Patricio Morales

Acrosome intact and acrosome-reacted human sperm can initiate binding to the zona pellucida.

Mammalian sperm must be acrosome reacted before penetrating the zona pellucida. In some species the sperm undergo the acrosome reaction before binding to the zona pellucida and in other species only acrosome intact sperm can initiate binding to the zona. In this study we addressed the question of acrosomal status and sperm-zona binding with human gametes. Sperm acrosome reactions were induced by treatment with human follicular fluid or N-(6-amino-hexyl)-5-chloro-naphthalene sulfonamide (W-7). The sperm suspensions, containing various percentages of acrosome-reacted sperm, were then incubated with human oocytes for 1 min. The acrosomal status of the sperm population bound to the zona was similar to the acrosomal status of the population of sperm in suspension (R2 = 0.77), regardless of the treatment to induce acrosome reactions. Our interpretation of these results is that both acrosome intact and acrosome-reacted human sperm can initiate binding to the zona pellucida. However, we reported earlier (N. L. Cross, P. Morales, J. W. Overstreet, and F. W. Hanson, 1988, Biol. Reprod. 38, 235-244) that the human zona pellucida is able to induce acrosome reactions. Thus, to exclude the possibility that sperm had undergone the acrosome reaction on the zona within 1 min of binding, sperm were suspended in a nominally calcium-free Tyrodes medium (0 Ca-mTyr) before incubation with oocytes (this medium was supplemented with SrCl2 and spermine to support sperm motility and zona binding). In 0 Ca-mTyr, the proportion of acrosome-reacted sperm on the zona was still highly correlated with the proportion of reacted sperm in suspension, indicating that the sperm were reacted before binding. Evidence that 0 Ca-mTyr effectively inhibited acrosome reactions induced by the zona pellucida was derived from experiments in which sperm were treated with human follicular fluid or control medium and the suspensions were diluted with either 0 Ca-mTyr or control medium.4+ Human oocytes were added for 1 min (pulse) at which time some oocytes were fixed and other oocytes were transferred to sperm-free medium and incubated for 35 min (chase) before fixation. Sperm diluted in control medium, pretreated with either human follicular fluid or control medium, showed a similar increase (40%) in the percentage of acrosome reactions among the zona-bound sperm after the chase. Sperm diluted in 0 Ca-mTyr did not show an increase in the percentage of acrosome-reacted sperm on the zona pellucida after the chase.(ABSTRACT TRUNCATED AT 400 WORDS)

A new procedure for determining acrosomal status of very small numbers of human sperm.

The acrosome of human sperm cannot be easily distinguished by light microscopy. Although several techniques are now available to label the acrosomal region of human sperm and report acrosomal status, they generally require large numbers of sperm. We describe here a new procedure in which sperm are collected and treated on small-pore filters. The acrosomal region is then labeled using fluoresceinated lectin. The main advantage of this method is that it enables the study of the acrosomal status of sperm in samples with very low sperm concentration.

N,N'-Dithiobisphthalimide, a disulfide aromatic compound, is a potent spermicide agent in humans.

Several studies have shown that users of vaginal preparations containing nonoxynol-9 (N-9) are at a high risk for sexually transmitted diseases, including HIV. Therefore, there is a great interest in identifying compounds that can specifically inhibit sperm without damaging the vaginal lining, possess a powerful spermicide activity, and can be used in contraceptive vaginal preparations to replace N-9. In this work, we studied the spermostatic and/or spermicidal activity of five non-detergent, disulfide compounds on human sperm, HeLa cells, and Lactobacillus acidophilus. The motility and viability of human sperm in semen and culture medium was evaluated after treatment with different concentrations of the disulfide compounds (2.5 – 100 µM). In addition, we evaluated the cytotoxic effect on HeLa cells and L. acidophilus. We identified compound 101, N,N-dithiobisphthalimide (No. CAS 7764-30-9), as the most effective molecule. It has a half maximal effective concentration (EC50) of 8 µM and a minimum effective concentration (defined as the concentration that immobilizes 100 percent of the sperm in 20 sec) of 24 µM. At these concentrations, compound 101 does not affect the viability of the sperm, HeLa cells, or L. acidophilus. Our results indicate that dithiobisphthalimide has a potent spermostatic, irreversible effect with no toxic effects on HeLa cells and L. acidophilus.

Mechanisms of filtration of morphologically abnormal human sperm by cervical mucus

It is well known that cervical mucus restricts penetration of morphologically abnormal human sperm, both in vitro and in vivo. However, the mechanisms of such restriction are not well understood. Using videomicrography to simultaneously analyze the motions and morphology of individual human sperm, we analyzed differential penetration of normal and abnormal sperm into fresh human cervical mucus. Abnormal sperm swam slower in mucus than the normal sperm, but their flagellar beat parameters were not commensurately different. Multivariate statistical analysis of the relationship between individual sperm velocity and flagellar beat parameters indicated that the heads of the abnormal sperm experienced greater resistance from the mucus than did normal heads. Differential mucus resistance, more than altered motile vigor, appears to be responsible for the restriction of abnormal sperm during migration through mucus.

Sperm capacitation in the human female reproductive tract**Supported by the National Institutes of Health grants HD-15149 and HD-03402.

Cette etude montre que les spermatozoides humains peuvent etre capacites in vivo dans le mucus cervical. En effet, ces spermatozoides, a linverse de ceux capacites in vitro dans des milieux de culture enrichis en albumine, sont capables de penetrer les ovocytes humains immatures et lovocyte depelluride dhamster. Ces faits montrent que le processus de capacitation des spermatozoides dont la complexite demeure extreme differe certainement in vivo et in vitro