Mammalian sperm chemotaxis and its association with capacitation

Much progress has been made in recent years in establishing mammalian sperm chemotaxis and understanding sperm capacitation. Thus far, chemotaxis to follicular fluid has been established by a variety of means in human and mouse spermatozoa. It was found that only a small fraction of a given sperm population (averaging around 10%) is chemotactically responsive and that this fraction constitutes capacitated (ripe) spermatozoa. Both the chemotactic responsiveness and the capacitated state are transient (with a lifetime of 50 min to 4 h) and they occur only once in the sperm's lifetime. It has been proposed that the role of sperm chemotaxis in mammals (at least in humans) is selective recruitment of capacitated spermatozoa for fertilizing the egg, and that the role of the continuous replacement of chemotactic/capacitated spermatozoa is to prolong the time during which capacitated spermatozoa are available in the female reproductive tract. The sperm chemoattractants have not been identified, but they appear to be heat‐stable peptides. Although the molecular mechanism and the in vivo location of sperm chemotaxis are not known, a number of possible mechanisms and locations are discussed. Dev. Genet. 25:87–94, 1999. © 1999 Wiley‐Liss, Inc.     

Le chimiotactisme des spermatozoïdes a-t-il un rôle dans la fécondation?

Sperm chemotaxis to follicular fluid has been established by a variety of means in human and mouse spermatozoa. It was found that only a small fraction of a given sperm population (averaging around 10%) is chemotactically responsive and that this fraction constitutes capacitated (ripe) spermatozoa. Both the chemotactic responsiveness and the capacitated state are transient (with a lifetime between 50 min and 4 h) and they occur only once in the sperm’s lifetime. It has been proposed that the role of sperm chemotaxis in mammals (at least in man) is selective recruitment of capacitated spermatozoa for fertilizing the egg, and that the role of the continuous replacement of chemotactic/capacitated spermatozoa is to prolong the duration of time over which capacitated spermatozoa would be available in the female reproductive tract. The sperm chemoattractants have not been identified but they appear to be heat-stable peptides. Thein vivo location of sperm chemotaxis is not known; a number of possible locations are discussed.     

Chemotactic response of frozen-thawed bovine spermatozoa towards follicular fluid

The aim of this study was to verify whether cattle spermatozoa respond by chemotaxis to follicular fluid (FF). The experimental conditions were defined to maintain a frozen-thawed sperm population with great motility and capacitation, and lesser sperm agglutination. Several sperm preparation conditions were studied: sperm separation from the seminal plasma by Sephadex column or migration-sedimentation, incubation under capacitating conditions in the presence or absence of a superficial layer of mineral oil, and different pH of the culture medium. The percentage of motile and agglutinated spermatozoa was determined in plate dishes under inverted phase contrast microscope. The percentage of capacitated spermatozoa was calculated as the difference between the percentages of acrosome reacted spermatozoa with and without lysophosphatidylcholine stimulation. The most ideal experimental conditions to evaluate chemotaxis in frozen-thawed cattle spermatozoa were: to separate the cells from the seminal plasma by migration-sedimentation and to incubate them under oil, in culture medium at pH 7.2, for less than 2h. The chemotaxis assays were conducted with spermatozoa treated as mentioned above which were confronted to several dilutions of FF (1:10(3), 1:10(4), 1:10(5), 1:10(6)) in a chemotaxis chamber by videomicroscopy and computer image analysis. A subpopulation of capacitated spermatozoa ( approximately 10%) that responded chemotactically to a concentration gradient generated by FF (1:10(4) to 1:10(5)) was observed. Since cryopreserved spermatozoa are regularly used to artificially inseminate the cows, the sperm chemotactic response towards FF would be potentially used to diagnose the bull sperm sample or to select the spermatozoa in the most functional state.     

Do human eggs attract spermatozoa?

A key process in human fertilization is bringing the two gametes together, so that the complex molecular events involved in sperm and egg interaction can begin. Does nature allow fertilization to occur only as a consequence of a chance collision, or is there a precontact sperm-egg communication? This review summarizes the bioassays used in testing human spermatozoa for chemotaxis, emphasizing the necessity to distinguish between chemotaxis and other accumulation-causing processes, and the results obtained. It demonstrates that human sperm chemotaxis to a follicular factor(s) does occur, at least in vitro, and that only capacitated spermatozoa are chemotactically responsive. Substances that have been proposed as attractants for human spermatozoa are reassessed. The potential role of sperm chemotaxis in vivo is discussed. Faulty precontact sperm-egg communication may be one of the causes of male infertility, female infertility, or both. On the other hand, interfering with human sperm chemotaxis may represent an exciting new approach to contraception. BioEssays 21:203–210, 1999. © 1999 John Wiley & Sons, Inc.     

Progesterone from the cumulus cells is the sperm chemoattractant secreted by the rabbit oocyte cumulus complex

Sperm chemotaxis in mammals have been identified towards several female sources as follicular fluid (FF), oviduct fluid, and conditioned medium from the cumulus oophorus (CU) and the oocyte (O). Though several substances were confirmed as sperm chemoattractant, Progesterone (P) seems to be the best chemoattractant candidate, because: 1) spermatozoa express a cell surface P receptor, 2) capacitated spermatozoa are chemotactically attracted in vitro by gradients of low quantities of P; 3) the CU cells produce and secrete P after ovulation; 4) a gradient of P may be kept stable along the CU; and 5) the most probable site for sperm chemotaxis in vivo could be near and/or inside the CU. The aim of this study was to verify whether P is the sperm chemoattractant secreted by the rabbit oocyte-cumulus complex (OCC) in the rabbit, as a mammalian animal model. By means of videomicroscopy and computer image analysis we observed that only the CU are a stable source of sperm attractants. The CU produce and secrete P since the hormone was localized inside these cells by immunocytochemistry and in the conditioned medium by enzyme immunoassay. In addition, rabbit spermatozoa express a cell surface P receptor detected by western blot and localized over the acrosomal region by immunocytochemistry. To confirm that P is the sperm chemoattractant secreted by the CU, the sperm chemotactic response towards the OCC conditioned medium was inhibited by three different approaches: P from the OCC conditioned medium was removed with an anti-P antibody, the attractant gradient of the OCC conditioned medium was disrupted by a P counter gradient, and the sperm P receptor was blocked with a specific antibody. We concluded that only the CU but not the oocyte secretes P, and the latter chemoattract spermatozoa by means of a cell surface receptor. Our findings may be of interest in assisted reproduction procedures in humans, animals of economic importance and endangered species.     

Behavioral mechanism during human sperm chemotaxis: involvement of hyperactivation

When mammalian spermatozoa become capacitated they acquire, among other activities, chemotactic responsiveness and the ability to exhibit occasional events of hyperactivated motility--a vigorous motility type with large amplitudes of head displacement. Although a number of roles have been proposed for this type of motility, its function is still obscure. Here we provide evidence suggesting that hyperactivation is part of the chemotactic response. By analyzing tracks of spermatozoa swimming in a spatial chemoattractant gradient we demonstrate that, in such a gradient, the level of hyperactivation events is significantly lower than in proper controls. This suggests that upon sensing an increase in the chemoattractant concentration capacitated cells repress their hyperactivation events and thus maintain their course of swimming toward the chemoattractant. Furthermore, in response to a temporal concentration jump achieved by photorelease of the chemoattractant progesterone from its caged form, the responsive cells exhibited a delayed turn, often accompanied by hyperactivation events or an even more intense response in the form of flagellar arrest. This study suggests that the function of hyperactivation is to cause a rather sharp turn during the chemotactic response of capacitated cells so as to assist them to reorient according to the chemoattractant gradient. On the basis of these results a model for the behavior of spermatozoa responding to a spatial chemoattractant gradient is proposed.     

Demonstration of receptor-mediated chemotaxis by human spermatozoa. A novel quantitative bioassay

A novel in vitro technique is described for measuring the chemotactic activity of soluble substances for human spermatozoa. This new bioassay has demonstrated that the synthetic chemotactic peptide N-formyl-Met-Leu-Phe elicits a potent, specific (i.e., receptor-mediated) chemotactic effect on human spermatozoa with an EC50 of 3.2 X 10(-10) M. Quantitative chemotactic studies on human spermatozoa with nine N-formylated-peptide analogs have shown a rank order of peptide potency indistinguishable (p less than 0.001) from that obtained in binding and chemotactic studies with rabbit neutrophils. The competitive antagonist Boc (t-butoxycarbonyl)-Phe-Leu-Phe-Leu-Phe, 10(-6) M, completely inhibited the chemotaxis elicited by f-Met-Leu-Phe, 10(-9) M, and was able to shift by one order of magnitude the molar concentration required by f-Met-Leu-Phe-Phe and f-Met-Leu-Phe to elicit the maximal response. The ability of N-formylated peptides to function as sperm chemoattractants reveals a high degree of correlation with binding, chemotaxis, and lysosomal enzyme release previously employed to define the neutrophil chemotactic receptor. This first unequivocal demonstration of substances having a receptor-mediated chemotactic effect for human male gametes suggests that human spermatozoa may indeed have the ability to respond chemotactically to appropriate environmental signals.     

Increased velocity and induction of chemotactic response in mouse spermatozoa by follicular and oviductal fluids.

The dynamic parameters of mouse sperm cells exposed to follicular and oviductal fluids were assessed. Spermatozoa were tracked on a chemotactic Zigmond chamber and recorded using a videomicroscopy system. The results were evaluated with computer-supported image analysis. Follicular fluid at a dilution of 10(-4) markedly increased the proportion of spermatozoa with high velocity, and stimulated chemotactic behaviour. The highest velocities were observed in sperm cells exposed to oviductal fluid, and a greater proportion of these cells had high velocity compared with those exposed to follicular fluid. Chemotaxis was induced in spermatozoa exposed to oviductal fluid at dilutions of 10(-3) and 10(-5). These results suggest the presence of temporal subpopulations of responsive spermatozoa, considering the distance travelled towards both follicular and oviductal fluids and the proportion of sperm cells migrating towards the gradient in the highest distance ranges. This is the first report on the effect of isolated follicular and oviductal fluids on dynamic parameters and chemotaxis of mouse spermatozoa. The findings support previous work showing that the motility and directionality of mouse sperm cells is increased by factors in the microenvironment of the egg. Although the significance of these factors in vivo is unknown, it is possible that there is a relay mechanism involving sequential activity of both oviductal and follicular fluids to direct the male gametes towards the egg.     

Appearance of chemotactic responsiveness to elastin peptides by developing fetal bovine ligament fibroblasts parallels the onset of elastin production

We studied chemotaxis to elastin peptides by bovine ligamentum nuchae fibroblasts to determine whether there is a developmental association between chemotactic responsiveness to elastin and expression of the elastin phenotype. Undifferentiated ligament cells demonstrate chemotactic responsiveness to platelet-derived growth factor and fibronectin, known chemoattractants for fibroblasts, but do not show chemotaxis to elastin peptides. After matrix-induced differentiation, however, young cells display a positive chemotactic response to elastin that persists even after the cells are removed from the matrix substratum. Matrix-induced chemotaxis to elastin could be inhibited selectively by incorporation of bromodeoxyuridine into DNA of undifferentiated cells before (but not after) contact with inducing matrix. These results show that the appearance of chemotaxis to elastin peptides parallels the onset of elastin synthesis and suggests that the acquisition of chemotactic responsiveness to elastin and expression of the elastin phenotype are affected by the same inducing elements or processes and may be closely coupled in development.     

The alterative effect of isobutylmethylxanthine in hypofunctional human neutrophil chemotaxis

The effects of isobutylmethylxanthine on the locomotion of chemotactically normofunctional and hypofunctional human neutrophils were compared. The hypofunctional cells were obtained from the peripheral blood of selected subjects with juvenile periodontitis. The chemically oriented migration (i.e. chemotaxis) of these cells was less than 50% of normal. Isobutylmethylxanthine caused a doubling of the CHO.Met.Leu.Phe.OH-induced chemotactic responsiveness of hypofunctional cells, while no effect of the drug was detected with respect to random migration. Isobutylmethylxanthine had no effect on chemotaxis or random migration in the normofunctional cell populations. Since isobutylmethylxanthine inhibits cAMP catabolism, these data suggest that the cyclic nucleotide plays a role in intracellular transduction mechanisms which affect chemotaxis.     

Phospholipase Cγ Activation, Phosphotidylinositol Hydrolysis, and Calcium Mobilization are Not Required for FGF Receptor-Mediated Chemotaxis

Basic fibroblast growth factor (FGF) is a potent angiogenic factor that stimulates several cell types to migrate along a chemotactic gradient. Most chemoartractant receptors appear to share a common mechanism that involves activation of phospholipase C (PLC), hydrolysis of phosphotidylinositol, and mobilization of intracellular calcium. We transf ected two different cell lines with either human FGF receptor-1 cDNA or chimeric FGF receptor cDNA. Ligand stimulation induced chemotaxis, activation of PLOγ, phosphotidylinositol hydrolysis, and calcium mobilization in both wild-type receptor cell lines. No such response was elicited in control cells. Mutation of the two fibroblast growth factor receptors at residue 766, replacing tyrosine with phenylalanine, made the receptors incapable of associating with and activating PLCγ following ligand stimulation. These mutant receptors also failed to mediate phosphotidylinositol hydrolysis and calcium mobilization. However, cells transfected with the mutant fibroblast growth factor receptors were as chemotactically responsive to the appropriate ligand as were cells transfected with the wild-type receptors. These findings demonstrate that the ability of the fibroblast growth factor receptor to promote chemotaxis is not dependent on increased activation of PLCγ, increased hydrolysis of phosphotidylinositol, or increased global mobilization of calcium.     

Phospholipase Cγ Activation,Phosphotidylinositol Hydrolysis,and Calcium Mobilization are Not Required for FGF Receptor-Mediated Chemotaxis

Basic fibroblast growth factor (FGF) is a potent angiogenic factor that stimulates several cell types to migrate along a chemotactic gradient. Most chemoartractant receptors appear to share a common mechanism that involves activation of phospholipase C (PLC), hydrolysis of phosphotidylinositol, and mobilization of intracellular calcium. We transf ected two different cell lines with either human FGF receptor-1 cDNA or chimeric FGF receptor cDNA. Ligand stimulation induced chemotaxis, activation of PLOγ, phosphotidylinositol hydrolysis, and calcium mobilization in both wild-type receptor cell lines. No such response was elicited in control cells. Mutation of the two fibroblast growth factor receptors at residue 766, replacing tyrosine with phenylalanine, made the receptors incapable of associating with and activating PLCγ following ligand stimulation. These mutant receptors also failed to mediate phosphotidylinositol hydrolysis and calcium mobilization. However, cells transfected with the mutant fibroblast growth factor receptors were as chemotactically responsive to the appropriate ligand as were cells transfected with the wild-type receptors. These findings demonstrate that the ability of the fibroblast growth factor receptor to promote chemotaxis is not dependent on increased activation of PLCγ, increased hydrolysis of phosphotidylinositol, or increased global mobilization of calcium.     

Analysis of the monocyte chemotactic response to lysophosphatidylcholine: role of lysophospholipase C

Previously, we reported that lysophosphatidylcholine (lyso-PtdCho), a component of oxidized low-density lipoprotein, was a monocyte chemoattractant (M.T. Quinn et al. (1988) Proc. Natl. Acad. Sci. USA 85, 2805-2809). Monocyte chemotaxis was also stimulated by lyso-platelet activating factor but not by platelet activating factor itself. In the present studies, we used other analogs of lyso-PtdCho to determine structural and metabolic features required for chemotactic activity. Although both D- and L-lyso-PtdCho stimulated chemotaxis, suggesting a lack of stereospecificity, studies using propanediol and ethanediol analogs of lyso-PtdCho suggested that a free hydroxyl moiety or an ester-linked fatty acid vicinal to the phosphocholine group of the lysophospholipid was required for the expression of activity. Incubation of [3H]choline-labeled lyso-PtdCho with monocytes resulted in the formation of labeled PtdCho, glycerophosphocholine (GPC), phosphocholine, and free choline, while resident peritoneal macrophages, cells which we show do not respond chemotactically to lyso-PtdCho, metabolized the labeled substrate to generate only labeled PtdCho and GPC; no labeled phosphocholine was found, suggesting a possible role for lysophospholipase C activity in the monocyte chemotactic response. Although monoacylglycerol, the product of lysophospholipase C hydrolysis of lyso-PtdCho, was not chemotactic for monocytes, diacylglycerol demonstrated chemotactic activity, suggesting that the subsequent acylation to diacylglycerol may be involved in the monocyte chemotactic response to lyso-PtdCho. Indeed, monocytes incorporated [3H]glycerol from [3H]glycerol-labeled lyso-PtdCho into di- and triacylglycerol. Based on these results, a model is proposed whereby the monocyte chemotactic response to lyso-PtdCho involves a sequence of metabolic steps which includes hydrolysis of lyso-PtdCho to monoacylglycerol and phosphocholine by lysophospholipase C followed by acylation of monoacylglycerol to diacylglycerol. Diacylglycerol would then act as an intracellular second messenger that could activate or facilitate the chemotactic response.     

Angiotensin II is chemotactic for a T-cell subset which can express migration inhibition factor activity in murine schistosomiasis mansoni

In murine schistosomiasis mansoni, ova induce a delayed-type hypersensitivity, granulomatous response in which angiotensins are produced. Angiotensin II (AII) elicits a chemotaxis for splenic mononuclear cells derived from these infected animals. The effect of AII upon the migration of a T-lymphocyte subset was defined functionally to further delineate this observation. A chemotaxis chamber was developed that permitted collection of large numbers of viable cells which migrate in response to AII. In a direct migration inhibition factor (MIF) assay, MIF activity was demonstrated with 100-fold fewer chemotactically attracted cells as opposed to whole splenic leukocytes. The MIF activity was eliminated by treatment of the cells with anti-Lyt 1.1 or-Thy 1.2 serum and complement. This observation was particularly interesting since migrated and whole spleen cell populations comprised equal numbers of T cells. Incubation of spleen cells with AII prior to assay did not alter MIF activity. These findings suggest that AII is chemotactic for at least one important T-cell subset relevant to the granulomatous response.     

Involvement of protein kinase C in signal transduction during fibroblast chemotaxis to platelet-derived growth factor and a fragment of fibronectin

The involvement of protein kinase C in chemotaxis of normal dermal fibroblasts to a mitogenic and a non-mitogenic attractant was investigated. Neomycin, an inhibitor of phosphoinositide metabolism, H7, staurosporine, and sphingosine, inhibitors of protein kinase C, as well as amiloride, an inhibitor of the Na+/H+-antiport, all abrogated chemotaxis of fibroblasts to platelet-derived growth factor and to a chemotactically active fragment of fibronectin. Down-regulation of protein kinase C by phorbol ester likewise diminished the capacity of fibroblasts to move directionally to these attractants. Therefore, all three of these signal transduction steps are required for the chemotactic response of this type of cells.     

PDGF AA homodimers are potent chemoattractants for fibroblasts and neutrophils, and for monocytes activated by lymphocytes or cytokines.

The A-chain homodimers of the platelet-derived growth factor (PDGF AA) are widely expressed in normal and transformed cells. The mitogenic properties of PDGF AA are well established; however, the chemotactic potential of PDGF AA remains controversial. We now show that PDGF AA is a strong chemoattractant for human monocytes, granulocytes, and fetal bovine ligament fibroblasts. However, highly purified (greater than 98%) monocytes require the addition of lymphocytes or IL-1 for chemotactic responsiveness to PDGF AA but not for full chemotactic activity with formyl-methionyl-leucyl-phenylalanine (fMLP) or C5a. These results indicate that PDGF AA is a potent chemoattractant. These results also indicate that monocytes require activation either by lymphocytes or exogenous cytokines in order to respond chemotactically to PDGF AA but not to fMLP or C5a and suggest roles of the lymphocyte and cytokine in the chemotactic response of the monocyte to PDGF AA in vivo.     

Role of monoclonal antibody J-23 in inducing acrosome reactions in capacitated mouse spermatozoa.

A monoclonal antibody (J-23) to the 15 kDa component on the sperm head, the acceptor, which functions in zona binding, was shown to induce the acrosome reaction in capacitated cells, but not in fresh cells. The antibody recognized its epitope in the acrosomal cap region of fresh spermatozoa and in the equatorial region on washed and capacitated spermatozoa. However, equatorial expression did not depend on the acrosome reaction, since washing fresh spermatozoa increased the percentage with equatorial fluorescence, but did not increase the percentage with reacted acrosomes. The data indicate that the acrosome reaction can be induced in capacitated spermatozoa in the absence of zona glycoproteins.     

Human sperm chemotaxis: is progesterone a chemoattractant?

Follicular fluid (FF) induces sperm chemotaxis in human spermatozoa. Progesterone also causes sperm accumulation. However, sperm accumulation can be caused by chemotaxis, chemokinesis, and trapping of various kinds. It has been suggested that progesterone also induces chemotaxis in human spermatozoa. In view of the physiological significance of sperm chemotaxis in human fertilization and its potential clinical implications, it is important to determine unequivocally whether chemotaxis is induced by progesterone and, if so, whether progesterone in FF is the chemoattractant. To resolve these questions we looked for characteristic changes in the direction of sperm swimming toward pure progesterone as well as toward FF before and after progesterone removal. Progesterone caused sperm accumulation and hyperactivation-like motility, but it caused very few changes in the direction of sperm swimming that are characteristic of chemotaxis. Removal of progesterone (and other steroids) from FF by charcoal treatment abolished the sperm hyperactivation-like motility but not sperm chemotaxis. These results suggest that while progesterone might be a weak chemoattractant, it is not the major chemoattractant in FF. Progesterone probably causes human sperm accumulation mainly by inducing hyperactivation-like motility and, as a consequence, sperm trapping.     

A chemotactic factor for rat thymocytes may regulate T-lymphocyte migration toward the thymic microenvironment

Using a modified Boyden chamber assay, extracts or culture supernatants of rat thymic stromal cells, or thymocytes were examined by chemotactic activity to rat leukocytes. Rat thymocytes responded chemotactically to the aqueous extract as well as to culture supernatants of thymic stromal cells. However, neither the extract and culture medium from concanavalin A-stimulated thymocytes nor any component of rat serum has shown such an activity. The thymic extract was fractionated into three molecular species with chemotactic activity for thymocytes. The thymocyte chemotactic factor(s) (TCFs) in the extract was distinct from known lymphocytic chemotactic factors, such as interleukin-1 (IL-1), IL-2, C5a, and the culture supernatant of stimulated thymocytes. In vitro, TCFs could attract, in addition to thymocytes, bone marrow cells, fetal liver cells, and nylon-wool nonadherent lymphocytes from peripheral blood and spleen. Lymph node cells, neutrophils, macrophages, and B cells from peripheral blood could not respond to TCFs. Thymocytes also responded to the extract of splenic stromal cells. Unlike the thymic extract, however, the splenic extract was chemotactically active for lymphocytes from lymph nodes but not for bone marrow cells. These results indicate that thymic stromal cells secrete a chemotactic factor(s) for a relatively immature type of T-lineage cells, which may by a thymus-homing progenitor T cell, while spleen may contain an attractant for a relatively mature type of T-lineage cells.     

An Epididymis-Specific Secretory Protein HongrES1 Critically Regulates Sperm Capacitation and Male Fertility

Mammalian sperm capacitation is an essential prerequisite to fertilizion. Although progress had been made in understanding the physiology and biochemistry of capacitation, little is known about the potential roles of epididymal proteins during this process. Here we report that HongrES1, a new member of the SERPIN (serine proteinase inhibitor) family exclusively expressed in the rat cauda epididymis and up-regulated by androgen, is secreted into the lumen and covers the sperm head. Co-culture of caudal sperms with HongrES1 antibody in vitro resulted in a significant increase in the percentage of capacitated spermatozoa. Furthermore, the percentage of capacitated spermatozoa clearly increased in rats when HongrES1 was down-regulated by RNAi in vivo. Remarkably, knockdown of HongrES1 in vivo led to reduced fertility accompanied with deformed appearance of fetuses and pups. These results identify HongrES1 as a novel and critical molecule in the regulation of sperm capacitation and male fertility.