Nicholas L. Cross

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)

Studies of the voltage-dependent polyspermy block using cross-species fertilization of amphibians☆

Fertilization of frog eggs by frog sperm is inhibited if the eggs membrane potential is positive (N.L. Cross and R.P. Elinson, 1980, Dev. Biol. 75, 187-198); however, fertilization of salamander eggs by salamander sperm does not depend on membrane potential (M. Charbonneau, M. Moreau, B. Picheral, J.P. Vilain, and P. Guerrier, 1983, Dev. Biol. 98, 304-318). Since salamander sperm can fertilize frog eggs, we have investigated whether this cross-fertilization is voltage dependent. If, during insemination with Notophthalmus sperm, Xenopus eggs were voltage clamped between +7 and +20 mV, fertilization proceeded in 7/10 (70%) of the clamped eggs, compared to 38/48 (79%) of the neighboring eggs. In control experiments in which voltage-clamped Xenopus eggs were inseminated with Xenopus sperm, fertilization proceeded in only 1/10 (10%) of the clamped eggs, compared to 59/60 (98%) of the neighbors. Similar results were obtained with cross-fertilization experiments between Notophthalmus sperm and Rana eggs. These experiments indicate that the voltage dependence of fertilization depends on the species of sperm.

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.

Sperm binding activity of the zona pellucida of immature mouse oocytes

Immature oocytes taken from ovarian follicles are sometimes used in studies of sperm-zona interaction in species for which it is difficult to obtain ovulated eggs. As yet, however, there has been no quantitative comparison of the sperm binding capacities of immature and ovulated oocytes. We report here that in mice there is no significant difference in the numbers of sperm which bind to the zonae pellucidae of immature and ovulated oocytes in vitro. These results support the use of immature oocytes in studies of sperm-zona interaction. We have also analyzed the sources of variability in sperm binding assays, and we make suggestions for the most efficient design of experiments.

A novel form of sperm detachment from eggs of Urechis caupo

Abstract Urechis sperm bind to the eggs extracellular envelope via acrosomally derived adhesive material. Sperm initially adhere tightly, but within minutes they loosen from the egg envelope. Using transmission electron microscopy, we have determined that the sperms binding material disperses during the detachment phase. Dispersion appears to be caused by the egg and to be necessary for sperm detachment, because sperm bound to glutaraldehyde-fixed eggs do not detach, nor does their binding material disperse. The binding material also fails to disperse when sperm are artificially acrosome reacted in the absence of eggs, using an ionophore. Our results suggest a form of sperm detachment not previously described: an egg-induced removal of the binding material.

Correlation of acrosomal status and sperm performance in the sperm penetration assay*†*Presented in part at the Forty-Third and Forty-Fourth Annual Meetings of The American Fertility Society, September 28 to 30, 1987, Reno, Nevada, and October 10 to 13, 1988, Atlanta, Georgia.†Supported in part by grant HD 19587 to N.L.C. from the National Institutes of Health, Bethesda, Maryland.

The sperm of some infertile men are unable to penetrate zona pellucida-free hamster oocytes but gain that ability after treatment with human follicular fluid (hFF). We asked whether altered incidences of acrosome reacted sperm explained these observations. Patient sperm failing to penetrate oocytes had fewer acrosome reactions than did healthy males, but the percentage reacted was not correlated with oocyte penetration. Sperm incubated 3 hours, then exposed to hFF, exhibited increased penetrations for 7 of 10 males, without an increase in percentage reacted sperm. Sperm incubated 22 hours before hFF treatment had penetrating ability enhanced 250- to 1000-fold, but the percentage reacted increased only sixfold. We conclude that factors other than the percentage reacted sperm are the major determinants of penetration capacity.

Detection of antisperm antibodies: their localization to human sperm antigens that are transferred to the surface of zona-free hamster oocytes during the sperm penetration assay**Supported by National Institutes of Health grants HD-15149 and HD-19587.

The authors have developed an extension of the sperm penetration assay for detecting serum immunoglobulins to sperm antigens that are transferred to the plasma membrane of a sperm-penetrated hamster oocyte. After the hamster oocytes have been scored for sperm penetration by observing for the presence of swollen sperm heads, they are incubated in serum followed by either a 20-minute treatment with rhodamine-conjugated protein A (which binds to most subclasses of IgA, IgG, and IgM) or a 2-hour incubation in guinea pig serum (complement). Positive fluorescence indicates that the serum contains antibodies to sperm antigens that were transferred to the surface of an oocyte during gamete fusion. Complement-mediated lysis indicates that the immunoglobulin that is bound can also fix complement. The advantages of this assay for detection of serum antisperm antibodies are that it is an extension of a widely used assay, is rapid and requires readily available reagents and equipment, can detect most subclasses of IgA, IgG, and IgM, detects antibodies to those sperm antigens that may be transferred to the oocyte during fertilization, and indicates whether the detected antisperm antibodies can mediate complement-dependent lysis of the fertilized oocyte.