Mary C. Mahony

Modulation of sperm tail protein tyrosine phosphorylation by pentoxifylline and its correlation with hyperactivated motility

OBJECTIVE To assess the effect of pentoxifylline on human sperm functions that are crucial to fertilization. DESIGN Prospective, controlled study. SETTING Academic tertiary care institute. PATIENT(S) Healthy male sperm donors. INTERVENTION(S) The effects of pentoxifylline (3.6 mM) on hyperactivated motility, sperm binding to the zona pellucida, and sperm protein tyrosine phosphorylation were evaluated. MAIN OUTCOME MEASURE(S) Hyperactivated motility was assessed by computer-assisted motion analysis, and tight binding of sperm to homologous zonae pellucidae was examined using the hemizona assay. Sperm protein phosphorylation was evaluated using indirect immunofluorescence with an antibody to phosphotyrosine (PY20). RESULT(S) Pentoxifylline significantly stimulated hyperactivated motility at 1 hour and 4 hours; it also significantly increased sperm binding to the zona pellucida and enhanced sperm tail tyrosine phosphorylation at 4 hours under capacitating conditions. There was a statistically significant correlation between hyperactivated motility and sperm tail protein phosphorylation. CONCLUSION(S) Pentoxifylline stimulates sperm functions that are essential to achieving fertilization under in vitro conditions in sperm obtained from fertile men. The enhancement of hyperactivated motility is associated with the stimulation of sperm tail tyrosine phosphorylation, suggesting a causal relation and the involvement of a modulatory effect after cyclic adenosine monophosphate-dependent phosphorylation of intermediate proteins.

Effects of hydrogen peroxide on human spermatozoa

PurposeReactive oxygen species (ROS) have been reported widely to cause deleterious effects on sperm viability and function due to peroxidation of membrane lipids. However, their action appears more selective at low concentrations; recent evidence indicates that the superoxide anion can promote capacitation and induce hyperactivated motility (HA) in human spermatozoa and that hydrogen peroxide (H2O2)may participate in capacitation of hamster spermatozoa. The objective of these studies was to investigate the direct effects of H2O2on functions crucial to fertilization in human spermatozoa.MethodsIn these prospective studies, we examined the dose-and time-dependent effects of H2O2on sperm membrane-mediated events (binding to the zona pellucida and changes in intracelłular calcium concentration [Ca2+]i,motility patterns, and acrosome reaction). Sperm from fertile donors were used in the experiments under capacitating conditions after separation of the motile fraction by wash/swim-up. [Ca2+]iwas measured by the fluorescent fura-2 indicator, and sperm-zona pellucida binding was assessed with the hemizona assay (HZA). Hyperactivated motility was evaluated by computerized analysis, and the percentage of acrosomereacted sperm was detected by FITC-Pisum sativumlectin and indirect immunofluorescence.ResultsIn the HZA, H2O2did not influence sperm-zona pellucida binding at low concentrations (0.05 mMand 0.1 mM),but significantly reduced binding at 0.2 mM (P<0.004 vs controls). H2O2significantly decreased HA in a dose-dependent manner (P<0.0001) and had a significant effect (P<0.01) on acrosome reaction (stimulatory effect at 0.01 mM).H2O2did not affect basal[Ca2+]i;however, H2O2 (0.1mMthrough 10 mM)decreased the initial phase of progesterone-induced (P4: 1 μM)enhancement of [Ca2+]iin a dose-and time-dependent fashion. Preincubation of sperm with catalase (20 μg/ml) potentiated the P4-induced increase of [Ca2+]i.H2O2did not significantly modify [Ca2+]iincrease in response to inomycin (10 μM ).ConclusionsThese experiments show that H2O2directly affects sperm functions crucial to fertilization in a dose-and time-dependent fashion. Low concentrations maintain capacitation, whereas higher concentrations have deleterious effects, as determined by the end points of the capacitation process. The latter effects are probably dependent on modifications of plasma membrane and intraceliular homeostasis by the oxidative process.

Clinical significance of human sperm-zona pellucida binding

OBJECTIVE To assess the relationship between sperm morphology and motion parameters and sperm-zona pellucida (ZP) binding capacity under hemizona assay (HZA) conditions and to determine the discriminatory power of the HZA for the prediction of in vitro sperm fertilizing ability. DESIGN Prospectively designed study. SETTING Academic tertiary centers. PATIENT(S) One hundred ninety-six couples undergoing IVF therapy participated in this study. INTERVENTION(S) Hemizona assay and IVF results were determined for each couple. MAIN OUTCOME MEASURE(S) Computerized sperm motion analysis, sperm morphology (strict) criteria), and HZA results were correlated with fertilization outcome. RESULT(S) Among sperm parameters from the original ejaculates, morphology was the best predictor of sperm-ZP binding ability; hyperactivated motility was the best predictor of HZA results after swim-up separation of the motile sperm fractions. The HZA index provided the highest discriminatory power for fertilization success/failure, with an overall accuracy of 86%. CONCLUSION(S) Sperm morphology and hyperactivated motility showed a high correlation with the capacity of sperm to achieve tight binding to the ZP. The excellent positive and negative predictive values of the HZA for fertilization outcome provide additional support for the use of this functional bioassay in the decision-making process within the assisted reproduction setting.

Inhibition of human sperm-zona pellucida tight binding in the presence of antisperm antibody positive polyclonal patient sera

Patient sera previously characterized as containing high levels of IgG and IgA antisperm antibodies that bound to the sperm surface, most specifically the head region, were evaluated for their effect on sperm-zona pellucida tight binding as assessed by the hemizona assay (HZA). Of the ten patient sera tested, 7 reduced zona binding by approximately half. Two of the most strongly inhibitory (greater than 70% inhibition) were examined for their effect on the prefertilization maturation of sperm. The patient sera did not affect sperm motion characteristics, or development of hyperactivated motility. However, in the presence of these sera some impedance was noted in calcium uptake after stimulation with human follicular fluid and in the acrosome reaction after calcium ionophore induction. Whether these two sera specifically affect sperm-zona pellucida binding or non-specifically affect the normal progression of capacitation remains to be eludicated.

Fucoidin inhibits the zona pellucida-induced acrosome reaction in human spermatozoa

We recently reported that fucoidin (a polymer of predominantly sulfated L-fucose) significantly inhibits tight binding of human sperm to the human zona pellucida in vitro and that several oligosaccharides obtained after acid hydrolysis possess sperm-zona pellucida binding inhibitory activity equal to the original fucoidin. This inhibition may be specific to sperm-zona interactions or may be the consequence of the interruption of capacitation, a series of biochemical and physiological events leading to final sperm maturation, that must occur for successful fertilization. Completion of capacitation is most often determined by assessing two end-points of the process: acquisition of hyperactivated motility and ability to complete the acrosome reaction. Here, we examined the effects of fucoidin on these two end-points of capacitation in vitro. Fucoidin did not affect the proportion of sperm with hyperstimulated motility. Neither did fucoidin cause an increase in sperm that had spontaneously acrosome-reacted at 4.5 hours compared to controls as evaluated by indirect immunofluorescence using the acrosomal marker, monoclonal antibody, T-6. Comparable percentages of sperm had completed the acrosome reaction when exogenously stimulated by calcium ionophore A23187 with and without the addition of fucoidin. However, in the presence of fucoidin, stimulation of the acrosome reaction by acid solubilized human zonae pellucidae was significantly inhibited. These data indicate that fucoidin does not impede the normal progression of capacitation. These results provide strong evidence to support the hypothesis is that the inhibitory effect of fucoidin is at the level of the sperm membrane since inhibition can be bypassed by increasing intracellular calcium directly with a calcium ionophore.

Estrogen and progesterone receptor MRNA are expressed in distinct pattern in male primate reproductive organs

PurposeThe role(s) of estrogens (E) and progesterone (P) in male reproductive physiology remain unclear. Estrogens are used in the treatment of prostatic cancer. Progestins have been used to control excessive sexual behavior in men, and proposed as a male contraceptive. Previous immunohistochemical studies have shown that E receptors (ER) are present in the reproductive tract of male nonhuman primates.MethodWe examined the expression pattern of ER and progesterone receptor (PR) mRNA in adult primate male reproductive tract. mRNA was extracted from male pituitary, testis, prostate and different regions of the epididymis of three intact adult cynomolgous monkeys. Ovarian, myometrial and spleen mRNA were used as controls. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify ER and PR mRNA; β-actin mRNA was used as a reference. Primers for ER, PR and β-actin were designed using the most conserved areas in the corresponding human cDNA sequences, and the identity of the PCR products was verified using Southern hybridization. Semiquantitative analysis of ER and PR mRNA content in different parts of the male reproductive tract was carried out by spiking the PCR reaction with33P-dCTP, and amplifying the samples for 20 cycles with the β-actin primers, whereas 30 cycles were used for ER and PR.ResultsThe results are expressed as cpm ratios of ER or PR/β-actin. All the male reproductive organs studied revealed a strong signal for ER and PR mRNA. The results of the semiquantitative analysis indicate that the expression of both ER and PR was highest in testis (mean±SE 6.4±1.3 and 0.5±0.1, respectively). The mean figures for prostate were 0.5 and 0.4, respectively. The mean content of ER and PR in the different areas of epididymis was 0.5 and 0.1, respectively. The epididymal ER mRNA was highest in the corpus region (ER/β-actin 0.7), the ratio being 0.4 for the caput and cauda regions. The expression pattern of PR mRNA was different, and the caput of epididymis being the most intense (0.2). Surprisingly, the pituitary content of ER and PR mRNA was close to that seen in the ovary, the mean±SE values being 7.6±0.5 and 1.3±0.1, respectively.ConclusionsWe, therefore, conclude that male monkey reproductive tract contains mRNA for ER and PR, and there appears to be regional variation in their expression. Thus the role(s) of Es and P in male reproductive physiology, specifically in sperm maturation, warrants further investigations.

Evaluation of human sperm-zona pellucida tight binding by presence of monoclonal antibodies to sperm antigens

Characterized WHO monoclonal antibodies (MAbs) to human sperm antigens were evaluated as to whether they inhibited sperm-zona pellucida tight binding as assessed by the hemizona assay (HZA). Of the 26 MAbs tested, only one inhibited zona binding. The whole sperm-specific MAb inhibited zona binding by 70%. The MAb also caused strong agglutination. Two procedures, Sephadex column chromatography and papain digestion, were used to determine whether agglutination or steric hindrance was a factor in the capability of MAb to inhibit zona binding. However, inhibition remained comparable to previous results. The MAb did not prevent capacitation, nor calcium influx and the resulting increase in hyperactivated motility and acrosome reaction. Since its inhibitory influence is not due to agglutination factors, steric hindrance or prevention of normal pre-fertilization maturation, the MAb may be blocking a portion of the zona binding receptor and may be useful in elucidating sperm antigens important to sperm-egg interaction. The approach used in this study allows definition of sperm surface antigens involved in zona pellucida binding.

Pentoxifylline stimulates various sperm motion parameters and cervical mucus penetrability in patients with asthenozoospermia

Summary. Pentoxifylline (PTX) was incubated in vitro with human spermatozoa to examine its effects on sperm motility characteristics and bovine cervical mucus penetrability (BCMP). Sperm motion parameters were assessed by computer‐assisted motion analysis (CASA) using HTM‐IVOS and BCMP was evaluated using the Penetrak kit. In vitro incubation with PTX (1 mg ml−1; 3.6 mm, 30 min) did not significantly change percentage motility, average path velocity (VAP), straight‐line velocity (VSL) or beat cross frequency (BCF) of spermatozoa from normozoospermic or asthenozoospermic samples. However, it significantly increased curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and hyperactivated motility (HA), and significantly decreased linearity (LIN) of spermatozoa from both samples. Pentoxifylline was found to increase BCMP scores for spermatozoa from asthenozoospermic samples, but did not affect scores for spermatozoa from normozoospermic samples. Bovine cervical mucus penetrability (BCMP) was found to be positively and significantly correlated with the percentage motility of both non‐PTX‐treated and PTX‐treated spermatozoa for asthenozoospermic samples. These results demonstrated that PTX enhanced several motion sperm parameters as well as BCMP in asthenozoospermic samples and suggest a potential use of the methylxanthine in infertile patients with motility defects undergoing artificial insemination.

Increase of intracellular calcium is not a cause of pentoxifylline-induced hyperactivated motility or acrosome reaction in human sperm

OBJECTIVE To investigate the effects of the phosphodiesterase inhibitor pentoxifylline on hyperactivated motility and acrosome reaction in human sperm and to determine whether its stimulatory effects occur via increased intracellular calcium levels. DESIGN Prospective study. SETTING Academic tertiary care facility. PARTICIPANT(S) Healthy male donors. INTERVENTION(S) The effects of pentoxifylline on hyperactivated motility, acrosome reaction, and intracellular calcium were studied and compared with the effects of progesterone. Thapsigargin, a known mobilizer of intracellular calcium, also was used as positive control. MAIN OUTCOME MEASURE(S) Hyperactivated motility was assessed by computer-assisted sperm motion analysis using the HTM-IVOS, acrosome reaction was evaluated with the fluorescent probe fluorescein isothiocyanate-labeled Pisum sativum agglutinin, and intracellular calcium was determined by fura-2 using spectrofluorometry. RESULT(S) Pentoxifylline significantly increased both hyperactivated motility and acrosome reaction. Enhancement of hyperactivated motility by pentoxifylline in the capacitation medium persisted for up to 5 hours after pentoxifylline was washed from the medium. It also enhanced the percentage of acrosome-reacted spermatozoa after 4 hours of incubation. These effects occurred in the presence of a marginally significant decrease in intracellular calcium. CONCLUSION(S) Pentoxifylline stimulates hyperactivated motility and acrosome reaction in spermatozoa from fertile men. Its stimulatory effects occur through mechanism(s) other than increase in intracellular calcium.

Regional Distribution of 5α-Reductase Type 1 and Type 2 mRNA Along the Human Epididymis

OBJECTIVE To determine the regional distribution and relative expression of 5alpha-reductase type 1 and type 2 mRNA within the human testis and regions of the epididymis. DESIGN Prospective observational study. SETTING University academic medical center. PATIENT(S) Two young adult male organ donors. INTERVENTION(S) None MAIN OUTCOME MEASURE(S) The distribution of 5alpha-reductase type 1 and type 2 mRNA in the testis and regions of the epididymis was detected by Northern blot analysis. The relative abundance of each 5alpha-reductase mRNA was evaluated using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in which cyclophilin mRNA, a house-keeping gene product, was coamplified as the reference standard. RESULT(S) Northern blot analysis revealed the 5alpha-reductase type 2 transcript in the midcaput, distal caput, corpus, and proximal cauda of the epididymis, but the transcript was undetectable in the testis, proximal caput, and distal cauda region. No transcript for the type 1 isozyme was detected by Northern blot. The more sensitive RT-PCR showed low levels of type 1 mRNA in the testis and epididymis, with the highest abundance in the proximal caput. Type 2 mRNA of 5alpha-reductase was most abundant in the midcaput, was decreased in the more distal regions, and was more abundant than type 1 mRNA in all epididymal regions except for the proximal caput. CONCLUSION(S) Both 5alpha-reductase type 1 and type 2 mRNAs are present in the human epididymis. The type 2 isozyme mRNA is predominant, being more highly expressed than the low-abundance type 1 mRNA.