Depression currently affects about 280 million people worldwide and its prevalence has been increasing dramatically, especially among the young and people of reproductive age, which consequently leads to an increase in antidepressant consumption. Antidepressants are associated with sexual dysfunction in both men and women; however, their role in male fertility has been scarcely studied. Fluoxetine and sertraline, two serotonin reuptake inhibitors (SSRIs), are among the most prescribed antidepressants worldwide. To determine their possible effects, human sperm cells were exposed to either sertraline or fluoxetine at concentrations previously found in blood and seminal fluid of patients undergoing treatment. Spermatozoa were incubated for up to 24 h at 37°C and 5% CO 2 , and important functional parameters such as sperm motility, viability, mitochondrial membrane potential, cellular reactive oxygen species (ROS) production, chromatin/DNA integrity, acrosome status, and tyrosine phosphorylation were assessed. At low levels, fluoxetine consistently decreased progressive motility throughout time while promoting fluctuations in ROS levels and sperm capacitation. Nevertheless, it did not affect viability, mitochondrial membrane potential, acrosome reaction nor chromatin/DNA integrity. Sertraline, on the other hand, had little to nonsignificant impact at low doses, but affected almost all tested parameters at supratherapeutic concentrations. Altogether, our results suggest that both antidepressants may impair sperm function, possibly through different mechanisms of action, but fluoxetine is the only exhibiting mild negative effects at doses found in vivo .
Humans ; Male ; Spermatozoa/drug effects* ; Fluoxetine/pharmacology* ; Sperm Motility/drug effects* ; Sertraline/pharmacology* ; Reactive Oxygen Species/metabolism* ; Antidepressive Agents/pharmacology* ; Membrane Potential, Mitochondrial/drug effects* ; Sperm Capacitation/drug effects* ; Selective Serotonin Reuptake Inhibitors/pharmacology* ; Cell Survival/drug effects* ; Acrosome Reaction/drug effects*

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*

To interact with the egg, the spermatozoon must undergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosomal exocytosis, near or on the egg, a process that allows the sperm to penetrate and fertilize the egg. In the present study, we investigated the involvement of cyclic adenosine monophosphate (cAMP)-dependent processes on acrosomal exocytosis. Inhibition of protein kinase A (PKA) at the end of capacitation induced acrosomal exocytosis. This process is cAMP-dependent; however, the addition of relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l^-1) analog induced significant inhibition of the acrosomal exocytosis. The induction of acrosomal exocytosis by PKA inhibition was significantly inhibited by an exchange protein directly activated by cAMP (EPAC) ESI09 inhibitor. The EPAC selective substrate activated AE at relatively low concentrations (0.02-0.1 μmol l^-1), whereas higher concentrations (>5 μmol l^-1) were inhibitory to the AE induced by PKA inhibition. Inhibition of PKA revealed about 50% increase in intracellular cAMP levels, conditions under which EPAC can be activated to induce the AE. Induction of AE by activating the actin severing-protein, gelsolin, which causes F-actin dispersion, was inhibited by the EPAC inhibitor. The AE induced by PKA inhibition was mediated by phospholipase C activity but not by the Ca²⁺-channel, CatSper. Thus, inhibition of PKA at the end of the capacitation process induced EPAC/phospholipase C-dependent acrosomal exocytosis. EPAC mediates F-actin depolymerization and/or activation of effectors downstream to F-actin breakdown that lead to acrosomal exocytosis.
8-Bromo Cyclic Adenosine Monophosphate/pharmacology* ; Acrosome/metabolism* ; Acrosome Reaction/drug effects* ; Calcimycin/pharmacology* ; Cyclic AMP/metabolism* ; Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors* ; Exocytosis/drug effects* ; Guanine Nucleotide Exchange Factors/metabolism* ; Humans ; Male ; Protein Kinase Inhibitors/pharmacology* ; Signal Transduction/drug effects* ; Spermatozoa/metabolism* ; Thapsigargin/pharmacology*

Objective: To elucidate the possible role of human lysozyme-like protein 4 (LYZL4) in fertilization and characterize its enzymatic properties. METHODS: The localization of LYZL4 in human spermatozoa was investigated by immunofluorescence staining, the sources of LYZL4 on the sperm surface examined by RT-PCR, and the role of LYZL4 in fertilization assessed by the zona-free hamster egg penetration test. The recombinant plasmid pPIC9K-LYZL4 was constructed and its expression induced with methanol after transformed into competent Pichia pastoris GS115. The recombinant LYZL4 protein (rLYZL4) was purified from the fermentation supernatant and subsequently identified by Western blot. The hyaluronan binding ability of rLYZL4 was determined by ELISA and the muramidase activity, hyaluronidase activity, and free radical scavenging ability examined by spectrophotometric methods. RESULTS: Immunodetection with a specific antiserum localized LYZL4 on the acrosomal membrane of mature spermatozoa, which was exclusively secreted from the testis and epididymis as shown by RT-PCR. Immunoneutralization of LYZL4 significantly decreased the number of human spermatozoa bound to zona-free hamster eggs in a dose-dependent manner in vitro. The recombinant protein was expressed successfully by the P. pastoris strain GS115. Purified rLYZL4 exhibited a potent hyaluronan binding ability and a strong free radical scavenging ability but no muramidase or hyaluronidase activity. CONCLUSIONS LYZL4 secreted from the testis and epididymis is localized on the acrosomal membrane of mature spermatozoa and plays a role in sperm-egg binding as well as in binding hyaluronan and scavenging free radicals, which suggests that it might be a multi-functional molecule contributive to sperm protection and sperm-egg binding.
Acrosome ; enzymology ; Animals ; Blotting, Western ; Cricetinae ; Enzyme-Linked Immunosorbent Assay ; Epididymis ; Female ; Fertilization ; physiology ; Free Radical Scavengers ; metabolism ; Humans ; Hyaluronic Acid ; metabolism ; Male ; Muramidase ; analysis ; physiology ; Pichia ; Plasmids ; metabolism ; Recombinant Proteins ; analysis ; metabolism ; Sperm-Ovum Interactions ; physiology ; Spermatozoa ; enzymology ; Testis

Stress affects the male reproductive system and can cause sub-fertility or infertility. Although Phyllanthus emblica L. (PE) extract has been shown to have high antioxidant capacity and protective properties in damaged tissue, the preventive effects of PE extract on testicular function from stress-related impairment have never been demonstrated. This study aimed to investigate the effects of PE aqueous leaf extract on testicular impairment and protein marker changes in rats suffering from chronic stress. Adult male rats were divided into four groups: a control group, a chronic stress (CS) group, and two groups with CS that received different doses of PE extract (50 or 100 mg/kg body weight (BW)). In the treatment groups, the animals were given PE extract daily before stress induction for 42 consecutive days. Stress was induced through immobilization (4 h/d) followed by forced cold swimming (15 min/d). Sperm quality and the histology of the testes and caudal epididymis were examined, as were levels of serum corticosterone, testosterone, and malondialdehyde (MDA). The expressions of testicular steroidogenic acute regulatory (StAR) and tyrosine-phosphorylated proteins were investigated using immuno-Western blot analysis, as these proteins are assumed to play important roles in spermatogenesis and androgen synthesis. The results showed that PE (50 mg/kg BW) significantly increased sperm concentration and testosterone levels, while decreasing corticosterone levels, MDA levels, sperm head abnormalities, and acrosome-reacted sperm in CS rats. In addition, PE at both doses was found to diminish testicular histopathology in the CS rats. We also found that 50 mg/kg BW of PE significantly improved StAR protein expression and altered the intensities of some tyrosine-phosphorylated proteins in testis. We conclude that PE leaf extract at 50 mg/kg BW can prevent testicular damage in rats with CS.
Acrosome Reaction ; Animals ; Antioxidants/pharmacology* ; Corticosterone/blood* ; Epididymis/metabolism* ; Male ; Malondialdehyde/blood* ; Phosphoproteins/metabolism* ; Phosphorylation ; Phyllanthus emblica/chemistry* ; Plant Extracts/pharmacology* ; Plant Leaves/chemistry* ; Rats ; Rats, Sprague-Dawley ; Sperm Count ; Spermatogenesis/drug effects* ; Spermatozoa/drug effects* ; Stress, Physiological ; Testis/drug effects* ; Testosterone/blood* ; Tyrosine/chemistry*

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

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

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

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

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