Sperm Penetration Of Hamster Eggs Research Articles

Cofilin is correlated with sperm quality and influences sperm fertilizing capacity in humans.

Spermatozoa should undergo a series of biochemical modifications in female reproduction tract, which is collectively called sperm capacitation. The capacitated spermatozoa can bind to the egg zona pellucida, resulting in the occurrence of acrosome reaction which enabled spermatozoa penetrate into the egg. The formation of actin plays an important role in these processes. Actin polymerized during sperm capacitation, but the polymers dispersed before acrosome reaction. In this study, we take our focus on actin-binding protein, cofilin. Our results showed that the % and intensity of sperm expressing cofilin in normal sperm were significantly higher than in abnormal sperm, and the sperm expressing cofilin was correlated with sperm quality. Furthermore, treatment with anti-cofilin antibody increased the percentage of sperm capacitation and inhibited progesterone- or A23187- induced acrosome reaction in a dose-dependent manner. The presence of 100ng/mL anti-cofilin antibodies markedly blocked the sperm penetration of zona-free hamster eggs. Besides, immunofluorescence results revealed that cofilin was colocalized with F-actin in the midpiece of spermatozoa; however, phospho-cofilin was expressed in the tail rather than in the midpiece of spermatozoa, which was not colocalized with F-actin in spermatozoa. Moreover, western blot revealed that phospho-cofilin increased in sperm capacitation, and the total cofilin and cofilin in insoluble fraction increased in acrosome reaction; immunofluorescence results showed that the amount of cofilin in acrosome increased in sperm capacitation. In conclusion, our study revealed that cofilin expression in human sperm is correlated with sperm quality and the alterations of cofilin and phospho-cofilin in fertilization affects sperm capacitation, acrosome reaction, and spermatozoa-oocyte fusion.

Inhibition of sperm capacitation and fertilizing capacity by adjudin is mediated by chloride and its channels in humans

Does adjudin disrupt chloride ion (Cl⁻) ion transport function in human sperm and impede sperm capacitation and fertilizing ability in vitro? In this study the results indicate that adjudin is a potent blocker of Cl⁻ channels: disrupting Cl⁻ ion transport function results in a decline in sperm capacitation and fertilizing ability in humans in vitro. Although our previous studies have demonstrated that adjudin exerts its effect by disrupting sertoli-germ cell adhesion junctions, most notably apical ectoplasmic specialization by targeting testin and actin filament bundles that disrupts the actin-based cytoskeleton in sertoli cells, it remains unclear whether adjudin impedes Cl⁻ ion transport function in the human sperm. Semen samples were obtained from 45 fertile men (aged 25-32). Spermatozoa were isolated from the semen in the human tube fluid (HTF) medium by centrifugation through a discontinuous Percoll gradient, and incubated with adjudin at 10 nM-10 µM and/or other reagents under capacitating conditions for 0-5 h. We evaluated the effect of adjudin and different reagents on sperm functions with which they were incubated at 37 °C. Sperm motility and hyperactivation were analyzed by a computer-assisted sperm analysis (CASA) system. Sperm capacitation and the acrosome reaction were assessed by chlortetracycline fluorescence staining. Sperm fertilizing ability was evaluated by sperm penetration of zona-free hamster egg assay, and cellular cAMP levels in spermatozoa were quantified by the EIA kit. The proteins tyrosine, serine and threonine phosphorylation in the presence or absence of adjudin were analyzed by means of a immunodetection of spermatozoa, especially, compared the effect of adjudin on sperm hyperactivation and capacitation in the complete HTF medium with the Cl⁻-deficient HTF medium as well as the various Cl⁻ channel blockers. Adjudin significantly inhibited sperm hyperactivation but not sperm motility. Adjudin-induced inhibition of sperm capacitation was reversible, and it was found to block the rhuZP₃β- and progesterone-induced acrosome reaction in a dose-dependent manner. Adjudin also blocked sperm penetration of zona-free hamster eggs, and significantly inhibited both forskolin-activated transmembrane adenylyl cyclase and soluble adenylyl cyclase activities leading to a significant decline in the cellular cAMP levels in human spermatozoa. Adjudin failed to reduce sperm protein tyrosine phosphorylation but it did prevent sperm serine and threonine protein phosphorylation. Interestingly, adjudin was found to exert its inhibitory effects on sperm capacitation and capacitation-associated events only in the complete Cl⁻-HTF medium but not Cl⁻-deficient medium, illustrating the likely involvement of Cl⁻. Adjudin inhibits the fertility capacity of human sperm is mediated by disrupting chloride ion and its transport function. This study has examined the effect of adjudin only on human sperm capacitation and fertilizing ability in vitro and thus has some limitations. Further investigations in vivo are needed to confirm adjudin is a potent male contraceptive. Our studies demonstrated that adjudin inhibition of capacitation is reversible and its toxicity is low, opening the door for the examination of adjudin as a mediator of male fertility control. Adjudin may be a safe, efficient and reversible male antifertility agent and applicable to initial clinical trials of adjudin as a male antifertility agent in humans. STUDING FUNDING/COMPETING INTEREST(S): This work was supported by the National Basic Research Program of China (2006CB504002), the Nature Science Foundation of China (Nos. 81000244 and 81170554), Zhejiang Project of Science and Technology (2011C23046), the Nature Science Fund of Zhejiang province (Nos.Y2100058 and Y2090236), the key Science and Technology Innovation Team of Zhejiang Province (No.2012R10048-07) and the National Institutes of Health (NICHD U54 HD029990 project 5), USA. The authors declare no conflict of interest.

CFTR is essential for sperm fertilizing capacity and is correlated with sperm quality in humans

Our previous studies have demonstrated the cystic fibrosis transmembrane conductance regulator (CFTR) is important for capacitation and male fertility in mouse and guinea pig spermatozoa. However, the exact function of CFTR on human sperm fertilizing capacity, and correlation with sperm quality has not been established. The present study may shed light on some unexplained male infertility, and on a possible new method for diagnosis of male infertility and strategy for male contraception. To assess the effect of CFTR on human sperm fertilizing capacity, we examined sperm capacitation and the acrosome reaction using chlortetracycline staining, analyzed sperm hyperactivation by computer-assisted semen analysis (CASA), measured intracellular cAMP levels using ElA and evaluated sperm penetration of zona-free hamster eggs assay in fertile men. The percentage of spermatozoa expressing CFTR from fertile, healthy and infertile men (mainly teratospermic, asthenoteratospermic, asthenospermic and oligospermic) was conducted by indirect immunofluorescence staining. Progesterone significantly facilitated human sperm capacitation and ZP3 triggered the acrosome reaction, both were significantly inhibited by CFTR inhibitor-172 (CFTRinh-172; 10 nM-1 microM) in a dose-dependent manner. The presence of 100 nM CFTRinh-172 markedly depressed intracellular cAMP levels, sperm hyperactivation and sperm penetration of zona-free hamster eggs. In addition, the percentage of spermatozoa expressing CFTR in the fertile men was significantly higher than healthy and infertile men categories (P < 0.01). CFTR is essential for human sperm fertilizing capacity and the impairment of CFTR expression in spermatozoa is correlated with a reduction of sperm quality. These results suggest that defective expression of CFTR in human sperm may lead to the reduction of sperm fertilizing capacity.

Relationship of acrosin activity to sperm function tests.

Acrosin activity of spermatozoa from infertile patients and fertile volunteers was measured. Acrosin activity of spermatozoa from asthenozoospermic patients and patients with unexplained infertility was lower than that from fertile volunteers. We utilize the zona-free hamster egg-sperm penetration test to select candidates for conventional in vitro fertilization among unexplained infertile patients. The zona-free hamster egg sperm penetration test, however, requires several hours and special equipment which are not used in the clinical setting. It is preferable that other sperm function tests or a combination of them can replace this test. Thus three distinct tests of sperm function, namely, acrosin activity, Penetrak test, hypo-osmotic swelling test, were compared with the hamster test, using spermatozoa from patients with unexplained infertility. A combination of the Penetrak test and measurement of acrosin activity could predict the results of the zona-free hamster egg sperm penetration test with 88.2% accuracy. Thus, the hamster test should be done when either Penetrak test or measurement of acrosin activity showed abnormal values.

Final Report on the Safety Assessment of Maleic Acid1

Maleic Acid is a dicarboxylic acid that functions as a fragrance ingredient and pH adjuster in cosmetics - it is used in a few cosmetic product formulations at low concentrations. Maleic Acid is commonly used in research studies to induce Fanconi syndrome in rats and dogs in an attempt to study the mechanism of this disease. One such study found decreased glomerular filtration rate in rats given 9.0 mmol/kg, but not with 1.5 mmol/kg, Maleic Acid intraperitoneally. Preincubation with 0.75 mmol/L of Maleic Acid reduced sperm penetration of golden hamster eggs to zero. Maleic Acid failed to induce any significant increases in revertant count in strains TA1535, TA1537, TA98, and TA100 at concentrations up to 7500 mu g/plate. A concentration of 2.0 x 10(-2) M Maleic Acid did show a positive pattern in a DNA synthesis inhibition test. Maleic Acid at 10%, pH 1.0, applied for 30 s on rabbit eyes, caused permanent opacity. A 1% solution, pH 1.0, applied for 2 min caused cloudiness of the cornea, but no lasting injury, and a 5% solution, also at pH 1.0, had a similar but more intense effect, with recovery delayed 6 to 7 days. Application of 10 mu l Maleic Acid (pH not stated) to the volar forearm and labia majora of 21 female Caucasians produced an inflammatory response at 24 and 48 h, which varied from minimal erythema to marked erythema with marked vesiculation. Maleic Acid at 20% (pH not stated) applied to one forearm daily for a period of 6 weeks to 50 human subjects produced acute vesicular dermatitis in 17 subjects, who were dropped from the study. Only five of the remaining subjects accommodated to the treatment, the rest had varying degrees of inflammation or hyperirritable skin. Although Maleic Acid itself may be a dermal and/or ocular irritant, its use as a pH adjustor in cosmetic formulations dictates that most of the acid will be neutralized into various maleate salts. Therefore, the concentration of free Maleic Acid is expected to be low, and dermal or systemic toxicity is not expected to be a concern. The safety of Maleic Acid as a pH adjustor should not be based on the concentration of use, but on the amount of free Maleic Acid that remains after neutralizing the formulation. There is no reason to expect this ingredient to induce any toxicity when used for this purpose. The Cosmetic Ingredient Review (CIR) Expert Panel concluded that Maleic Acid is safe for use in cosmetic formulations as a pH adjustor in the practices of use as described in this safety assessment.

Morphology and the sperm penetration assay

Objective To assess the ability of Kruger morphology to predict the outcome of the sperm penetration assay (SPA). Design A retrospective review using univariate and multivariate analysis, t tests, and logistic regression was performed to examine the correlation between Kruger morphology and the optimized zona-free hamster egg sperm penetration assay (O-HESPA). Setting University hospital. Patient(s) One hundred fifteen private-practice patients who underwent semen analysis, including Kruger morphology and O-HESPA, as part of an infertility evaluation between 1997–2000 were retrospectively reviewed. Intervention(s) Retrospective analysis of the sperm penetration assay. Main outcome measure(s) Univariate and multivariate analysis, Student’s t test, and logistic regression were performed to analyze Kruger morphology, count, and viability and their relationship to SCI result. Result(s) Univariate analysis demonstrated a statistically significant correlation between SCI and sperm count, viability, and Kruger morphology. Multivariate analysis demonstrated statistically significant correlations between SCI and count and viability. There was no correlation between Kruger morphology and SCI. Logistic regression was performed on the SCI results, using count, Kruger morphology, and viability. Sperm count was found to be the only variable that was statistically significant with respect to SCI results. Conclusion(s) This study demonstrated that Kruger morphology assessment cannot be used to predict the results of SCI or replace it in the management of the infertile couple.

Kruger morphology predicts outcome of sperm penetration assay: true or false?

Objective: To assess the ability of Kruger morphology to predict the outcome of the Sperm Penetration Assay (SPA). Design: Sperm Capacitation Index (SCI) and Strict Kruger morphology have both been proven to correlate with in vitro fertilization rates. Each of these tests has been utilized to direct patient management. However, the correlation between these two tests has never been clearly established. We hypothesized that Kruger Morphology is predictive of SCI outcomes, as smaller studies have demonstrated this correlation. A retrospective review using univariate and multivariate analysis, T-tests, and logistic regression was performed to examine the correlation between all semen parameters and the results of the Optimized Zona-Free Hamster Egg Sperm Penetration Assay (O-HESPA). Materials/Methods: A retrospective review of 115 patients who underwent semen analysis with Kruger morphology and O-HESPA between 1997–2000 was performed. All semen analyses were performed by one andrologist, all O-HESPA and SCI studies were performed by the same reference laboratory. Routine semen analysis included: total sperm count, motility, viability, and Kruger morphology. The Log of the count and % viability were used in the analysis. The significance of Kruger morphology was assessed as a continuous variable and as a discontinuous variable designated as: very poor (0–4), abnormal (5–13), and normal (≥14). SCI was recoded into a dichotomous variable classifying SCI as either a normal (10) or abnormal (≤10). Student’s t-test and logistic regression was performed analyzing Kruger, count, and viability and their relationship to SCI result. Results: When univariate analysis was performed there was a statistical significance between SCI and sperm count (p = 0.001), sperm viability (p = 0.040), and Kruger morphology (p = 0.031). Multivariate analysis found statistically significant correlations between SCI and sperm count (p = 0.002) and sperm viability (0.051). However, the significance between Kruger morphology and SCI was lost (p = 0.128). When SCI was analyzed as a dichotomous variable, 57 patients were identified as having a SCI ≤ 10, and 58 patients with SCI >10. Logistic regression was performed on the SCI results, using log count, Kruger morphology quartiles, and log viability. Log of sperm count was found to be the only variable with statistical significance with respect to SCI results (p = 0.001). Conclusions: Although we had hypothesized that Kruger morphology would be predictive of SCI results, after performing comprehensive statistical analysis, no such correlation was found. T-tests and univariate analysis shows a significant correlation between sperm count, viability and Kruger morphology as they relate to SCI. Logistic regression or multivariate analysis demonstrated that the significance between sperm count and its relationship with SCI still existed. This study found that Kruger morphology could not be used in lieu of SCI. Surprisingly, we found that as sperm count increases the likelihood of a normal SPA increases. This study demonstrates that Kruger morphology assessment cannot be utilized to predict the results of or replace SCI in the management of the infertile couple. Supported by: Male Reproduction Special Funds. Department of Urology, Beth Israel Medical Center.

Pokeweed antiviral protein: a potential nonspermicidal prophylactic antiviral agent

Objective: To investigate the effects of pokeweed antiviral protein (PAP), a 29-kDa anti-human immunodeficiency virus (HIV) protein purified from the leaves of Phytolacca americana, on human sperm function. Design: Prospective, controlled study. Setting: Reproductive biology department. Patient(s): Seven sperm donors. Intervention(s): Human sperm and female genital tract epithelial cells were exposed to PAP ranging in concentration from 1 to 1,000 μg/mL. Main Outcome Measures: Effect of PAP on sperm motility, kinematics, and sperm penetration through bovine mucus, as well as binding, penetration, and fusion of zona-free hamster eggs. Results: Exposing human sperm to PAP (IC 50 p24 = 14 ± 2 nM) did not affect sperm motility and kinematics over a dose range of 1 to 1,000 μg/mL. Treating sperm with either 100 or 1,000 μg/mL of PAP had no effect on cervical mucus penetrability, nor did it affect sperm binding, penetration, and fusion of zona-free hamster eggs. PAP was noncytotoxic to genital-tract epithelial cells. Conclusions: The broad-spectrum antiviral agent PAP was nontoxic to human sperm and female genital tract epithelial cells even at a concentration 2,000 times higher than its IC 50 value against HIV-1. PAP has particular clinical usefulness both as a nonspermicidal intravaginal microbicide and as a prophylactic antiviral agent that can inactivate infective viruses and virus-infected cells in semen before assisted reproductive technology procedures are undertaken.

Specific localization in the equatorial region of gp20, a 20 kDa sialylglycoprotein of the capacitated human spermatozoon acquired during epididymal transit which is necessary to penetrate zona-free hamster eggs.

In this study we set out to characterize gp20, a 20 kDa glycoprotein of the human sperm surface, first identified by us by radiolabelling the sialic acid residues of the sperm surface [R. Focarelli et al. (1995), Mol. Hum. Reprod., 2, 2755-2759]. The protein was partially purified from pooled sperm samples of several healthy donors and used to raise a specific antiserum to study its localization in the reproductive system. When tested with freshly ejaculated spermatozoa, the anti-gp20 antibody intensely stained the head and midpiece. However, on capacitated spermatozoa the antigen was restricted to a sharp zone in the equatorial region. The antibody did not bind to differentiating germ cells but the antigen was present in epididymal epithelial cells and also in seminal plasma. Anti-gp20 exerted a blocking effect in a test for sperm penetration of zona-free hamster eggs, thus suggesting that gp20 is involved in the early stages of fertilization.

Expression of CD15 (Lewisx) antigen on human sperm and its role in sperm-egg interaction.

The carbohydrate epitope 3-fucosyl-N-acetyllactosamine (CD15) is a constituent of cell surface glycoconjugates that has been implicated in cell-cell adhesion mediated by carbohydrate-specific ligands. The present study was designed to investigate whether CD15 is present on human sperm and whether it plays a role in human sperm-egg interaction. Fluorescent flow cytometry was used to quantitate the binding of monoclonal antibodies (mAb) to sperm-bound CD15 and CD46 antigens on acrosome-intact (AI) and acrosome-reacted (AR) sperm. The location of the binding site of these mAbs was assessed by fluorescence microscopy. The effects of anti-CD15 and anti-CD46 mAbs on gamete interaction were tested utilizing both homologous (human zona binding and penetration) and heterologous (zona-free hamster egg binding and penetration) systems. The mean percentage of capacitated sperm which bound anti-CD15 or anti-CD46 mAbs was low (4.8% and 5.1%, respectively). Exposure to calcium ionophore A23187 (CaI) resulted in an increase in anti-CD15 (38.6 +/- 4%) and anti-CD46 (83.4 +/- 2%) binding to sperm. Both anti-CD15 and anti-CD46 binding sites were localized by fluorescence microscopy on the sperm acrosomal region. In four experiments, the percent of zona-free hamster eggs penetrated by human sperm were medium control 93% (62/66), irrelevant mAb 74% (70/94), anti-CD46 0% (0/107), and anti-CD15 10% (9/90). One hundred percent (6/6) of human zona were penetrated by human sperm exposed to medium control, 88% (8/9) following exposure to irrelevant mAb, 0% (0/11) following exposure to anti-CD46, and 50% (5/10) following exposure to anti-CD15. The mean (+/- SD of tightly bound sperm to hamster eggs were medium control 57 +/- 18%, irrelevant mAb 64 +/- 16%, anti-CD46 37 +/- 13%, and anti-CD15 19 +/- 10%. The corresponding values for human zona were: medium control 118 +/- 14%, irrelevant mAb 61 +/- 11%, anti-CD46 39 +/- 18%, and anti-CD15 99 +/- 19%. CD15 antigen is expressed on human sperm that have undergone acrosomal loss. mAb to CD15 was shown to inhibit significantly sperm binding and penetration of zona-free hamster eggs and penetration of human zona pellucida. These findings suggested that sperm-egg interaction may be mediated in part by the CD15 antigen. Capsule: Acrosome-reacted human sperm bind monoclonal antibodies specific for CD15 (Lewis(x)) epitope. The binding sites were located on the sperm head. Anti-CD15 antibody impaired both the binding and penetration of zona-free hamster eggs and the penetration of human zona by human sperm.

The effect of peritoneal fluid from patients with endometriosis on human sperm function in vitro

OBJECTIVE: Our purpose was to evaluate the effect of peritoneal fluid from women with endometriosis on sperm motility and function in an in vitro model. STUDY DESIGN: Peritoneal fluid was collected at laparoscopy from patients with and without endometriosis. Human donor sperm was diluted with this fluid, and its effect on sperm function and motility was measured with the zona-free hamster egg sperm penetration assay and computer-assisted semen analysis. RESULTS: The mean number of eggs penetrated by the sperm mixed with peritoneal fluid from patients with endometriosis was significantly fewer than the number penetrated by the sperm mixed with fluid from control patients (22.9 ± 5.31 vs 44.4 ± 4.96, p < 001, Student t test, n = 20). When evaluated by computer-assisted semen analysis, sperm mixed with peritoneal fluid from patients with endometriosis showed a significant decrease in mean swimming velocity compared with sperm mixed with peritoneal fluid from control patients (54.0 ± 1.77 vs 59.2 ± 105, p = 002, Student t test, n = 20). A significant increase in the fraction of sperm swimming at slower velocities was also found. A trend toward a positive correlation between eggs penetrated and sperm velocity was seen, but statistical significance was not achieved (correlation coefficient 0.4392, p = 0053, n = 20). CONCLUSION: These data suggest that substances found in the peritoneal fluid of patients with endometriosis could contribute to infertility through impairment of both sperm function and motion kinematics. (Am J Obstet Gynecol 1996;174:1779-85.)

Exposure of human sperm to low-calcium medium enhances fertilizing ability.

This investigation was conducted to evaluate whether exposure of human sperm to low calcium enhances the fertilizing ability. When sperm from normal semen samples were preincubated in low-calcium mBWW medium for 1 h, the percentages of live acrosome-reacted sperm after 4 and 6 h of culture were significantly higher than control (5.0 +/- 0.5 vs. 3.2 +/- 0.3% and 6.0 +/- 0.5 vs. 4.2 +/- 0.3%, p < .05). The penetration rate (%P) and fertility index (FI) in zona-free hamster egg sperm penetration assay significantly increased, compared to control (%P: 66.4 +/- 9.0 vs. 42.7 +/- 8.1%; FI: 1.69 +/- 0.19 vs. 1.34 +/- 0.11; p < .01). When low-calcium treatment was used for human in vitro fertilization and embryo transfer (IVF-ET) for male infertility, the fertilization and pregnancy rates were significantly higher than those in control. The results indicate that preincubation of human sperm in low-calcium medium enhances their fertilizing ability. This method is a simple and useful for preparing sperm from subfertile men for human IVF-ET.

Effects of nicotine on sperm attachment and penetration of zona-free hamster eggs.

A previous study from this laboratory showed that nicotine in vitro has deleterious effects on sperm motion characteristics. This study was conducted to evaluate the effects of nicotine on the ability of human spermatozoa to attach and penetrate zona-free hamster eggs. Spermatozoa from fertile donors, washed free of seminal plasma, were incubated with medium (control) and 0.1, 1, 5, and 10 mM concentrations of nicotine (concentrations estimated to approximate residual concentrations of nicotine in the testes of heavy smokers) for 18 h at 37 degrees C in a humid 5% carbon dioxide incubator. The sperm preparations were then mixed with enzymatically denuded hamster eggs and incubated for 3 h at 37 degrees C. The oocytes were examined by phase-contrast microscopy to enumerate the rates of sperm attachment and penetration. The data were analyzed by a paired t test and repeated measures analysis of variance using the arcsine transformation of the percentages. The percentages of eggs with attached spermatozoa significantly declined in a dose-dependent manner, the highest inhibition being at 10 mM (F = 24). The rate of sperm penetration was even more significantly decreased with the increase in nicotine concentrations in the following order: 10 mM (F = 56) > 5 mM (F = 30) > 1 mM (F = 44) > 0.1 mM (F = 12). Nicotine concentrations of 0.1 mM and above negatively affected sperm penetration of zona-free hamster eggs.

Study on the fertility of epididymal sperms

One approach to the treatment of obstructive azoospermic cases in whom either vasovasostomy cannot be performed or the vas deferens is absent is to construct an artificial spermatocele in the epididymis. By using this method to recover sperms for use in AIH, we have succeeded in achieving pregnancy in only 2 of 35 cases, leading us to investigate the fertility of the recovered sperms. Sperm fertility was investigated by the hypoosmotic swelling (HOS) test, observation by confocal laser-scanning microscope (CLSM), flow cytometry and hamster egg sperm penetration test. The percentage of swollen sperm determined by the HOS test was 15.0-83.3%, with a mean of 47.6 +/- 16.1%, significantly lower than the values of the normal group and the infertile male group, excluding cases of obstructed azoospermia. Examination by confocal laser-scanning microscope of samples stained with PI and FITC-PSA or PI and FITC-Con A stain revealed viable and dead spermatozoa as well as viable and dead acrosome-reacted spermatozoa. In addition, fertility was evaluated from the distribution of spermatozoa in each area from the flow cytometry percentage. This method was shown previously to be useful for the evaluation of fertility as it demonstrated the presence of numerous spermatozoa in the fertile area of cases who did not succeeded in pregnancy. The hamster egg sperm penetration test yielded a fertility rate of 0-25% with a mean of 8.2 +/- 10.0%, which was significantly lower than the value of the normal group, but was not significantly different from the infertile group. The findings of this study indicated that the fertility of epididymal sperms is low, thereby pointing to the need for studies to improve the materials used in the artificial spermatocele as well as the method of sperm recovery. Furthermore, our findings suggest that flow cytometry may be used to select the epididymal sperms with the highest fertility for sperm recovery.

Effect of sperm-associated antibodies on human sperm ability to bind to zona pellucida and to penetrate zona-free hamster oocytes

Antibodies directed against sperm membrane antigens located mainly over the sperm heads were eluted from the sperm cell fraction of autoimmune ejaculates and transferred to antibody-negative spermatozoa of fertile donors. The ability of these antibody-coated spermatozoa to bind to the human zona pellucida and to penetrate zona-free hamster oocytes was evaluated in vitro. The majority of the sperm-eluted samples contained both IgA and IgG antibodies. In order to evaluate the effect of each class of antibody on the analyzed sperm functions, each isotype was specifically absorbed before transfer. Sperm-binding to salt-stored zona pellucida, as assayed by FITC and TRITC labeling of antibody-free and antibody-coated spermatozoa incubated with the same zona, was consistently reduced by 60–85% by the five eluted samples tested. Removal of either IgA or IgG antibodies from the eluted samples did not change the overall effect. Sperm penetration of zona-free hamster eggs was variously affected by sperm-associated antibodies. Of the 8 samples of sperm-eluted antibodies tested, only 4 had a significant effect on sperm penetration. Three of them decreased the penetration by 67–78%, while the fourth exhibited a modest increasing effect of 39%. These four samples contained antibodies of the two isotypes. In the samples with a decreasing effect, the elimination of one or another of the two isotypes restored the ability of the sperm to penetrate the hamster oocytes. These results suggest that sperm-associated antibodies may have different effects on zona-binding and gametic fusion events that lead to fertilization. Whereas IgA and IgG antibodies taken together or separately decreased sperm binding to the human zona pellucida, the two classes of antibodies must be associated in order to impair sperm penetration into zona-free hamster oocytes.

Sperm surface fibronectin. Expression following capacitation.

The Arg-Gly-Asp (RGD) amino acid sequence plays a role in many cell-to-cell and cell-to-matrix adhesion systems, as a recognition sequence for cell membrane receptors termed integrins. Receptors of the VLA subfamily of integrins recognize fibronectin, laminin, and collagen. Given the authors' findings that fibronectin-derived, RDG-containing peptides competitively inhibit sperm-oolemmal adhesion and penetration in both heterologous (human-hamster) and homologous (hamster-hamster) gamete interactions, the expression of fibronectin on the surface of fresh, capacitated, and acrosome-reacted human spermatozoa was studied. The majority of fresh spermatozoa did not display fibronectin on their plasma membrane (0 to 16% positive), as demonstrated by the lack of binding of both monoclonal and polyclonal anti-fibronectin antibodies. In contrast, a significantly greater proportion of spermatozoa (varying between 18% to 100% for different donors) incubated overnight under capacitating conditions reacted with anti-fibronectin antibodies. The induction of an acrosome reaction with progesterone did not alter the proportion of sperm displaying fibronectin or its distribution on the sperm surface. A physiologic role of fibronectin in sperm-oolemmal interaction was suggested by the effects of anti-fibronectin antibodies on sperm oolemmal adhesion and penetration of hamster eggs by human spermatozoa, which were both significantly reduced (P less than 0.001).

IL-7 and IL-8 inhibit gamete interaction in the zona-free hamster egg sperm penetration assay

Current thought in reproductive endocrinology suggests hat endometriosis-associated subfertility may be the result of an adverse influence of activated immunocompetent cells on fertilization and embryo development. Inflammatory ediators such as interleukin-1 and tumour necrosis actor have been implicated in the pathophysiology of this process. The purpose of this study was to assess the effect of two recently characterized cytokines, interleukin-7 (IL-7 ) and interleukin-8 (IL-8), on gamete interaction in the perm penetration assay (SPA). Donor sperm were preincubated or 4 h with 0.5, 5, 50, or 500 ng ml-1 of human ecombinant IL-7 or IL-8. Sperm penetration was determined by an experienced gametologist by the presence of decondensed sperm heads or pronuclei formation. A dose-dependent inhibition of gamete interaction was observed following coincubation with either IL-7 or IL-8. These data offer the possibility that IL-7 and IL-8 may play a role in the pathogenesis of immunocompetent cell-associated subfertility.

Presence and Role of c-myc Proto-oncogene Product in Mammalian Sperm Cell Function1

The presence and role of c-myc protein was investigated in mature sperm cells of the human, mouse, and rabbit. The monoclonal antibodies against c-myc protein (c-myc) reacted with the acrosomal region of the sperm of these mammalian species in the indirect immunofluorescence technique. The c-myc monoclonal antibody (MCA) recognized c-myc protein of 62 and 64 kDa on Western blots of lithium diiodosalicylate-solubilized sperm preparations of these species. The c-myc MCA showed a dose-dependent inhibition of human sperm penetration of zona-free hamster eggs, inhibition of murine in vitro fertilization, and reduced in vivo fertilization in rabbits. There was no effect of the antibody on percent sperm motility, though the antibody significantly affected various motility characteristics such as mean and maximum amplitude of lateral head displacement and curvilinear velocity involved in hyperactivation phenomenon of human sperm cells. These results suggest that c-myc or c-myc-like protein is present in mature sperm cells and may have a role in sperm cell function especially in capacitation and/or acrosome reaction.

Effect of Smoking on Concentration, Motility and Zona-Free Hamster Test on Human Sperm

The effect of smoking on sperm concentration, motility, and zona-free hamster egg sperm penetration assay (SPA) was evaluated in 293 smokers of more than 10 cigarettes/day and 382 nonsmokers. Prerequisites for inclusion in the study were sperm concentration of greater than 10 x 10(6)/ml, sperm motility of greater than 30%, and normal morphology of greater than 60%. Although a lower sperm concentration was found in smokers, no significant difference was observed in sperm motility and SPA.

The Predictive Value of Zona-Free Hamster Egg Sperm Penetration Assay for Failure of Human in Vitro Fertilization and Subsequent Successful Zona Drilling

VAZQUEZ-LEVIN, MONICA; GARRISI, G. JOHN; KAPLAN, PAUL; GORDON, JON; SANDLER, BENJAMIN; NAVOT, DANIEL Author Information