilinear progressiveness ratio (PRc), an index of the degree of lateral oscillation about that trajectory. Uterine sperm from fertile males were progressive, with straight trajectories and little lateral oscillation. There were no consistent differences in any motility parameter between uterine sperm from tw32/+ and congenic +/+ males. Uterine sperm from sterile tw32/t0 males were extremely slow and showed very little progressive movement, which could explain their lack of transport to the oviduct. For all fertile males, isthmic oviductal sperm differed significantly from uterine sperm in every motility parameter except Vc: isthmic sperm were less consistent in swimming speed, and less progressive with less straight trajectories and more lateral movement. One or more of these motility characteristics may be related to hyperactivation. A large proportion of isthmic sperm from tw32/+ males had nonlinear trajectories (LI less than .50); these nonlinear sperm were faster than nonlinear isthmic sperm from congenic +/+ males. These motility characteristics of isthmic sperm from tw32/+ males may be related to hyperactivation, or to their previously observed abnormal transport within the oviduct.

Animals↗

PubMed1988

Interspecies differences in the stability of mammalian sperm nuclei assessed in vivo by sperm microinjection and in vitro by flow cytometry.

To assess the structural stability of mammalian sperm nuclei and make interspecies comparisons, we microinjected sperm nuclei from six different species into hamster oocytes and monitored the occurrence of sperm nuclear decondensation and male pronucleus formation. The time course of sperm decondensation varied considerably by species: human and mouse sperm nuclei decondensed within 15 to 30 min of injection, and chinchilla and hamster sperm nuclei did so within 45 to 60 min, but bull and rat sperm nuclei remained intact over this same period of time. Male pronuclei formed in oocytes injected with human, mouse, chinchilla, and hamster sperm nuclei, but rarely in oocytes injected with bull or rat sperm nuclei. However, when bull sperm nuclei were pretreated with dithiothreitol (DTT) in vitro to reduce protamine disulfide bonds prior to microinjection, they subsequently decondensed and formed pronuclei in the hamster ooplasm. Condensed rat spermatid nuclei, which lack disulfide bonds, behaved similarly. The same six species of sperm nuclei were induced to undergo decondensation in vitro by treatment with DTT and detergent, and the resulting changes in nuclear size were monitored by phase-contrast microscopy and flow cytometry. As occurred in the oocyte, human sperm nuclei decondensed the fastest in vitro, followed shortly by chinchilla, mouse, and hamster and, after a lag, by rat and bull sperm nuclei. Thus species differences in sperm nuclear stability exist and appear to be related to the extent and/or efficiency of disulfide bonding in the sperm nuclei, a feature that may, in turn, be determined by the type(s) of sperm nuclear protamine(s) present.

Animals↗

PubMed1997

Expression of CD15 (Lewisx) antigen on human sperm and its role in sperm-egg interaction.

PROBLEM: The carbohydrate epitope 3-fucosyl-N-acetyllactosamine (CD15) is a constituent of cell surface glycoconjugates that has been implicated in cell-cell adhesion mediated by carbohydrate-specific ligands. The present study was designed to investigate whether CD15 is present on human sperm and whether it plays a role in human sperm-egg interaction. METHODS: Fluorescent flow cytometry was used to quantitate the binding of monoclonal antibodies (mAb) to sperm-bound CD15 and CD46 antigens on acrosome-intact (AI) and acrosome-reacted (AR) sperm. The location of the binding site of these mAbs was assessed by fluorescence microscopy. The effects of anti-CD15 and anti-CD46 mAbs on gamete interaction were tested utilizing both homologous (human zona binding and penetration) and heterologous (zona-free hamster egg binding and penetration) systems. RESULTS: The mean percentage of capacitated sperm which bound anti-CD15 or anti-CD46 mAbs was low (4.8% and 5.1%, respectively). Exposure to calcium ionophore A23187 (CaI) resulted in an increase in anti-CD15 (38.6 +/- 4%) and anti-CD46 (83.4 +/- 2%) binding to sperm. Both anti-CD15 and anti-CD46 binding sites were localized by fluorescence microscopy on the sperm acrosomal region. In four experiments, the percent of zona-free hamster eggs penetrated by human sperm were medium control 93% (62/66), irrelevant mAb 74% (70/94), anti-CD46 0% (0/107), and anti-CD15 10% (9/90). One hundred percent (6/6) of human zona were penetrated by human sperm exposed to medium control, 88% (8/9) following exposure to irrelevant mAb, 0% (0/11) following exposure to anti-CD46, and 50% (5/10) following exposure to anti-CD15. The mean (+/- SD of tightly bound sperm to hamster eggs were medium control 57 +/- 18%, irrelevant mAb 64 +/- 16%, anti-CD46 37 +/- 13%, and anti-CD15 19 +/- 10%. The corresponding values for human zona were: medium control 118 +/- 14%, irrelevant mAb 61 +/- 11%, anti-CD46 39 +/- 18%, and anti-CD15 99 +/- 19%. CONCLUSION: CD15 antigen is expressed on human sperm that have undergone acrosomal loss. mAb to CD15 was shown to inhibit significantly sperm binding and penetration of zona-free hamster eggs and penetration of human zona pellucida. These findings suggested that sperm-egg interaction may be mediated in part by the CD15 antigen. Capsule: Acrosome-reacted human sperm bind monoclonal antibodies specific for CD15 (Lewis(x)) epitope. The binding sites were located on the sperm head. Anti-CD15 antibody impaired both the binding and penetration of zona-free hamster eggs and the penetration of human zona by human sperm.

Acrosome↗

PubMed1993

A role for the WH-30 protein in sperm-sperm adhesion during rouleaux formation in the guinea pig.

Mammalian spermatozoa participate in specific cell adhesion phenomena during their development and functional lifespan; this includes interaction with Sertoli cells, the zona pellucida, and the oolemma. In some species such as the guinea pig, an additional sperm-sperm adhesion occurs during epididymal maturation which results in the formation of rouleaux in which the sperm heads are stacked one upon the other and the periacrosomal plasma membranes of adjacent sperm are linked by periodic cross-bridges. In this study, we have used a monoclonal antibody to investigate the role of the WH-30 protein on the sperm surface in the formation of the junctional zones between adjacent guinea pig sperm in rouleaux. WH-30 monoclonal antibodies caused a dose- and time-dependent dissociation of rouleaux and an increase in the percentage of single, acrosome-intact sperm; there were no effects on sperm motility (maintained at 80-90%) or ultrastructure during the 120-min incubations. The maximal effect of about 80% single sperm was obtained with a 1:4 dilution of the WH-30 hybridoma supernatant or 5-50 micrograms/ml of purified WH-30 IgG. In contrast, incubation of sperm in AH-20 IgG, myeloma cell supernatants, or purified, nonspecific mouse IgG1 had no effect on rouleaux. Treatment of sperm with a WH-30 Fab fragment resulted in almost complete dissociation of rouleaux without any observed effect on sperm motility or acrosomal status. Surface labeling of sperm followed by immunoprecipitation and SDS-PAGE revealed that the WH-30 antibody recognizes a single polypeptide of 43-45 kDa. Using immunofluorescence, the WH-30 protein was localized over the entire surface of the sperm head (whole-head pattern), and immunogold labeling showed that WH-30 is localized in the glycocalyx on both the dorsal and ventral surfaces of the periacrosomal and postacrosomal plasma membranes. These results indicate that the WH-30 protein on the sperm surface is a cell adhesion protein which is involved in the molecular interactions that maintain guinea pig sperm in rouleaux.

Animals↗

PubMed2001

Significance of peristaltic squeezing of sperm bundles in the silkworm, Bombyx mori: elimination of irregular eupyrene sperm nuclei of the triploid.

Silkworm (Lepidoptera) males produce dimorphic sperm: nucleate eupyrene sperm and anucleate apyrene sperm. The eupyrene sperm are ordinary sperm to fertilise the eggs, while the function of apyrene sperm remains uncertain. After meiosis, 256 sperm cells are enclosed by a layer of cyst cells, forming a sperm bundle. We have previously documented that the nucleus of eupyrene sperm anchors to the head cyst cell, which locates at the anterior apex of the bundle, by an acrosome tubule-basal body assembly. Neither the basal body attachment to the nucleus nor the acrosome is seen in apyrene sperm, and the nuclei remain in the middle region of the bundle. Peristaltic squeezing starts from the anterior of the bundles in both types of sperm, and cytoplasmic debris of the eupyrene sperm, and both the nuclei and debris of apyrene sperm, are eliminated at the final stage of spermatogenesis. Since the irregularity of meiotic division in apyrene sperm is known, we used triploid silkworm males that show irregular meiotic division even in eupyrene spermatocytes and are highly sterile. The irregular nuclei of the triploid are discarded by the peristaltic squeezing just as those of the apyrene sperm. Transmission electron microscopic observations disclose the abnormality in the acrosome tubule and in the connection to the basal body. The peristaltic squeezing of sperm bundles in the silkworm appears to be the final control mechanism to eliminate irregular nuclei before they enter female reproductive organs.

Animals↗

PubMed2007

Sperm competition games: optimal sperm allocation in response to the size of competing ejaculates.

Sperm competition theory predicts that when males are certain of sperm competition, they should decrease sperm investment in matings with an increasing number of competing ejaculates. How males should allocate sperm when competing with differently sized ejaculates, however, has not yet been examined. Here, we report the outcomes of two models assuming variation in males' sperm reserves and males being faced with different amounts of competing sperm. In the first 'spawning model', two males compete instantaneously and both are able to assess the sperm competitive ability of each other. In the second 'sperm storage model', males are sequentially confronted with situations involving different levels of sperm competition, for instance different amounts of sperm already stored by the female mating partner. In both of the models, we found that optimal sperm allocation will strongly depend on the size of the male's sperm reserve. Males should always invest maximally in competition with other males that are equally strong competitors. That is, for males with small sperm reserves, our model predicts a negative correlation between sperm allocation and sperm competition intensity, whereas for males with large sperm reserves, this correlation is predicted to be positive.

Animals↗

PubMed2005

The evolution of sperm-allocation strategies and the degree of sperm competition.

The prevailing viewpoint in the study of sperm competition is that male sperm-allocation strategies evolve in response to the degree of sperm competition an ejaculate can expect to experience within a given mating. If males cannot assess the degree of sperm competition their ejaculate will face and/or they are unable to facultatively adjust sperm investment in response to perceived levels of competition, high sperm allocation (per mating) is predicted to evolve in the context of high sperm competition. An implicit assumption of the framework used to derive this result is that the degree of sperm competition is unaffected by changes in sperm-allocation strategies. We present theory based on an alternative perspective, in which the degree of sperm competition and the sperm-allocation strategy are coupled traits that coevolve together. Our rationale is that the pattern of sperm allocation in the population will, in part, determine the level of sperm competition by affecting the number of ejaculates per female in the population. In this setting, evolution in sperm-allocation strategies is driven by changes in underlying environmental parameters that influence both the degree of sperm competition and sperm allocation. This change in perspective leads to predictions that are qualitatively different from those of previous theory.

Animals↗

PubMed2004

How the sperm lost its tail: the evolution of aflagellate sperm.

The typical sperm is comprised of a head, midpiece and flagellum. Around this theme there is an enormous diversity of form--giant sperm, multi-flagellate sperm and also sperm that lack flagella entirely. Explaining this diversity in sperm morphology is a challenging question that evolutionary biologists have only recently engaged in. Nonetheless, one of the selective forces identified as being an important factor in the evolution of sperm form is sperm competition, which occurs when the sperm of two or more males compete to fertilize a female's ova. In species with a truly monandrous mating system, the absence of sperm competition means that the selection pressure on males to produce motile sperm may be relaxed. Potentially aflagellate sperm are less costly to produce, both in terms of energy and time. Thus, selection may therefore favour the loss of the sperm flagellum and any other motile mechanisms in monandrous taxa. A review of the literature revealed that 36 taxonomic groups, from red algae to fish, were found independently to have evolved aflagellate sperm. I review what is known about the mating systems of each of these taxa and their nearest sister taxa. A sister-group analysis using this information provided weak evidence suggesting that the evolution of aflagellate sperm could be linked to the removal of selective pressures generated by sperm competition.

Animals↗

PubMed1997

Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 minutes after injection as a result of the sperm-oocyte interaction.

We have previously shown that sperm plasma membrane damage makes the sperm plasma membrane permeable and the sperm nucleus accessible for low molecular weight molecules such as eosin and dithiothreitol. In the present study, we investigated whether this damage is associated with a passive release of the sperm-associated oocyte activating factor (SAOAF) from the spermatozoon and, if so, its time sequence. In a first study, human oocytes remaining unfertilized after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and freshly ovulated mouse oocytes were injected with a whole spermatozoon or a sperm head respectively. They were randomly allocated to one of three groups: oocytes in group 1 were injected with a spermatozoon immobilized or sperm head detached immediately prior to the injection; oocytes in group 2 were injected with a spermatozoon immobilized or sperm head detached 2-4 h before injection; oocytes in group 3 were injected with a spermatozoon or sperm head that had been subjected to heat treatment. The activation rate of oocytes injected with a spermatozoon or sperm head was the same for groups 1 and 2, and significantly higher than in group 3 (P < 0.001). In a second series of experiments, human oocytes remaining unfertilized after IVF or ICSI were injected with a sperm head that was subsequently removed from the ooplasm 20-30 min after injection. The activation rates were compared to that of oocytes injected with heat-treated spermatozoa which subsequently were removed from the ooplasm. We found that the removal of the spermatozoon 30 min after injection did not prevent oocyte activation. Our data indicate that the initial damage to the sperm plasma membrane induced at immobilization, although essential for the onset of sperm nuclear swelling after ICSI, does not by itself lead to the release of SAOAF from the spermatozoon. We postulate, however, that SAOAF is released during the sperm nuclear swelling phase, which is induced by the so-called sperm nucleus decondensing factor (SNDF) of the oocyte.

Animals↗

PubMed1987

Effects of exposing bovine sperm to bovine serum albumin, or freeze-thawing, on sperm-bound amidase activity.

Isolation of a self-selected population of motile spermatozoa is possible by using a gradient of bovine serum albumin (BSA). We determined if exposure to BSA altered the sperm or if isolated sperm differed from nonisolated cells in terms of motility or activity of sperm-bound amidase, either before or after subsequent cryopreservation. Exposure of sperm to 6% BSA in egg yolk Tris extender induced changes in the plasma and acrosomal membranes of sperm that resulted in exposure and activation of sperm-bound amidase (P less than .01). In experiment 2, semen extended in egg yolk Tris was cooled to 5 degrees C or layered onto a solution of 6% BSA in extender at 37 degrees C, from which the sperm that had swum into the BSA solution were recovered 2 h later and cooled to 5 degrees C. Sperm in both treatments were cryopreserved. The percentage of progressively motile sperm was determined visually and by track motility. Activity of sperm-bound amidase exposed to substrate was evaluated. After recovery of sperm from the 6% BSA solution, 81% were progressively motile as compared to 59% in the starting samples (P less than .01). However, the amount of exposed sperm-bound amidase also was greater (P less than .05); this was a deleterious change. Immediately after thawing, more (P less than .01) sperm were motile in samples of isolated sperm than for nonisolated cells (43 vs 24%), but after incubating the thawed sperm for 1 h at 37 degrees C there was no difference.(ABSTRACT TRUNCATED AT 250 WORDS)

Amidohydrolases↗

PubMed2004

Macaque sperm release ESP13.2 and PSP94 during capacitation: the absence of ESP13.2 is linked to sperm-zona recognition and binding.

ESP13.2 coats the entire surface of macaque sperm and remains until sperm become capacitated (Yudin et al., 2003: Biol Reprod 69: 1118-1128). Capacitation of macaque sperm is synchronized by treatment with dibutyrl cAMP (dbcAMP) and caffeine. ESP13.2 and PSP94 constituted approximately 95% of the proteins released from the sperm surface following treatment with caffeine + dbcAMP. Caffeine and dbcAMP alone induce different patterns of ESP13.2 release. As determined by ELISAs of supernatants and immuno-fluorescent labeling of sperm heads, caffeine alone and caffeine + dbcAMP induced comparable release of ESP13.2, while dbcAMP-treated sperm did not differ from controls. Sperm treated with caffeine + dbcAMP showed a reduction of ESP13.2 from the entire surface, while caffeine treatment alone induced removal of ESP13.2 from the sperm head and midpiece. As confirmed with immunofluorescence, ESP13.2 could be added back to the surfaces of sperm that had been previously exposed to caffeine. Treatment with caffeine significantly increased the number of sperm that bound tightly to the zona pellucida as compared with controls (42 +/- 9 and 13 +/- 3 sperm/zona, respectively; P < or = 0.01). This increase in binding was inhibited by "adding back" ESP13.2 to the sperm surface (12.8 +/- 3; P < or = 0.01). Alexa-conjugated anti-ESP13.2 Ig labeling of live sperm showed that only sperm lacking ESP13.2 over the head were capable of tight binding to the zona. Our results suggest that ESP13.2 masks zona pellucida ligands on the sperm surface and its release, as part of capacitation, is required for sperm-zona interaction.

Amino Acid Sequence↗

PubMed1996

In utero and lactational exposure of the male Holtzman rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin: decreased epididymal and ejaculated sperm numbers without alterations in sperm transit rate.

Decreased daily sperm production (DSP) and cauda epididymal sperm number (CESN) are some of the most sensitive effects of in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. The reduction in CESN cannot be fully accounted for by decreased spermatogenesis. To explain the decrease in CESN it was hypothesized that TCDD exposure increases the rate of sperm transit through the excurrent duct system, thereby decreasing the number of sperm in the system at any given time. Pregnant Holtzman rats were administered a single dose of TCDD (1.0 microgram/ kg,po) or vehicle on gestation day 15 and offspring were weaned on postnatal day (PND) 21. On PND 50, testicular sperm were labeled in five males per litter, from 30 control and 26 TCDD-exposed litters, by injecting 15 microCi [3H]thymidine into each testis, under general anesthesia. Sperm movement through the excurrent duct system was monitored daily 35-64 days post [3H]thymidine injection. On PNDs 92-93, TCDD exposure significantly decreased DSP/testis, corpus and cauda epididymis sperm numbers, vas deferens sperm number, and ejaculated sperm number by 28, 30, 36, 39, and 46%, respectively. The decreases in sperm number in the distal excurrent duct system were greater than the decrease in DSP, consistent with the hypothesis that TCDD exposure causes an effect other than decreased DSP that reduced epididymal and ejaculated sperm numbers. However, in utero and lactational TCDD exposure did not alter radiolabeled sperm transit time through the whole epididymis (15 days). With TCDD exposure causing no obvious alteration in sperm transit rate, a plausible explanation for the sperm loss is an increase in sperm phagocytosis in the excurrent duct system.

Animals↗

PubMed2005

Simple determination of human sperm DNA fragmentation with an improved sperm chromatin dispersion test.

OBJECTIVE: To improve the sperm chromatin dispersion (SCD) test and develop it as a simple kit (Halosperm kit) for the accurate determination of sperm DNA fragmentation using conventional bright-field microscopy. DESIGN: Method development, comparison, and validation. SETTING: Medical genetics laboratory, academic biology center, and reproductive medicine centers. PATIENT(S): Male infertility patients attending the Reproductive Medicine Center. A varicocele patient and a group of nine fertile subjects. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): [1] The quality of chromatin staining in relaxed sperm nuclear halos and tail preservation; [2] SCD scoring reproducibility; [3] comparison with the sperm chromatin structure assay in 45 samples; [4] frequency of sperm with DNA fragmentation after incubation with increasing doses of the nitric oxide donor sodium nitroprusside and in sperm samples for 9 fertile men, 46 normozoospermic patients, 23 oligoasthenoteratozoospermic patients, and a subject with varicocele. RESULT(S): The sperm nuclei with DNA fragmentation, either spontaneous or induced, do not produce or show very small halos of DNA loop dispersion after sequential incubation in acid and lysis solution. The improved SCD protocol (Halosperm kit) results in better chromatin preservation, therefore highly contrasted halo images can be accurately assessed using conventional bright-field microscopy after Wright staining. Moreover, unlike in the original SCD procedure, the sperm tails are now preserved, making it possible to unequivocally discriminate sperm from other cell types. The chi2 test did not detect significant differences in the mean number of sperm cells with fragmented DNA as scored by four different observers. The intraobserver coefficient of variation for the estimated percentage of spermatozoa with fragmented DNA ranged from 6% to 12%. There was good correlation between the SCD and the sperm chromatin structure assay DNA fragmentation index (intraclass correlation coefficient R: 0.85; percent DNA fragmentation index mean difference: 2.16 significantly higher for SCD). Using the Halosperm kit, a dose-dependent increase in sperm DNA damage after sodium nitroprusside incubation was detected. The percentage of sperm cells with fragmented DNA in the fertile group was 16.3 +/- 6.0, in the normozoospermic group, 27.3 +/- 11.7, and in the oligoasthenoteratozoospermic group, 47.3 +/- 17.3. In the varicocele sample, an extremely high degree of nuclear disruption was detected in the population of sperm cells with fragmented DNA. CONCLUSION(S): The improved SCD test, developed as the Halosperm kit, is a simple, cost effective, rapid, reliable, and accurate procedure, for routinely assessing human sperm DNA fragmentation in the clinical andrology laboratory.

Chromatin↗