Fluorometric study of rabbit sperm head membrane phospholipid asymmetry during capacitation and acrosome reaction using Annexin-V FITC.

This study was conducted to evaluate phosphatidylserine translocation in head plasma membrane of Percoll-gradient purified of rabbit cauda epididymal sperm during capacitation and acrosome reaction (AR) using Annexin-V. Propidium iodide was used as control to reject dead or dying cells. The presence and distribution of Annexin-V binding sites were analyzed using flow fluorocytometry and confocal microscopy. After 6 h of incubation of sperm in capacitation medium, the number of cells positively stained with Annexin-V showed a small but significant increment. The Annexin-V binding sites produced during capacitation were found mainly in the post-acrosomal region of the sperm head plasma membrane. After AR induction with progesterone, the localization of phosphatidylserine was changed and the Annexin-V binding sites were found almost only in the acrosomal region, but with higher number of binding sites in the equatorial area. On the contrary, after AR induction with A23187, phosphatidylserine translocation, although predominant over the acrosomal region, was also observed in the post-acrosomal region. Plasma membrane destabilization during capacitation and AR may be important for sperm-oocyte fusion.

[Evaluation and preparation of gametes]. / Evaluación y preparación de gametos.

The aim of this paper is to review some topics related to the management and evaluation of the male and female gametes to be used in any assisted reproduction procedure. First, the international criteria for the quantitative morphological and biochemical evaluation of the semen samples are presented, both for the initial analysis and when chosen procedure is performed. Next, we discuss the biochemical characteristics of the solutions used to in vitro induce capacitation and acrosome reaction, indispensable events previous to the in vivo or in vitro interaction of the gametes. We also present the methodology for the in vitro management of the spermatozoa and to evaluate the efficiency of these cells to achieve in vitro acrosome reaction, as well as the utilization of the zone-free hamster egg penetration test to evaluate the human sperm capacity to penetrate the female gamete. Finally, the morphological characteristics of the cumulus oophorus-corona radiate-zona pellucida-vitellus complexes are analyzed, as well as the biochemical and hormonal composition aspiration, in order to assess the quality of the recovered oocytes, their probabilities to be fertilized and their potential to develop into subsequent stages which culminate with the birth of full term, viable and healthy babies.

[Kinetics of the proteolytic activity of intact human spermatozoa]. / Estudio cinético de la actividad proteolítica del espermatozoide humano intacto

Seminal plasma free intact human washed spermatozoa have hydrolytic activity on N-benzoil-D-L-arginine-naftilamide (BANA). Kinetic activities of this enzymatic activity are similar to those described for acrosine. Therefore, it is likely that it would be the same enzyme. BANA- hydrolytic activity might be liberated to the medium by incubation of pH lower than 4.0 and reincorporates to the membrane after adjustment of the medium to alkaline pH. pH- dependant enzyme liberation curve is similar to titration curve of sialic acid attached to the membrane. Attachment of the enzyme to the spermatic membrane diminishes its specific activity. However, if spermatozoa are previously treated with neuraminidase, its membrane afinity is inhibited. It is thought that this enzyme in the spermatozoon is present as protein ionically bound to the membrane and that sialic acid participates in this union. It is postulated that drastic treatments such as the use of ultrasound or detergents produce redistribution of specific proteins.

PIP: Intact human spermatozoa free of seminal plasma and washed have a hydrolytic action on N-benzoyl-D-L-arginine-beta-naphtylamide(BANA). The kinetic properties of this enzyme activity are similar to those described with reference to acrosin, which suggests the possibility that the same enzyme is involved. The hydrolytic action on BANA can be relea sed into the medium by incubation at pH lower than 4.0 and be returned to the membrane when the pH of the medium is changed to alkaline. The release curve of the pH-dependent enzyme is similar to the titration curve of sialic acid attached to the membrane. When the enzyme is attached to the sperm membrane, its specific activity decreases, but if the spermatozoa are subjected to prior treatment with neuraminidase the membrane-enzyme affinity is inhibited. The findings show that the enzyme in the spermatozoon is ion-bound to the membrane, and that sialic acid plays a role in this bond. It is suggested that drastic treatment, such as ultrasonic waves or the use of detergents, causes a redistribtuion of specific proteins, and that copper IUDs may be effective contraceptives by inhibiting the peptidase activity of spermatozoa.