Acrosome reaction of boar spermatozoa in homologous in vitro fertilization

Ejaculated spermatozoa from four different boars were used to evaluate the acrosome reaction during in vitro fertilization with homologous ovulated oocytes. The acrosome reaction was assessed according to a peroxidase-labeling peanut agglutinin method and a triple-stain technique. An increase in the proportion of living sperm with reacted acrosomes was observed after preincubation and 2 hr of coincubation (P < 0.05). The percentage of true acrosome-reacted sperm remained reasonably constant throughout coincubation. In vitro penetration rates of oocytes varied among boars, but no relationship was found between fertilization rates of oocytes and maximum percentages of acrosome reacted living sperm. © 1993 Wiley-Liss, Inc.     

Proteomic analysis of the mouse sperm acrosome - towards an understanding of an organelle with diverse functionality

The acrosome located within the mammalian sperm head is essential for successful fertilization, as it enables the sperm to penetrate the extracellular layers of the oocyte and fuse with oolemma. However, the mammalian acrosomal vesicle is no longer considered to contain only hydrolytic enzymes. Using label-free nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS) proteomics, we identified a total of 885 proteins in the acrosome isolated from spermatozoa obtained from cauda epididymis of free-living house mice Mus musculus musculus contains a total of 885 proteins. Among these, 334 proteins were significantly enriched in the acrosome thus representing 27.3% of the whole proteome of the intact sperm. Importantly, we have detected a total of nine calycins while eight of them belong to the lipocalin protein family. In mice, lipocalins are involved in multi-level chemical communication between individuals including pheromone transport and odor perception. Using an indirect immunofluorescence assay, we demonstrated that lipocalin 5 (LCN5) is expressed in the mouse germ cells, and after completing spermatogenesis, it remains localized in the sperm acrosome until the last step of the extratesticular maturation, the acrosome reaction. The presence of lipocalins in the acrosome and acrosome-reacted sperm suggests their original role as chelators of organic and potentially toxic compounds resulting from ongoing spermiogenesis. Along with this evidence, detected mitochondrial (e.g., a subunit of the cytochrome c oxidase MTCO1) and proteasomal proteins (subunits of both 20 S core proteasome [PSMA2, PSMBs] and 19 S regulatory particle [PSMDs]) in acrosomes provide further evidence that acrosomes could also function as `waste baskets` after testicular sperm maturation.     

A mutation in the lipase H (LIPH) gene underlie autosomal recessive hypotrichosis

Hereditary hypotrichosis is a rare autosomal recessive disorder characterized by sparse hair on scalp and rest of the body of affected individuals. Two forms of such hypotrichosis LAH and AH have been mapped on chromosome 18q12.1 and 3q27, respectively. Mutations in desmogelin 4 (DSG4) gene have been reported to underlie LAH. Recently, a deletion mutation in Lipase H (LIPH) gene, located at AH locus, has been identified in two ethnic groups of Russian population. In the present study, a four generation Pakistani family with AH phenotype has been mapped to chromosome 3q27. Sequence analysis of candidate gene LIPH revealed a novel five base pair deletion mutation (c.346–350delATATA) in exon 2 of the gene leading to frameshift and downstream premature termination codon. The mutation reported in the family, presented here, is the second mutation identified in LIPH gene. The identification of a genetic defect in LIPH suggests that this enzyme regulates hair growth.     

Lead chloride affects sperm motility and acrosome reaction in mice

Lead is highly toxic and persistent in the environment and, thus, a major concern for public health. In this study, the effects of lead chloride (PbCl₂) on mouse epididymal sperm were evaluated. Male mice were subcutaneously injected with 74 and 100 mg PbCl₂/kg body weight for four consecutive days. Sperm was collected from the epididymis and several parameters of sperm function, such as sperm density, motility, viability, mitochondrial function, acrosome integrity and morphology, were evaluated. Furthermore, DNA fragmentation was assessed by the terminal deoxylnucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) assay and chromatin integrity was evaluated by sperm chromatin structure assay (SCSA). In order to assess direct effects on existing sperm population, we sacrificed one group for each condition at day 5. The effects of lead upon one entire spermatogenic cycle were evaluated on day 35. Both lead concentrations used in this work affected sperm motility, although no significant differences were observed in sperm viability, mitochondrial function and DNA/chromatin integrity. However, a decrease in the percentage of intact acrosomes was also observed, mirroring a lead-induced premature acrosome reaction. Thus, the results obtained indicate that, together with impaired motility, the effect of lead toxicity on acrosome integrity, leading to premature reaction, may compromise the ability of sperm to fertilize the oocyte.     

ACROSOME BREAKDOWN IN LEPTODACTYLUS CHAQUENSIS (AMPHIBIA ANURA) SPERMATOZOA*

Acrosome breakdown in Leptodactylus chaquensis is described: during this process acrosome enlarges, becomes round-shaped and finally disrupts. Low tonicity media (0.025 M sucrose and 1/10 Holtfreter's solutions) favor acrosome breakdown and sperm fertility loosing. High tonicity media (0.250 M sucrose and Holtfreter's solutions) maintain acrosomes in an unreacted stage and sperm fertilizing capacity is preserved. Sperm motility does not seem to be a sufficient condition for the sperm to fertilize and also does not seem to be related with acrosome breakdown. The presence of lectins in the incubation media does not modify the time-course of acrosome breakdown.     

Zona-penetration in vitro test for evaluating boar sperm fertility

We investigated the capacity of porcine sperm-zona binding and penetration by using bioassay to differentiate between spermatozoa from fertile and subfertile boars. Semen was collected from Large White boars grouped into categories of fertile and subfertile (n=5 per each group) according to the results of artificial insemination. Boars in both groups showed similarly hyperactivated sperm motility at insemination (44.72 and 43.03% respectively) regardless of the lower percentage of progressive motility observed in the ejaculates of subfertile boars. At in vitro insemination, a high proportion of the sperm population (43.76%) in the subfertile boars was without acrosomes, while in the fertile boars this proportion was only 24.35%. The sperm penetration rate of fertile boars reached 66.03% while that of subfertile boars was only 25.08%. In conclusion, the results of our study showed that the penetration rate by boar spermatozoa of the zona pellucida can be used to predict fertility and/or as an in vitro standard for describing porcine semen characteristics.     

Influence of Boar and Semen Parameters on Motility and Acrosome Integrity in Liquid Boar Semen Stored for Five Days

Ninety ejaculates from a total of 76 AI boars were extended in Beltsville Thawing Solution (BTS). Boar identity, breed, weight of the ejaculate and sperm concentration were registered. Motility and acrosome integrity were assessed after storage at 16–18°C for 6, 30, 54, 78, and 102 h. Storage time had a significant influence on both motility (p < 0.01) and acrosome integrity (p < 0.001). The Least Square Means for percentage of motility showed a small decline from 79.8% after 6 h of storage to 78.4% at 102 h. Motility at 78 and 102 h was significantly different from motility at 6 h (p < 0.05). The percentage of sperm cells with normal acrosomes declined throughout the experiment. The Least Square Means for 6, 30, 54, 78, and 102 h of storage were 93.9%, 90.6%, 88.0%, 84.8%, and 78.2%, respectively. The decrease in acrosome integrity from one storage time to the next was highly significant throughout the trial (p < 0.001). There was a significant influence of boar (p < 0.001) and sperm concentration (p < 0.01) on motility, while acrosome integrity was affected only by boar (p < 0.001). Breed of the boars and weight of the ejaculate did not influence the dependent variables.     

Effect of the knobbed acrosome defect in bovine sperm on IVF and embryo production

An IVF and culture system was used to determine the effect of the knobbed acrosome defect in bovine spermatozoa on fertilization and early embryonic development. Three bulls affected with knobbed acrosomes were identified as K+ (flattened acrosome), K2+ (indented acrosome) or K3+ (deep indentation of the acrosome) based on the predominant type of acrosomal aberration present in sperm of the respective bulls. After swim-up, all semen traits, except for acrosome morphology, were similar between bulls with varying degrees of the knobbed acrosome defect and a control bull, C. The mean number of spermatozoa bound to the zona pellucida was lower (P< 0.05) for the bulls with the knobbed acrosome defects (40.3 +/- 2.3, 29.5 +/- 1.6, 14.6 +/- 1.3, respectively, for Bulls K+, K2+ and K3+) than for Bull C (52.3 +/- 2.3). The percentages of zonae pellucidae penetrated by spermatozoa from Bulls K+ (51.2%), K2+ (49.5%) and K3+ (37.1%) were lower than that of Bull C (84.5%). No sperm with knobbed acrosome defects were found to have penetrated the zona pellucida. Fertilization rates for bulls with the knobbed acrosome defect, K+ (63.0%), K2+ (62.7%) and K3+ (22.6%), were significantly lower than that of the control bull (82.8%). Percentages of cleaved embryos, morulae and blastocysts produced were also lower for the bulls with knobbed acrosomes than that of the control bull. Results indicate that sperm with the knobbed acrosome defect had a reduced ability to bind to the zona pellucida, depending upon the severity of the defect, and that these aberrant spermatozoa did not penetrate the zona pellucida. The apparently normal spermatozoa coexisting in the inseminate of bulls with a high percentage of knobbed spermatozoa were also functionally deficient; oocytes penetrated by these spermatozoa had a reduced potential for fertilization, and resulting zygotes had a reduced ability for cleavage and embryonic development to the blastocyst stage. The results of the present study do not support the hypotheses that the knobbed acrosome defect is compensable.     

Partial Deletion of Chromosome 8 β-defensin Cluster Confers Sperm Dysfunction and Infertility in Male Mice

β-defensin peptides are a family of antimicrobial peptides present at mucosal surfaces, with the main site of expression under normal conditions in the male reproductive tract. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. We show here that homozygous deletion of a cluster of nine β-defensin genes (DefbΔ9) in the mouse results in male sterility. The sperm derived from the mutants have reduced motility and increased fragility. Epididymal sperm isolated from the cauda should require capacitation to induce the acrosome reaction but sperm from the mutants demonstrate precocious capacitation and increased spontaneous acrosome reaction compared to wild-types but have reduced ability to bind the zona pellucida of oocytes. Ultrastructural examination reveals a defect in microtubule structure of the axoneme with increased disintegration in mutant derived sperm present in the epididymis cauda region, but not in caput region or testes. Consistent with premature acrosome reaction, sperm from mutant animals have significantly increased intracellular calcium content. Thus we demonstrate in vivo that β-defensins are essential for successful sperm maturation, and their disruption leads to alteration in intracellular calcium, inappropriate spontaneous acrosome reaction and profound male infertility.     

Effects of a high semen-collection frequency on the quality of sperm from ejaculates and from six epididymal regions in boars

This study examines the effect of semen-collection rhythm on the sperm maturation process in boar epididymis. Three post-pubertal boars were submitted to a high semen-collection frequency (stressed boars) during 4 days, and three males were kept as a control group (control boars). Semen samples coming from six epididymal regions and from the ejaculate of each male were evaluated for sperm concentration, sperm vitality, sperm motility and sperm morphology. In each epididymal region, either fluid resorption or fluid secretion was determined from the variation in sperm concentration. The pattern of fluid resorption-secretion along the epididymal duct differed significantly between the stressed and control boars. A high semen-collection frequency also affected the development of sperm motility and the sperm cytoplasmic droplet displacement along the epididymal duct. The incidence of some sperm abnormalities was also found to be higher in some epididymal regions and ejaculates of stressed boars. From the results of this study, it can be concluded that a high semen-collection frequency brings about an altered resorption and secretion pattern of the epididymal fluid, which results in defective sperm maturation and abnormal development of sperm motility.     

Gamete interaction in the white sturgeon Acipenser transmontanus: a morphological and physiological review

Synopsis Sturgeon gametes differ from those of most fish in that the sperm possess acrosomes that undergo exocytosis and filament formation while the eggs possess numerous micropyles. Acipenser transmontanus eggs are encased by multilayered envelopes that consist of outer adhesive jelly coats and three structured layers interior to the jelly. The glycoprotein jelly layer only becomes adhesive upon exposure to freshwater. The layer interior to the jelly, layer 3, is the other carbohydrate-containing component of the egg envelope. This layer consists of a water-insoluble glycoprotein that, upon freshwater exposure, is hydrolyzed by a trypsinlike protease to yield a water-soluble, lower molecular weight carbohydrate-containing component. This component can be identified in the surrounding medium when unfertilized eggs are incubated in freshwater. This egg water component elicits acrosome reactions only in homologous sperm. The A. transmontanus sperm acrosome reaction is a Ca⁺⁺ and/or Mg⁺⁺ dependent event that includes the formation of a 10 μ long fertilization filament. A. transmontanus fertilization can occur at low sperm per egg ratios; however, crossfertilization of A. transmontanus eggs with lake sturgeon, A. fluvescens, sperm results in a very low number of fertilized eggs, even at high sperm per egg ratios. The morphological, physiological, and biochemical phenomenon reviewed in this paper are related to the environment in which they occur. Also, the possible role of the acrosome and the presence of numerous micropyles are discussed.     

A new dispensable genetic locus of the terminus region involved in control of cell division in Escherichia coli

Summary Temperature-sensitive mutants defective in cell division were isolated after localised mutagenesis of the terminus region of the Escherichia coli chromosome. The defective gene in one of these mutants, dicA, was mapped at 34.9 min by linkage with manA and with three physically characterized Tn10 insertions. Temperature-sensitivity conferred by mutation dicA1 in a recA backround was suppressed by the presence of hybrid plasmids carrying the wild-type gene. In addition, the mutation was suppressed either by tranposon inactivation of a nearby gene, dicB, or by deletion of the entire dicA-dicB interval. These results define the dicA-dicB locus as a new dispensable genetic cluster involved in the control of cell division.     

Genetic analysis of hereditary multiple exostoses in Tunisian families: a novel frame-shift mutation in the <Emphasis Type="Italic">EXT1</Emphasis> gene

Hereditary multiple exostoses (HME) is an autosomal dominant orthopaedic disorder most frequently caused by mutations in the EXT1 gene. The aim of the present study is to determine the underlying molecular defect of HME in two multigenerational Tunisian families with 21 affected members and to examine the degree of intrafamilial variability. Linkage analysis was performed using three microsatellite markers encompassing the EXT1 locus and mutation screening was carried out by direct sequencing. In family 1, evidence for linkage to EXT1 was obtained on the basis of a maximum LOD score of 4.26 at θ = 0.00 with D8S1694 marker. Sequencing of the EXT1 revealed a heterozygous G > T transversion (c.1019G>T) in exon 2, leading to a missense mutation at the codon 340 (p.Arg340Leu). In family 2 we identified a novel heterozygous 1 bp deletion in the exon 1 (c.529_531delA) leading to a premature codon stop and truncated EXT1 protein expression (p.Lys177LysfsX15). This mutation was associated with the evidence of an intrafamilial clinical variability and considered to be a novel disease-causing mutation in the EXT1 gene. These findings provide additional support for the involvement of EXT1 gene in the HME disease.     

Disrupted Sperm Function and Fertilin β Processing in Mice Deficient in the Inositol Polyphosphate 5-Phosphatase Inpp5b

Inpp5b is an ubiquitously expressed type II inositol polyphosphate 5-phosphatase. We have disrupted the Inpp5b gene in mice and found that homozygous mutant males are infertile. Here we examine the causes for the infertility in detail. We demonstrate that sperm from Inpp5b^−/− males have reduced motility and reduced ability to fertilize eggs, although capacitation and acrosome exocytosis appear to be normal. In addition, fertilin β, a sperm surface protein involved in sperm-egg membrane interactions that is normally proteolytically processed during sperm transit through the epididymis, showed reduced levels of processing in the Inpp5b^−/− animals. Inpp5b was expressed in the Sertoli cells and epididymis and at low levels in the developing germ cells; however, mice lacking Inpp5b in spermatids and not in other cell types generated by conditional gene targeting, were fully fertile. The abnormalities in mutant sperm function and maturation appear to arise from defects in the functioning of Sertoli and epididymal epithelial cells. Our results directly demonstrate a previously unknown role for phosphoinositides in normal sperm maturation beyond their previously characterized involvement in the acrosome reaction. Inpp5b^−/− mice provide an excellent model to study the role of Sertoli and epididymal epithelial cells in the differentiation and maturation of sperm.     

Novel and traditional traits of frozen-thawed porcine sperm related to in vitro fertilization success

Cryopreserved semen allows the use of single ejaculates for repeated analyses, potentially improving IVF consistency by eliminating interejaculate variability observed with fresh semen. However, the freezing and thawing processes result in compromised sperm function and IVF success. Semen samples are often screened for motility before use for IVF. Samples that are below a designated motility threshold may be discarded. Our objectives were to determine if post-thaw sperm motility, other traits that may be indicative of sperm function, or a novel assay of oviduct binding were related to IVF success. Semen from 16 boars was cooled to 15 °C for overnight shipment before cryopreservation. Semen was thawed and motility was recorded microscopically and confirmed using computer-automated sperm assessment. Each sample was tested by IVF in two to three independent replicates. Regression and correlation analyses were employed to determine the interrelationships between sperm traits and the relationships between post-thaw motility, sperm-oviduct binding and IVF outcomes. Among the sperm traits examined, sperm acrosome integrity was negatively correlated with post-thaw motility (r² = 0.64) but not with IVF results. The number of sperm bound to oviduct aggregates was correlated with IVF polyspermy rates (r² = 0.62, P < 0.05) but less with overall IVF rates (r² = 0.31, P > 0.10). There was some relationship of post-thaw motility with IVF monospermic fertilization (P = 0.06, r² = 0.08) but not to other IVF outcomes. Our results indicate that post-thaw motility of frozen-thawed boar sperm is strongly related to acrosome integrity but has limited use for predicting IVF success. The number of sperm bound to oviduct cells was related to IVF polyspermy rates and may be more indicative of in vitro sperm function than traditional sperm motility and acrosome status evaluation.     

Semen characteristics of the swamp buffalo (Bubalus bubalis)

The semen characteristics were studied in 182 ejaculates collected with a bovine artificial vagina from five swamp buffalo (Bubalus bubalis) bulls. The mean values were: volume, 2.9 ml; general motility, 70.7%; live (unstained) sperm, 86.5%; abnormal sperm, 10.3%; intact acrosomes, 82.4%; sperm concentration, 1.06 × 10⁹cells/ml and total sperm/ejaculate, 3.18 × 10⁹cells/ml. Among the sperm abnormalities noted were “knobbed” acrosome, abaxial implantation, the “Dag” defect and the corkscrew midpiece. There were no significant (P > 0.05) monthly variations for any of the semen characteristics studied.     

Evolution of sperm morphology in potamid freshwater crabs (Crustacea: Brachyura: Potamoidea)

We investigated sperm cells and spermatophores of four species of Old World freshwater crabs belonging to three different genera of the subfamily Potaminae (family Potamidae). Characters previously believed to be apomorphic for the potamid subfamily Potamiscinae were also found to occur in the Potaminae. To infer the morphological ancestral character state combination of the Potamidae, ancestral character state analysis of four different sperm traits was performed, based on a 16S rDNA phylogeny of the investigated species. Comparing molecular phylogeny and character state distribution, several cases of convergent evolution could be identified. The densely packed, coenospermic spermatophores and the occurrence of a ‘tongue‐and‐groove’ connection between operculum and acrosomal zones are probably apomorphies for the whole Potamidae. The spermatozoa of Socotrapotamon socotrense show several unique characters. We also analysed the evolution of acrosome size. The sperm cells of the Potamidae and their sister‐group Gecarcinucidae only slightly overlap in acrosome size. Within the investigated species, the ‘East Asia’ subclade (subfamily Potamiscinae) developed significantly larger acrosomes than the subfamily Potaminae. Our results suggest that the use of brachyuran acrosome morphology for phylogenetic inference at the family level is strongly affected by small sample size, and by convergent character evolution. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010.     

Influence of partial deletion of the Y chromosome on mouse sperm phenotype

Two congenic strains of mice (control, B10.BR/SgSn; mutant, B10.BR-Ydel/Ms with partial deletion of the Y chromosome) were examined. In control males, 22.6% of spermatozoa had abnormal heads; in mutant males, there were 64.2%, the most common being heads with flat acrosomes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of mature sperm proteins, followed by acrosin assay and acrosome silver staining, revealed a reduced concentration of acrosin in acrosomal caps in 35.8% of the spermatozoa in mutant males. Electron microscope analysis showed that some of the round, early spermatids in the mutants had normally formed acrosomal caps but lacked the proacrosomal granule and had no, or only scarce, acrosomal material. These observations indicate that formation of the acrosomal cap is controlled separately from the synthesis of the acrosomal material and suggest that some factors linked on the Y chromosome are involved in the control of acrosome development.