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.

Spermatozoa normally encounter the egg at the fertilization site (in the Fallopian tube) within 24 hr after ovulation. A considerable fraction of the spermatozoa ejaculated into the female reproductive tract of mammals remains motionless in storage sites until ovulation, when the spermatozoa resume maximal motility and reach the fertilization site within minutes. The nature of the signal for sperm movement is not known, but one possible mechanism is attraction of spermatozoa to a factor(s) released from the egg. We have obtained evidence in favor of such a possibility by showing that human spermatozoa accumulate in follicular fluid in vitro. This accumulation into follicular fluid was higher by 30-260% than that observed with buffer alone and was highly significant (P < 10-8). Not all of the follicular fluids caused sperm accumulation; however, there was a remarkably strong correlation (P < 0.0001) between the ability of follicular fluid from a particular follicle to cause sperm accumulation and the ability of the egg, obtained from the same follicle, to be fertilized. These findings suggest that attraction may be a key event in the fertilization process and may give an insight into the mechanism underlying early egg-sperm communication.Spermatozoa normally meet the egg at the fertilization site (isthmic-ampullary junction of the Fallopian tube) within 24 hr after ovulation (1)(2)(3). In women, cervical spermatozoa have been reported to possess prolonged acrosomal integrity (in vivo), and this has led to the suggestion that the human cervix may serve as a site of sperm storage (4). In cattle, pigs, and rabbits it has been shown that a considerable fraction of the spermatozoa ejaculated into the female reproductive tract remain in the isthmus of the Fallopian tube until ovulation occurs; they then resume their motility and reach the fertilization site of the ampulla within minutes (2). The mechanisms for sperm selection and the synchronization between ovulation and sperm movement in the female reproductive tract are not known. One plausible mechanism for directed sperm movement would be the attraction of mammalian spermatozoa to factor(s) released from the egg. In vivo studies are currently difficult or impossible, and the many techniques available for measuring leukocyte chemotaxis in vitro (5) appear to be inadequate for studies on mammalian sperm chemotaxis, primarily because of the complexity and rapidity of sperm motion, which subsequently results in poor signal-to-noise ratios. On the other hand, microscopic methods have been useful for demonstrating chemoattraction of invertebrate spermatozoa to the egg (6, 7). Such studies in the mammal have not been reported to our knowledge. Here we examine the possibility of sperm attraction to follicular factor(s) by measuring sperm accumulation in follicular fluid in vitro. We show that human spermatozoa indeed accumulate in follicular fluid and that this accumulation correlates with the ability of the egg to be fertilized. MATERIALS AND METHODSPreparations o...

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.

We have developed a rapid and sensitive fluorimetric method, based on the formation of a fluorescent product from nitrosation of 2,3-diaminonaphthalene, for measuring the ability of bacteria to catalyze nitrosation of amines. We The concept that bacteria might participate in nitrosation (i.e., formation of an NNO bond to yield the N-nitroso derivative of amines) has been considered to some extent in most hypotheses concerning the possible role of endogenous nitrosation in cancer etiology (2,4,5,16,20,22,29). As recently as 1981, it was felt that the major contribution of bacteria to nitrosation was the reduction of nitrate to nitrite, resulting in an increase in the concentrations of nitrosating species (21). More recently, however, several groups have shown that bacteria can in fact participate directly in the nitrosation of amines, and this area has consequently gained renewed interest (4,5,16,17,18).A number of bacterial genera, including Neisseria, Pseudomonas, Escherichia, Klebsiella, Proteus, Alcaligenes, and Bacillus, have been investigated from several experimental perspectives and reported to have some ability to catalyze nitrosation (4,5,8,16,17,19,20,29). This catalysis is generally believed to be an anaerobic process which occurs in intact resting cells. It should probably be noted that, at this stage of research, it is not clear that all investigators have been examining the same catalytic activity.Typically, these studies have been concerned with the identification of bacterial strains (mostly clinical isolates) capable of nitrosation, rather than characterization of the reaction itself. We have thus begun a detailed investigation of this activity in Escherichia coli, which is well characterized from both biochemical and genetic perspectives. To facilitate the study of a number of bacterial strains under a wide range of reaction conditions, we have developed a new assay. This assay is based on the formation of a fluorescent product upon nitrosation of 2,3-diaminonaphthalene (31) as an alternative to the gas chromatographic-thermal energy analysis method (11) for following nitrosation of secondary amines.We have attempted to characterize the bacterial catalysis of nitrosation from a biochemical point of view by examining the effects of nitrite, nitrate, and electron donors on this process.

Spermatozoa normally encounter the egg at the fertilization site (in the Fallopian tube) within 24 hr after ovulation. A considerable fraction of the spermatozoa ejaculated into the female reproductive tract of mammals remains motionless in storage sites until ovulation, when the spermatozoa resume maximal motility and reach the fertilization site within minutes. The nature of the signal for sperm movement is not known, but one possible mechanism is attraction of spermatozoa to a factor(s) released from the egg. We have obtained evidence in favor of such a possibility by showing that human spermatozoa accumulate in follicular fluid in vitro. This accumulation into follicular fluid was higher by 30-260% than that observed with buffer alone and was highly significant (P < 10-8). Not all of the follicular fluids caused sperm accumulation; however, there was a remarkably strong correlation (P < 0.0001) between the ability of follicular fluid from a particular follicle to cause sperm accumulation and the ability of the egg, obtained from the same follicle, to be fertilized. These findings suggest that attraction may be a key event in the fertilization process and may give an insight into the mechanism underlying early egg-sperm communication.