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

Protein palmitoylation, one of post-translation modifications, refers to the addition of saturated 16-carbon palmitic acid to cysteine residues via the thioester bond. It plays key roles in various functional activities, such as the interaction, stability and location of proteins. Heat shock protein 90 (Hsp90), an important molecular chaperone, has been reported to be involved in sperm capacitation. However, it remains unclear whether protein palmitoylation exists in sperm and whether Hsp90 in sperm is palmitoylated under different physiological conditions. In this study, we examined whether the protein palmitoylation is present in mouse cauda epididymis sperm using acyl-biotin exchange method, predicted the potential palmitoylated sites of Hsp90 by the software CSS-Palm 4.0 and detected the palmitoylated Hsp90 in the mouse sperm from caput epididymis and cauda epididymis by immunoprecipitation. We found that some proteins, approximately 50, 65, 72, 85 and 130 kDa, were palmitoylated in mouse cauda epididymis sperm. Five sites in two Hsp90 isoforms were predicted to be palmitoylated. The results also showed that Hsp90 in mouse sperm was palmitoylated and its palmitoylation level was involved in different physiological conditions: the palmitoylation level of cauda epididymis sperm was higher than that of caput epididymis sperm; and the palmitoylation level after capacitation was much higher than that before capacitation. In conclusion, this study reveals that protein palmitoylation is present in mouse sperm and the palmitoylated Hsp90 is associated with different physiological conditions in sperm.
Animals ; Epididymis ; HSP90 Heat-Shock Proteins ; metabolism ; Lipoylation ; Male ; Mice ; Palmitic Acid ; chemistry ; Sperm Capacitation ; Spermatozoa ; metabolism

Plasma membrane hyperpolarization associated with activation of Ca²⁺-activated K⁺ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary γ2-subunit, LRRC52 (leucine-rich-repeat–containing 52), is known to mediate the pH-sensitive, sperm-specific K⁺ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical BK(Ca) activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting γ2 subunit, hLRRC52. As previously reported, Slo3 K⁺ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 K⁺ current, and internal alkalinization and Ca²⁺ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 K⁺ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular Ca²⁺. In contrast, elevation of intracellular Ca²⁺ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate Ca²⁺-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.
4-Aminopyridine ; Animals ; Cell Membrane ; Fertilization ; HEK293 Cells ; Humans ; Hydrogen-Ion Concentration ; Membrane Potentials ; Membranes ; Mice ; Potassium Channels, Calcium-Activated* ; Sperm Capacitation ; Sperm Motility ; Spermatozoa ; Tea

Oocytes retrieval, in vitro maturation (IVM) and fertilization (IVF) efficiency are inevitable steps towards in vitro production of embryos. In the present study, these parameters were investigated in the ovaries of prepubertal (n = 31) and pubertal (n = 61) black Bengal goats obtained from a slaughterhouse. Nuclear maturation was evaluated upon aspiration and following IVM in TCM-199 (Earle's salt with L-glutamine and sodium bicarbonate) for 27 h at 39degrees C under 5% CO2 in humidified air. The oocytes retrieval and efficiency (mean +/- SD) per prepubertal and pubertal goats were 5.2 +/- 0.6 and 6.8 +/- 0.6, and 77.3 +/- 0.1% and 80.5 +/- 0.6%, respectively. Anaphase I - telophase I stages differed significantly (7.3 +/- 0.8 vs. 2.6 +/- 0.2, p < 0.05) between the two groups of goats. After IVM, the percentages of metaphase II were significantly higher (66.3 vs. 60.3, p < 0.05) in pubertal goats than in their prepubertal counterparts. The percentages of normal in vitro fertilization (IVF) in Fert-Tyrode's albumin lactate pyruvate of pubertal goat oocytes did not differ between Percoll and swim-up sperm separation methods (36.7 +/- 0.9% vs. 32.7 +/- 1.3%, p > 0.05). Furthermore, sperm capacitation by heparin alone or in combination with ionomycin did not lead to a significant increase in the normal fertilization rate (34.8 +/- 1.7 vs. 32.2 +/- 1.5%, respectively) in the oocytes of pubertal goats. In conclusion, the ovaries of pubertal black Bengal goats obtained from the slaughterhouse could be used for in vitro embryo production. However, further optimization of the IVM and IVF techniques are necessary for satisfactory in vitro embryo production.
Animals ; Culture Techniques ; Embryonic Development/*physiology ; Female ; Fertilization in Vitro/*veterinary ; Goats/*embryology ; Male ; Oocyte Retrieval/veterinary ; Oocytes/*physiology ; Ovary/cytology ; Sexual Maturation ; Sperm Capacitation ; Tissue and Organ Harvesting/veterinary

The sperm plasma membrane is rich in polyunsaturated fatty acids and a variety of proteins, and its function is associated with sperm capacitation, acrosome reaction and sperm-egg fusion. Sperm fertilizability can be predicted by detecting the function of the sperm plasma membrane, which is performed mainly with the following five techniques: sperm hypoosmotic swelling test, Eosin gamma water test, sperm membrane lipid peroxidation determination, seminal plasma superoxide dismutase determination, and flow cytometry. The evaluation of the function of sperm plasma membrane can be applied in detecting semen quality, selecting semen centrifugation, assessing the quality and fertilizability of sex-sorted sperm, improving cryopreservation, and guiding the optimization of intracytoplasmic sperm injection. This review presents an update on the principles, methods and steps of the detection of sperm plasma membrane function, as well as an overview of its status quo and application.
Cell Membrane ; physiology ; Flow Cytometry ; Humans ; Male ; Semen Analysis ; Semen Preservation ; Sperm Capacitation ; Sperm Injections, Intracytoplasmic ; Sperm Motility ; Spermatozoa ; physiology

Ion channels in mammal sperm, including Ca2+, Na+, K+, Cl- and HCO3- channels, each play a key role in the process of sperm capacitation. Ca2+, HCO3- and ROS, as signal molecules, activate soluble adenylyl cyclase (sAC) with the cooperation of cyclic adenosine monophosphate (cAMP), Ca2+ and intracellular pH and, via a cross talk between the cAMP/protein kinase A (PKA) and tyrosine phosphatase signaling pathways, promote the biological effect of sperm capacitation.
Animals ; Humans ; Ion Channels ; metabolism ; Ions ; metabolism ; Male ; Mammals ; Signal Transduction ; Sperm Capacitation ; physiology

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

OBJECTIVETo explore the effects of fertilization methods and sperm sources in intracytoplasmic sperm injection (ICSI) on the developmental capacity of surplus embryos.

METHODSWe analyzed the blastocyst formation of the surplus embryos from 2 135 patients, who were divided according to fertilization methods into an IVF (n=1803) and an ICSI group (n=332), the former again allocated to a normal fertilization (n=1642) and a rescue fertilization group (n=161), and the latter, according to sperm sources, to an ejaculated (n=248), an epididymal (n=70) and a testicular sperm group (n=14). The rates of blastocyst formation and good-quality blastocysts were compared between different fertilization methods and sperm sources.

RESULTSA total of 1884 blastocysts (28.87%) formed from 6525 surplus embryos of the patients after sequential culture, of which 974 (51.70%) were good-quality ones. The blastocyst formation rate of surplus embryos was significantly higher in the IVF (30.14%) than in the ICSI group (21.40%, P < 0.05), the rate of good-quality blastocysts was also higher in the former (52.44%) than in the latter (45.54%), but with no significant difference (P > 0.05). The rates of blastocyst formation and good-quality blastocysts were significantly higher in the normal (31.04% and 53.28%) than in the rescue fertilization IVF group (20.38% and 38.54%, P < 0.05), and in the testicular sperm ICSI group (30.23% and 53.85%) than in either the epididymal (18.36% and 42.11%) or the ejaculated sperm ICSI group (21.76% and 45.70%) (P < 0.05).

CONCLUSIONThe development potential of surplus embryos was higher in IVF than in ICSI, in the normal than in the rescue fertilization IVF group, and in the testicular than in the epididymal and ejaculated sperm ICSI groups.

Blastocyst ; Embryonic Development ; Female ; Fertilization in Vitro ; methods ; Humans ; Male ; Oocytes ; Sperm Capacitation ; Sperm Injections, Intracytoplasmic ; methods ; Sperm Motility

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

OBJECTIVETo evaluate the role of sperm kinematic parameters in the hyperactivation of Guinea pig spermatozoa, and to confirm the index of their hyperactivated motility.

METHODSComputer-aided sperm analysis (CASA) was used to describe the kinesis parameters of the Guinea pig spermatozoa incubated for 1, 3, 5 and 7 hours.

RESULTSThe curvilinear velocity, average path velocity and amplitude of lateral head displacement were increased with time and reached the peak at 5 hours, while the straight linear velocity, linearity, straightness and beat cross frequency were gradually decreased with time and hit the bottom at 5 hours.

CONCLUSIONThe sperm movement pattern changes greatly before hyperactivation during the capacitation of Guinea pig spermatozoa.

Animals ; Guinea Pigs ; Kinetics ; Male ; Numerical Analysis, Computer-Assisted ; Sperm Capacitation ; physiology ; Sperm Motility ; physiology ; Spermatozoa ; physiology