OBJECTIVESTo evaluate the application of Coomassie brilliant blue (CBB) G250 staining for the detection of human sperm deformity rate, rate of intact acrosome and acrosome reaction.

METHODSThe smear of spermatozoa before and after capacitation and induced acrosome reaction with progesterone (P) were stained with 0.05% CBB G250 and Wright-Giemisa solution respectively, and visualized with light microscopy. The deformity rate of spermatozoa, rate of intact acrosome and acrosome reaction were calculated.

RESULTSThere was no any difference in detection of deformity rate of spermatozoa and rate of intact acrosome with CBB G250 and Wright-Giemisa staining(P < 0.05). The sperm population of acrosome reaction with induced P was divided by CBB staining into two types: positive staining with dark violet blue on acrosome cap and pale or negative staining on the same area. The rate of the latter was increasing with increasing inductive time, maybe representative of the rate of acrosome reaction. The mean rate was(75.1 +/- 3.8)% after induced for 1 h.

CONCLUSIONSCBB G250 staining is a reliable method for assessment of the human sperm morphology and acrosome reaction.

Acrosome ; metabolism ; Acrosome Reaction ; Humans ; Male ; Rosaniline Dyes ; chemistry ; metabolism ; Spermatozoa ; cytology ; enzymology ; Staining and Labeling

OBJECTIVETo analyze the quality characteristics of human spermatozoa with hyaluronic acid (HA) receptors and search for a new indicator for the assessment of sperm quality.

METHODSUsing sperm-HA binding assay with HA-coated slides, we determined the binding rate of motile sperm with HA receptors and analyzed its correlation with routine semen parameters, sperm membrane function, sperm fertilizing function and diminished/arrested sperm maturation.

RESULTSThe motile sperm with HA binding sites in the acrosomal region showed significantly higher acrosomal integrity ([95.4 +/- 3.9]%) and mitochondrial membrane potential (MMP) ([97.8 +/- 2.1]%) than those in the initial semen ([68.8 +/- 6.2]% and [72.8 +/- 7.4]%) (P < 0.01). The sperm-HA binding scores were correlated mildly with many routine semen parameters (r = 0.195-0.268, P < 0.05), positively with the acrosome reaction level after ionophore challenge (r = 0.666, P < 0.01) and normal sperm morphology (r = 0.417, P < 0.01), and negatively with sperm nucleoprotein immaturation (r = -0.266, P < 0.01), DNA fragmentation (r = -0. 308, P < 0.01) and excessive residual cytoplasm (r = -0.218, P < 0.05).

CONCLUSIONSperm with HA receptors in the acrosomal region exhibit significant advantages in plasma membrane structure, fertilizing potential and maturation. The sperm-HA binding assay, which is based on a relationship between sperm receptors for zona pellucida and HA, is likely to become a new independent indicator for assessing the multiple qualities of spermatozoa.

Acrosome ; metabolism ; Humans ; Hyaluronan Receptors ; metabolism ; Hyaluronic Acid ; Male ; Spermatozoa ; cytology ; metabolism ; physiology

Sexual reproduction is marked by the fusion of the sperm cell with the oocyte during fertilization to produce the diploid zygote, in which the lipids in the sperm plasma membrane play an important role. Due to the loss of most cell organelles and DNA transcription, spermatozoa lack protein expression and vesicular transport. However, the lipids of the sperm plasma membrane undergo complicated dynamic changes, which may facilitate the capacitation, binding with zona pellucida, acrosome reaction and fusion of the sperm cell with the oocyte. This paper summarizes the progress in the studies of the lipids in the sperm plasma membrane, their composition, structure, peroxidation, metabolism and role in fertilization.
Acrosome Reaction ; Animals ; Cell Membrane ; chemistry ; Fertilization ; Humans ; Male ; Membrane Lipids ; metabolism ; Sperm Capacitation ; Spermatozoa ; chemistry

OBJECTIVESTo study the inhibitory effect of KF-950 on human acrosin and sperm acrosome.

METHODSHuman acrosin was extracted and purified with 2% acetic acid, and its residual activity was evaluated by BAEE/ADH assay after treated with different concentrations of KF-950. ABC assay was used to observe the effect of KF-950 on human acrosome with Biotin-PSA as a probe.

RESULTS1. The activity of normal sperm acrosin was (37.65 +/- 4.47) U/L. 2. The residual activity was inversely related to the concentration of KF-950 (r = -0.998), and had a dose-response curve. The result could be described by Y = 7.57-1.895X. 3. With increase of KF-950 concentration and prolongation of action time, the staining rate of acrosome obviously dropped (P < 0.01).

CONCLUSIONSKF-950 directly inhibits acrosin activity and assumely injures sperm acrosome. It might be a new kind of highly effective inhibitor.

Acrosin ; antagonists & inhibitors ; Acrosome ; metabolism ; Humans ; Male ; Spermatozoa ; drug effects ; physiology

Protein kinase C (PKC) is localized in the equatorial segment and the principal piece of the tail of spermatozoa. Activator of PKC results in increasing flagellar motility of sperm that is blocked by PKC inhibitors such as staurosporine. A good correlation (r = 0.9, P < 0.001) is found between the content of PKC in sperm and sperm motility. Zona pellucida (ZP) stimulates the spermatozoa binding the acrosome reaction resulting in the release of hydrolytic enzymes and in the exposure of new membrane domains. ZP binding to receptors in the plasma membrane can regulate adenyl cyclase (AC) leading to elevation of cAMP and protein kinase A (PKA) activation. The PKA activates a voltage-dependent Ca2+ channel in the outer acrosomal membrane which releases Ca2+ from the interior of the acrosome to the cytosol. Activation of the PLC resulted from the rise in Ca2+ hydrolyze phosphatidyl inositol bisphosphate. The product activate PCK to open a voltage-dependent Ca2+ channel (L) in the plasma membrane, leading to the second (II) Ca2+ higher increase which result in membrane fusion and acrosome reaction. It is proposed that PKC would be involved in the regulation of motility and acrosome reaction of sperm.
Acrosome Reaction ; physiology ; Humans ; Male ; Protein Kinase C ; metabolism ; Sperm Motility ; physiology ; Spermatozoa ; enzymology ; physiology

OBJECTIVETo explore the expression patterns of the chemokine CXCL12 and its receptor CXCR4 in human sperm.

METHODSWe collected semen samples from 10 fertile men, performed density gradient centrifugation, and then determined the expressions of both CXCL12 and CXCR4 in the sperm by RT-PCR and immunofluorescence staining.

RESULTSRT-PCR revealed the mRNA expressions of CXCL12 (0.641 +/- 0.180) and CXCR4 (0.464 +/- 0.100) in the sperm. However, only CXCR4 rather than CXCL12 was expressed at the protein level, and the positive staining for CXCR4 was observed mainly in the posterior part of the acrosome.

CONCLUSIONCXCL12 and CXCR4 are involved as important molecules in regulating the function of human sperm.

Acrosome ; metabolism ; Centrifugation, Density Gradient ; Chemokine CXCL12 ; metabolism ; Humans ; Male ; Receptors, CXCR4 ; metabolism ; Signal Transduction ; Spermatozoa ; metabolism

Prior to fertilization sperm has to undergo an activation process known as capaciation, leading to the acrosome reaction. Till now, little is known about the mechanism for preventing premature capacitation in sperm although decapacitation factors from various sources have been thought to be involved. In this study, we report that NYD-SP27, an isoform of phospholipase C Zeta 1 (PLCZ1), is localized to the sperm acrosome in mouse and human spermatozoa by immunofluorescence using a specific antibody. Western blot and double staining analyses show NYD-SP27 becomes detached from sperm, as they undergo capacitation and acrosome reaction. The absence of HCO3-, a key factor in activating capacitation, from the capacitation-inducing medium prevents the loss of NYD-SP27 from sperm. The anti-NYD-SP27 antibody also prevents the loss of NYD-SP27 from sperm, reduced the number of capacitated sperm, inhibited the acrosome reaction induced by ATP and progesterone, and inhibited agonist-induced PLC-coupled Ca2+ mobilization in sperm, which can be mimicked by the PLC inhibitor, U73122. These data strongly suggest that NYD-SP27 is a physiological inhibitor of PLC that acts as an intrinsic decapacitation factor in sperm to prevent premature capacitation and acrosome reaction.
Acrosome ; drug effects ; metabolism ; Acrosome Reaction ; physiology ; Adult ; Animals ; Calcium ; metabolism ; Fluorescent Antibody Technique, Indirect ; Humans ; Immune Sera ; pharmacology ; Male ; Mice ; Middle Aged ; Phosphoinositide Phospholipase C ; immunology ; metabolism ; Sperm Capacitation ; drug effects ; physiology ; Spermatozoa ; drug effects ; metabolism

Fertilization is a process involving multiple steps that lead to the final fusion of one sperm and the oocyte to form the zygote. One of the steps, acrosome reaction (AR), is an exocytosis process, during which the outer acrosome membrane fuses with the inner sperm membrane, leading to the release of acrosome enzymes that facilitate sperm penetration of the egg investments. Though AR has been investigated for decades, the initial steps of AR in vivo, however, remain largely unknown. A well elucidated model holds the view that AR occurs on the surface of the zona pellucida (ZP), which is triggered by binding of sperm with one of the ZP glycosylated protein, ZP3. However, this model fails to explain the large number of 'falsely' acrosome-reacted sperms found within the cumulus layer in many species examined. With the emerging evidence of cross-talk between sperm and cumulus cells, the potential significance of AR in the cumulus oophorus, the outer layer of the egg, has been gradually revealed. Here we review the acrosome status within the cumulus layer, the cross-talk between sperm and cumulus cells with the involvement of a novel sperm-released factor, NYD-SP8, and re-evaluate the importance and physiological significance of the AR in the cumulus in fertilization.
Acrosome Reaction ; physiology ; Cell Communication ; Cumulus Cells ; metabolism ; Female ; Fertilization ; physiology ; Humans ; Male ; Membrane Proteins ; metabolism ; Oocytes ; metabolism ; Progesterone ; physiology ; Spermatozoa ; metabolism

The acrosome reaction is a prerequisite for fertilization, and its signaling pathway has been investigated for decades. Regardless of the type of inducers present, the acrosome reaction is ultimately mediated by the elevation of cytosolic calcium. Inositol 1,4,5-trisphosphate-gated calcium channels are important components of the acrosome reaction signaling pathway and have been confirmed by several researchers. In this study, we used a novel permeabilization tool BioPORTER^® and first demonstrated its effectiveness in spermatozoa. The inositol 1,4,5-trisphosphate type-1 receptor antibody was introduced into spermatozoa by BioPORTER^® and significantly reduced the calcium influx and acrosome reaction induced by progesterone, solubilized zona pellucida, and the calcium ionophore A23187. This finding indicates that the inositol 1,4,5-trisphosphate type-1 receptor antibody is a valid inositol 1,4,5-trisphosphate receptor inhibitor and provides evidence of inositol 1,4,5-trisphosphate-gated calcium channel involvement in the acrosome reaction in human spermatozoa. Moreover, we demonstrated that the transfer of 1,4,5-trisphosphate into spermatozoa induced acrosome reactions, which provides more reliable evidence for this process. In addition, by treating the spermatozoa with inositol 1,4,5-trisphosphate/BioPORTER^® in the presence or absence of calcium in the culture medium, we showed that the opening of inositol 1,4,5-trisphosphate-gated calcium channels led to extracellular calcium influx. This particular extracellular calcium influx may be the major process of the final step of the acrosome reaction signaling pathway.
Acrosome Reaction/physiology* ; Calcimycin/pharmacology* ; Calcium/pharmacology* ; Calcium Ionophores/pharmacology* ; Drug Delivery Systems ; Humans ; Inositol 1,4,5-Trisphosphate Receptors/metabolism* ; Male ; Progesterone/pharmacology* ; Spermatozoa/metabolism* ; Zona Pellucida/metabolism*

The aim of this study was to investigate whether a relationship exists between the presence of low numbers of leukocytes in normal ovulatory cervical mucus and sperm quality and lipid content after migration. The percentages of live, motile and morphologically normal spermatozoa, movement parameters assessed by computer-aided sperm analysis (CASA), and ionophore-induced acrosome reaction measured by flow cytometry were determined before and after migration. High-performance liquid chromatography with ultraviolet detection was used to measure the sperm lipid content, including the various diacyl subspecies. The number of leukocytes found in solubilized mucus samples was counted using a haemocytometric method. Overall, the presence of leukocytes in the cervical mucus samples did not significantly influence sperm motility and morphology, sperm kinematic parameters, or the sperm content in sphingomyelin or cholesterol. In contrast, after migration, the decrease in various sperm diacyls and the level of induced acrosome reaction was significantly less pronounced in mucus samples containing>or=10(4) leukocytes than in mucus samples with no or rare leukocytes whereas the level of induced acrosome reaction was higher. The present data suggest that the low level of leukocytes found in normal ovulatory cervical mucus could influence the process of sperm lipid remodelling/capacitation.
Acrosome Reaction ; physiology ; Cervix Mucus ; immunology ; metabolism ; Female ; Humans ; Leukocytes ; cytology ; Lipids ; Male ; Ovulation ; Sperm Motility ; physiology ; Spermatozoa ; cytology ; metabolism ; Tissue Donors