In humans, only a small fraction (2-12%) of a sperm population can respond by chemoattraction to follicular factors. This recent finding led to the hypothesis that chemotaxis provides a mechanism for selective recruitment of functionally mature spermatozoa (i.e., of capacitated spermatozoa, which possess the potential to undergo the acrosome reaction and fertilize the egg). This study aimed to examine this possibility. Capacitated spermatozoa were identified by their ability to undergo the acrosome reaction upon stimulation with phorbol 12-myristate 13-acetate. Under capacitating conditions, only a small portion (2-14%) of the spermatozoa were found to be capacitated. The spermatozoa were then separated according to their chemotactic activity, which resulted in a subpopulation enriched with chemotactically responsive spermatozoa and a subpopulation depleted of such spermatozoa. The level of capacitated spermatozoa in the former was -13-fold higher than that in the latter. The capacitated state was temporary (50 min < life span < 240 min), and it was synchronous with the chemotactic activity. A continuous process of replacement of capacitated/chemotactic spermatozoa within a sperm population was observed.Spermatozoa that had stopped being capacitated did not become capacitated again, which indicates that the capacitated state is acquired only once in a sperm's lifetime. A total sperm population depleted of capacitated spermatozoa stopped being chemotactic. When capacitated spermatozoa reappeared, chemotactic activity was restored. These observations suggest that spermatozoa acquire their chemotactic responsiveness as part of the capacitation process and lose this responsiveness when the capacitated state is terminated. We suggest that the role of sperm chemotaxis in sperm-egg interaction in vivo may indeed be selective recruitment of capacitated spermatozoa for fertilizing the egg.Human spermatozoa are attracted to follicular factors in vitro, and the attraction is correlated with egg fertilizability (1). The attraction results from chemotaxis and is accompanied by speed enhancement (ref. 2; for reviews, see refs. 3 and 4). Unlike the case of species with external fertilization in which most, if not all, of the sperm population is chemotactically responsive (for reviews, see refs. 5 and 6), in humans only a small fraction of the sperm population (2-12%) is chemotactically responsive at any given time (7). The identity of the responsive spermatozoa in humans changes with time by turnover: chemotactic spermatozoa lose their activity while others acquire it (7). This raised the possibility that spermatozoa are selectively chemotactic only at a certain physiological stage. The capacitated stage-i.e., the stage at which spermatozoa possess the potential to undergo the acrosome reaction (a release of proteolytic enzymes enabling sperm penetration through the egg coat) and to fertilize the egg (for recent reviews, see refs. 8-13)-seemed a reasonable possibility (3, 7). This study investigates this possibility an...

Human spermatozoa accumulate in vitro in diluted follicular fluids obtained from follicles from which the eggs have been fertilized. Using capillary assays under a variety of experimental conditions (ascending or descending gradients of follicular fluid, or no gradient at all) and microscopic assays in which individual spermatozoa could be followed, we found that the sperm accumulation in follicular fluid was the result of both sperm chemotaxis and chemokinesis and eventually hyperactivation-like motility. We determined the optimal conditions for sperm accumulation, which involved sperm preincubation (possibly to induce sperm capacitation) and proper dilution of follicular fluid. In all the assays, the net accumulation was low, probably reflecting the chemotactic responsiveness of only a small fraction of the sperm population at any given time. We partially fractionated follicular fluid in a Centricon microconcentrator (Amicon, Danvers, MA) and by acetone precipitation, and found that at least one of the chemotactic factors is a small (< 10-kDa) molecule that is probably nonhydrophobic. This is the first time that sperm chemotaxis and chemokinesis in response to a follicular factor(s) in mammals has been established and has been distinguished from other processes that might cause sperm accumulation. The physiological significance of these findings is discussed.

Recent studies have indicated that human spermatozoa respond to follicular fluid by attraction to chemotactic factor(s) in the fluid, accompanied by enhancement of motility and ultimately hyperactivation. In this study, we quantified the sperm response. We exposed spermatozoa to a gradient of a chemotactically active fraction of follicular fluid (denoted as "the attractant") and separated the spermatozoa that accumulated in the attractant and those that did not. We thus obtained two subpopulations: one enriched with chemotactically responsive spermatozoa, and one deficient in such spermatozoa. The fraction of the responsive spermatozoa out of the total sperm population was 2-12% at any measured time point. With time, the responsive spermatozoa lost their ability to be attracted, while such activity was gradually acquired by the subpopulation originally deficient in responsive spermatozoa. These results indicate that the identity of responsive spermatozoa is continuously changing. If the in vitro results are representative of the physiological conditions in vivo, they imply that the role of sperm chemotaxis combined with enhanced motility may be to select capacitated spermatozoa and bring them to the egg. Such a mechanism may, over an extended period of time, increase the prospect that an egg will meet capacitated spermatozoa as soon as it ovulates.

Mast cells respond to clustering of the type I Fc epsilon receptor (Fc epsilon RI) on their membranes by mediator secretion. Recently, a marked enhancement of tyrosine phosphorylation on several proteins has been observed as a result of antigen-induced Fc epsilon RI aggregation on these cells. We report here that the phosphatidyl inositide specific phospholipase C gamma 1 (PLC gamma 1) is one of the prime proteins that undergoes tyrosine phosphorylation as a result of this stimulus. This was determined by immunoprecipitation of phosphotyrosine containing proteins from detergent lysates of rat mucosal mast cells (rat basophilic leukemia cells, subline 2H3; RBL-2H3) and Western blotting analysis of the separated components. A fast appearance of phosphorylated tyrosine residues on PLC gamma 1 was observed, reaching its maximal intensity at approximately 1-3 min after stimulation and declined afterwards to basal levels. Moreover, the phosphorylation depended on maintaining the aggregated Fc epsilon RI as did other cellular responses (e.g. phosphatidyl inositides hydrolysis and secretion). The time course of both Fc epsilon RI induced phospholipase C gamma 1 activation, as monitored by the formation of inositol phosphates, and of the secretory response of the cells followed that of the PLC gamma 1 phosphorylation. Furthermore, the tyrphostin AG490, a protein tyrosine kinase inhibitor, caused similar inhibition of the Fc epsilon RI-induced PLC gamma 1 phosphorylation, inositol phosphates formation, and mediator secretion. Significantly, no tyrosine phosphorylation of PLC gamma 1 was induced by the Ca2+ ionophore, ionomycin, even at doses that cause optimal secretory response.(ABSTRACT TRUNCATED AT 250 WORDS)

The acrosome reaction (AR)^an essential step in mammalian fertilization^can occur, according to the consensus, only in capacitated spermatozoa. In apparent contrast, recent reports have demonstrated that human spermatozoa incubated in vitro in an albumin-free medium and therefore believed to be noncapacitated, do undergo the AR. With the aim of determining unequivocally whether or not capacitation is required for the AR and whether albumin is essential for capacitation, we compared the potential to undergo partial and complete AR (induced by phorbol myristate ester or by the Ca 2+ ionophore A23187) between human spermatozoa incubated in a capacitating medium, albumin-free medium, and non-capacitating medium. The results clearly demonstrate that capacitation is, after all, a prerequisite for both partial and complete AR. Albumin, on the other hand, is essential only for acquiring the capacity to undergo complete, not partial AR.z 1998 Federation of European Biochemical Societies.