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*

Sperm function and male fertility are closely related to pH dependent K⁺ current (KSper) in human sperm, which is most likely composed of Slo3 and its auxiliary subunit leucine-rich repeat-containing protein 52 (LRRC52). Onion peel extract (OPE) and its major active ingredient quercetin are widely used as fertility enhancers; however, the effect of OPE and quercetin on Slo3 has not been elucidated. The purpose of this study is to investigate the effect of quercetin on human Slo3 channels. Human Slo3 and LRRC52 were co-transfected into HEK293 cells and pharmacological properties were studied with the whole cell patch clamp technique. We successfully expressed and measured pH sensitive and calcium insensitive Slo3 currents in HEK293 cells. We found that OPE and its key ingredient quercetin inhibit Slo3 currents. Inhibition by quercetin is dose dependent and this degree of inhibition decreases with elevating internal alkalization and internal free calcium concentrations. Functional moieties in the quercetin polyphenolic ring govern the degree of inhibition of Slo3 by quercetin, and the composition of such functional moieties are sensitive to the pH of the medium. These results suggest that quercetin inhibits Slo3 in a pH and calcium dependent manner. Therefore, we surmise that quercetin induced depolarization in spermatozoa may enhance the voltage gated proton channel (Hv1), and activate non-selective cation channels of sperm (CatSper) dependent calcium influx to trigger sperm capacitation and acrosome reaction.
Acrosome Reaction ; Calcium ; Fertility ; HEK293 Cells ; Humans ; Hydrogen-Ion Concentration ; Male ; Onions ; Phosphatidylinositols ; Protons ; Quercetin ; Sperm Capacitation ; Spermatozoa

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*

The evaluation of infertility in males consists of physical examination and semen analyses. Standardized semen analyses depend on the descriptive analysis of sperm motility, morphology, and concentration, with a threshold level that must be surpassed to be considered a fertile spermatozoon. Nonetheless, these conventional parameters are not satisfactory for clinicians since 25% of infertility cases worldwide remain unexplained. Therefore, newer tests methods have been established to investigate sperm physiology and functions by monitoring characteristics such as motility, capacitation, the acrosome reaction, reactive oxygen species, sperm DNA damage, chromatin structure, zona pellucida binding, and sperm-oocyte fusion. After the introduction of intracytoplasmic sperm injection technique, sperm maturity, morphology, and aneuploidy conditions have gotten more attention for investigating unexplained male infertility. In the present article, recent advancements in research regarding the utilization of male fertility prediction tests and their role and accuracy are reviewed.
Acrosome Reaction ; Aneuploidy ; Chromatin ; DNA Damage ; Fertility* ; Humans ; Infertility ; Infertility, Male ; Male* ; Physical Examination ; Physiology ; Reactive Oxygen Species ; Semen Analysis ; Sperm Injections, Intracytoplasmic ; Sperm Motility ; Spermatozoa ; Zona Pellucida

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*

The CatSper channel is known as one of the most important Ca²⁺ channels on the cell membrane of mammalian sperm and plays a key role in the motility, hyperactivation and fertilization function of sperm. The CatSper protein, expressed exclusively in the principal piece of the sperm tail, is composed of CatSper1-4 and 5 auxiliary unitsβ,γ,δ and ε, and has an essential part in the functional and structural domains of Ca²⁺as well as in the spatiotemporal regulation of the P-Tyr protein, sperm hyperactivation, efficient sperm migration in the oviduct, egg penetration, and normal fertility. Recent studies show that functional deficiency of CatSper seriously affects sperm function,and the loss of any one of its 9 subunits may lead to male reproductive dysfunction. This paper outlines recent advances in the studies of the CatSperprotein, focusing on its expression, location, structure, and regulation,as well as itsinfluence on sperm hyperactivation and male reproduction.
Animals ; Calcium Channels ; chemistry ; physiology ; Humans ; Infertility, Male ; etiology ; Male ; Sperm Motility ; physiology ; Sperm Tail ; metabolism ; Sperm-Ovum Interactions ; physiology ; Spermatozoa ; physiology

UNLABELLEDObjective To explore the effects of sperm DNA integrity rate, acrosome integrity rate and acrosome reaction rate on the outcomes of rescue intracytoplasmic sperm injection (ICSI).

METHODSThis retrospective analysis was conducted among 97 infertile couples receiving rescue ICSI due to failure of in vitro fertilization procedures in our Reproductive Medicine Center. Of these 97 women, 41 had clinical pregnancy and 56 did not, and the effects of sperm DNA integrity rate (estimated by DNA fragmentation index, DFI), acrosome integrity rate and acrosome reaction rate on rescue ICSI outcomes were analyzed.

RESULTSNo significant difference was found in paternal age, testosterone value, testicular volume, FSH, female patient' age or the number of eggs retrieved between the two groups (P>0.05), but the infertility years was significantly shorter in the pregnancy group than in the non-pregnancy group (P<0.05). The fertilization rate and cleavage rate were similar between the two groups (P>0.05), but the good embryo rate was significantly higher in the pregnancy group (P<0.05). The sperm DNA integrity or acrosome reaction rate did not differ significantly between the two groups (P>0.05), but the acrosome integrity rate was significantly higher in the pregnancy group (P<0.05). The sperm DNA integrity rate, acrosome integrity or acrosome reaction rate were not correlated with the fertilization rate, cleavage rate or good embryo rate (P>0.05). The pregnancy rate, twin and single fetus rates were 42.3%, 10.3% and 32.0% in this cohort after recue ICSI, respectively.

CONCLUSIONRescue ICSI is an effective treatment after failed in vitro fertilization procedure, and sperm acrosome integrity rate is associated with the outcome of rescue ICSI.

Acrosome ; pathology ; Acrosome Reaction ; DNA Fragmentation ; Female ; Fertilization ; Fertilization in Vitro ; Humans ; Infertility ; Male ; Pregnancy ; Pregnancy Rate ; Retrospective Studies ; Sperm Injections, Intracytoplasmic

Davallialactone (DAVA) is a hispidin analogue derived from the medicinal fungus Phellinus baumii. We examined the effect of DAVA on in vitro fertilization (IVF) of pigs. Boar spermatozoa were incubated in fertilization medium with varying concentrations of DAVA, then sperm motility and reactive oxygen species (ROS) level were evaluated. Higher sperm motility was found following the addition of 0.5 or 1 µM DAVA after incubation than addition of other concentrations or controls. ROS level decreased significantly with the addition of DAVA. The rate of normal fertilization was higher in the presence of 1 µM DAVA (65.1%) than were those of other concentrations or controls (45.4~59.4%), and the highest total fertilization rate (mono- and polyspermic oocytes) was observed at 1 µM DAVA (83%). In conclusion, addition of DAVA to fertilization medium improved sperm motility, and reduced ROS level so as to potentially improve sperm-oocyte binding in IVF, suggesting the potential of a compound isolated from mushrooms in assisted reproductive technology for humans and animals.
Agaricales ; Animals ; Fertilization ; Fertilization in Vitro* ; Fungi ; Humans ; Reactive Oxygen Species ; Reproductive Techniques, Assisted ; Sperm Motility ; Sperm-Ovum Interactions* ; Spermatozoa* ; Swine*