Effect of antisperm antibodies present in human follicular fluid upon the acrosome reaction and sperm-zona pellucida interaction.

PROBLEM: To determine the ability of IgGs isolated from follicular fluids (hFFIgGs) to induce the acrosome reaction (AR) in human spermatozoa and to inhibit sperm-zona pellucida (ZP) interaction. METHOD OF STUDY: Incubation of capacitated spermatozoa with hFFIgGs (n = 40) and assessment of their effect on the AR or hemizona (HZ) assay in a condition that allows sperm-ZP interaction, avoiding acrosomal exocytosis. RESULTS: hFFIgGs from different women varied in their ability of inducing the AR. Those hFFIgGs with the highest AR-inducing capacity evoked the exocytotic response in most of the different sperm donors tested [high Induction Frequency (IF)]. Some of these antibodies were also able of inhibiting sperm binding to ZP [low HZ Index (HZI)]. A significant correlation was found between the IF and the HZI for each hFFIgG. CONCLUSIONS: Human follicular fluid contains antibodies capable of inducing the AR and inhibiting sperm-ZP binding, suggesting that they could be directed towards ZP receptors. hFFIgGs would constitute a tool for the identification of sperm entities involved in fertilization.

Identification of human sperm proteins involved in the interaction with homologous zona pellucida.

OBJECTIVE: To identify human sperm proteins involved in homologous zona pellucida (ZP) interaction. DESIGN: Prospective study. SETTINGS: Basic research laboratory. PATIENT(S): Semen samples from normozoospermic donors, tissue sections from surgical pieces, and ZP from nonfertilized oocytes. INTERVENTION(S): Antibodies for sperm proteins (HSE; high salt extract) were developed (anti-HSE) and partially characterized. Participation of sperm proteins on ZP-interaction was tested with the hemizona assay (HZA). Antigens were immunolocalized in sperm and tissues. MAIN OUTCOME MEASURE(S): Sperm and tissue immunostaining; Western blotting; and number of sperm bound to the ZP. RESULT(S): Anti-HSE antibodies recognized several polypeptides in HSE (9 to 200 kd). Specific antibodies for 49 and 66 kd proteins (p49, p66) were obtained. Both (anti-p49 and anti-p66) stained the head of ejaculated and capacitated sperm. In the HZA, sperm preincubation with a mixture of anti-p49 and anti-p66 (100 micro g/mL) resulted in a decrease in the number of spermatozoa bound to the ZP. Presence of p66 (10 micro g/mL) inhibited sperm-ZP interaction. In contrast, p49 did not alter sperm binding to the ZP. Immunohistochemical analysis showed that p66 is present in the epididymis. No staining was observed in testicular sections. CONCLUSION(S): We found that p66 is an epididymal protein that participates in human sperm interaction with homologous ZP.

Calcium requirements for human sperm function in vitro.

OBJECTIVE: To determine extracellular calcium (Ca(2+)) requirements for the maintenance of human sperm function in vitro. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): Normozoospermic volunteers provided fresh semen samples; follicular fluid (human FF) and oocytes were collected from women undergoing IVF-ET. INTERVENTION(S): Spermatozoa were incubated for /=0.1 mM of Ca(2+) were able to undergo the AR when exposed to human FF in the presence of 2.5 mM of Ca(2+). Calcium concentrations of >/=0.22 mM were sufficient to reach protein tyrosine phosphorylation levels and hyperactivated motility values similar to those of controls. Higher Ca(2+) concentrations (>/=0.58 mM) were required to produce maximum human FF-induced AR in previously capacitated cells and to obtain an adequate sperm-ZP binding. CONCLUSION(S): Different steps of the fertilization process have distinctive Ca(2+) requirements. Whereas 0.22 mM of Ca(2+) is sufficient for the development of some capacitation-related events, human FF-induced AR and sperm-ZP interaction require 0.58 mM of this cation.

Effect of incubating human sperm at room temperature on capacitation-related events.

OBJECTIVE: To determine the effect of human sperm incubation at room temperature (20 degrees C) upon capacitation-related events. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): Semen samples were obtained from normozoospermic volunteers. Human follicular fluid (hFF) was collected from women undergoing assisted reproductive treatment. INTERVENTION(S): Spermatozoa were incubated for up to 18 hours at 20 degrees C and/or 37 degrees C. MAIN OUTCOME MEASURE(S): Protein tyrosine phosphorylation patterns, development of hyperactivated motility, and induction of acrosome reaction (AR) in response to hFF. RESULT(S): Spermatozoa incubated for 18 hours at 20 degrees C showed an array of tyrosine phosphorylated proteins similar to noncapacitated cells. After incubation at 20 degrees C, the percentage of spermatozoa displaying hyperactivated motility and undergoing acrosomal loss in response to hFF was significantly lower when compared with cells kept the same time at 37 degrees C. Conversely, spermatozoa incubated overnight at 37 degrees C could respond to hFF, either at 37 degrees C or 20 degrees C. When preincubation at 20 degrees C was followed by sperm exposure to 37 degrees C, capacitation-related events could be activated. In capacitated cells (16 hours at 37 degrees C), 2-hour incubation at 20 degrees C led to a significant decrease in acrosome reaction inducibility, suggesting sperm decapacitation. CONCLUSION(S): Human sperm incubation at room temperature does not allow capacitation, although it does not affect hFF-induced acrosome reaction in capacitated cells. The blocking effect is overcome when spermatozoa are exposed to 37 degrees C.

Identificación de proteínas espermáticas humanas involucradas en la interacción espermatozoide-zona pellucida / aaa

La interacción inicial entre las gametas es mediada por proteínas de superficie de la cabeza del espermatozoide y de la matriz extracelular del ovocito, la zona pellucida (ZP). El presente trabajo tuvo como objetivo la identificación de antígenos de superficie de espermatozoides humanos potencialmente involucrados en el reconocimiento de la ZP. Para ello se obtuvo un extracto de proteínas periféricas de espermatozoides humanos por tratamiento de las células con solución de alta fuerza iónica (HSE - High Salt Extract) (Buffer Pipes 100mM, pH 7,4; NaCl1M, sacarosa 0,25 M). Se desarrollaron anticuerpos policlonales (anti-HSE) que reconocieron numerosas proteínas en HSE (9-200 KDa). Las proteínas fueron separadas por electroforesis en geles de poliacrilamida, transferidas a membranas de nitrocelulosa y utilizadas para adsorber inmunoglobulinas de anti-HSE. Con éste método se obtuvieron anticuerpos contra dos polipéptidos mayoritarios de 49 (p49) y 66 (p66) kDa. Ambos anticuerpos (anti-p49 y anti-p66) reconocieron epitopes localizados en la cabeza y el flagelo de espermatozoides eyaculados y capacitados...

Identificación de proteínas espermáticas humanas involucradas en la interacción espermatozoide-zona pellucida / aaa

La interacción inicial entre las gametas es mediada por proteínas de superficie de la cabeza del espermatozoide y de la matriz extracelular del ovocito, la zona pellucida (ZP). El presente trabajo tuvo como objetivo la identificación de antígenos de superficie de espermatozoides humanos potencialmente involucrados en el reconocimiento de la ZP. Para ello se obtuvo un extracto de proteínas periféricas de espermatozoides humanos por tratamiento de las células con solución de alta fuerza iónica (HSE - High Salt Extract) (Buffer Pipes 100mM, pH 7,4; NaCl1M, sacarosa 0,25 M). Se desarrollaron anticuerpos policlonales (anti-HSE) que reconocieron numerosas proteínas en HSE (9-200 KDa). Las proteínas fueron separadas por electroforesis en geles de poliacrilamida, transferidas a membranas de nitrocelulosa y utilizadas para adsorber inmunoglobulinas de anti-HSE. Con éste método se obtuvieron anticuerpos contra dos polipéptidos mayoritarios de 49 (p49) y 66 (p66) kDa. Ambos anticuerpos (anti-p49 y anti-p66) reconocieron epitopes localizados en la cabeza y el flagelo de espermatozoides eyaculados y capacitados...(AU)