Mechanics of sperm-egg interaction at the zona pellucida.

Mammalian sperm traverse several layers of egg vestments before fertilization can occur. The innermost vestment, the zona pellucida, is a glycoprotein shell, which captures and tethers the sperm before they penetrate it. We report here direct measurements of the force required to tether a motile human sperm as well as independent calculations of this force using flagellar beat parameters observed for sperm of several species on their homologous zonae. We have compared these sperm-generated forces with the calculated tensile strength of sperm-zona bonds, and found that a motile sperm can be tethered, at least temporarily, by a single bond. Therefore, sperm can be captured by the first bond formed and tethered permanently by a few. The sperm cannot subsequently penetrate the zona unless the bonds are first eliminated. However, premature elimination would simply allow the sperm to escape. Therefore, not only must the bonds be eliminated, but the timing of this must be regulated so that the sperm is already oriented toward the egg and beginning to penetrate as the bonds are broken.     

Use of salt-stored zonae pellucidae for assessing rabbit sperm capacitation for in vitro fertilization

Zonae pellucidae of tubal and follicular oocytes were collected and prepared for salt storage. Cumulus and corona radiata cells were removed from oocytes with hyaluronidase and a small bore pipette. The oocytes (referred to as zonae since vitelli were rendered nonfunctional) were stored in a salt solution at 4°C. Using in utero capacitated sperm, the penetrability of zonae from tubal and follicular oocytes stored immediately after collection was compared to controls, i.e., in vitro development of tubal ova to the 4-cell stage within 24 hr. The penetration rates were 100% (8 penetrated/ 8 inseminated), 77.8% (7 penetrated/ 9 inseminated), and 100% (10 fertilized/10 inseminated), respectively, and these were not statistically different. The mean (x ) numbers of sperm able to penetrate the zonae, into the perivitelline space (PVS) for tubal (34.0) and follicular (1.1) oocytes were significantly different (P < 0.01). However, following maturational incubation before salt storage, zonae of tubal and follicular origin showed no significant differences in penetrability of in utero capacitated sperm when assessed by percent penetration, or mean numbers of sperm cells reaching the PVS: tubal zonae, 100% (15/15), and follicular zonae, 100% (18/18), and mean number of sperm in the PVS (x [tubal zonae] = 12.4, and x [follicular zonae] = 11.8). The penetrability of tubal zonae with and without maturational incubation was compared, and no significant differences in penetrability by in utero capacitated sperm were present when assessed by percent penetration nonmatured 92.6% (25/27) and matured 93.3% (28/30) and mean number of sperm in the PVS (x [nonmatured] = 3.33 and x [matured] = 2.41). In vitro capacitation of ejaculated rabbit sperm by serum treatment was assessed by the penetration of salt-stored zonae, zonae-free hamster oocytes (ZFHO), and in vitro fertilization of freshly collected tubal oocytes. None of 60 salt-stored zonae and none of 31 tubal oocytes were penetrated, and these values were significantly (P < 0.005) smaller than the 9 of 78 (12%) zona-free hamster ova that were penetrated by sperm cells from the same sample. In vitro capacitation of ejaculated rabbit sperm by washing and preincubation was assessed by the penetration of salt-stored zonae, zona-free hamster oocytes (ZFHO), and fertilization of freshly collected tubal oocytes. Seventy-six of 80 salt-stored zonae were penetrated, and this was significantly (P < 0.005) greater than the 67 of 87 tubal oocytes fertilized and 30 of 35 ZFHO penetrated, which were not significantly different. The salt-stored zonae were more readily penetrated by capacitated sperm when compared to tubal oocytes. However, the ZFHO are more penetrable than salt-stored zonae and tubal oocytes when incompletely capacitated sperm is used. A useful role for this approach in studies dealing with sperm fertilizing ability is anticipated.     

Changes in the magnitude and distribution of forces at different Dictyostelium developmental stages

The distribution of forces exerted by migrating Dictyostelium amebae at different developmental stages was measured using traction force microscopy. By using very soft polyacrylamide substrates with a high fluorescent bead density, we could measure stresses as small as 30 Pa. Remarkable differences exist both in term of the magnitude and distribution of forces in the course of development. In the vegetative state, cells present cyclic changes in term of speed and shape between an elongated form and a more rounded one. The forces are larger in this first state, especially when they are symmetrically distributed at the front and rear edge of the cell. Elongated vegetative cells can also present a front-rear asymmetric force distribution with the largest forces in the crescent-shaped rear of the cell (uropod). Pre-aggregating cells, once polarized, only present this last kind of asymmetric distribution with the largest forces in the uropod. Except for speed, no cycle is observed. Neither the force distribution of pre-aggregating cells nor their overall magnitude are modified during chemotaxis, the later being similar to the one of vegetative cells (F(0) approximately 6 nN). On the contrary, both the force distribution and overall magnitude is modified for the fast moving aggregating cells. In particular, these highly elongated cells exert lower forces (F(0) approximately 3 nN). The location of the largest forces in the various stages of the development is consistent with the myosin II localization described in the literature for Dictyostelium (Yumura et al.,1984. J Cell Biol 99:894-899) and is confirmed by preliminary experiments using a GFP-myosin Dictyostelium strain.     

Maximal Force Characteristics of the Ca2+-Powered Actuator of Vorticella convallaria

The millisecond stalk contraction of the sessile ciliate Vorticella convallaria is powered by energy from Ca²⁺ binding to generate contractile forces of ～10 nN. Its contractile organelle, the spasmoneme, generates higher contractile force under increased stall resistances. By applying viscous drag force to contracting V. convallaria in a microfluidic channel, we observed that the mechanical force and work of the spasmoneme depended on the stalk length, i.e., the maximum tension (150–350 nN) and work linearly depended on the stalk length (～2.5 nN and ～30 fJ per 1 μm of the stalk). This stalk-length dependency suggests that motor units of the spasmoneme may be organized in such a way that the mechanical force and work of each unit cumulate in series along the spasmoneme.     

Force of an actin spring

Cellular movements are produced by forces. Typically, cytoskeletal proteins such as microtubules and actin filaments generate forces via polymerization or in conjunction with molecular motors. However, the fertilization of a Limulus polyphemus egg involves a third type of actin-based cellular engine--a biological spring. During the acrosome reaction, a 60-microm long coiled and twisted bundle of actin filaments straightens and extends from a sperm cell, penetrating the vitelline layer surrounding the egg. A subtle overtwist of 0.2 degrees /subunit underlies the mechanochemical basis for the extension of this actin spring. Upon calcium activation, this conformational strain energy is converted to mechanical work, generating the force required to extend the bundle through the vitelline layer. In this article, we stall the extension of the acrosome bundle in agarose gels of different concentrations. From the stall forces, we estimate a maximum force of 2 nN and a puncturing pressure of 1.6 MPa. We show the maximum force of extension is three times larger than the force required to puncture the vitelline layer. Thus, the elastic strain energy stored in the acrosome bundle is more than sufficient to power the acrosome reaction through the egg envelope.     

Neutrophil traction stresses are concentrated in the uropod during migration

We find that in contrast to strongly adherent, slow moving cells such as fibroblasts, neutrophils exert contractile stresses largely in the rear of the cell (uropod) relative to the direction of motion. Rather than the leading edge pulling the cell, the rear is both anchoring the cell and the area in which the contractile forces are concentrated. These tractions rapidly reorient themselves during a turn, on a timescale of seconds to minutes, and their repositioning precedes and sets the direction of motion during a turn. We find the total average root mean-squared traction force to be 28+/-10 nN during chemokinesis, and 67+/-10 nN during chemotaxis. We hypothesize that the contraction forces in the back of the neutrophil not only break uropodial adhesive contacts but also create a rearward squeezing contractility, as seen in amoeboid or amoeboidlike cells and the formation of blebs in cells, causing a flow of intracellular material to the fluidlike lamellipod. Our findings suggest an entirely new model of neutrophil locomotion.     

Structural conservation of mouse and rat zona pellucida glycoproteins. Probing the native rat zona pellucida proteome by mass spectrometry

The mammalian zona pellucida is an egg extracellular matrix to which sperm bind. Mouse zonae are composed of three glycoproteins (ZP1, ZP2, and ZP3), while rat zonae contain four (ZP1, ZP2, ZP3, and ZP4/ZPB). Mouse sperm bind to zonae comprised solely of mouse ZP2 and ZP3. In this report, we show that rat sperm also bind to these zonae, indicating that ZP2 and ZP3 contain a "minimum structure(s)" to which rodent sperm can bind, and ZP1 and ZP4/ZPB are dispensable in these two rodents. These data are consistent with our mass spectrometric analysis of the native rat zona pellucida proteome (defined as the fraction of the total rat proteome to which the zonae glycoproteins contribute) demonstrating that the rat zonae glycoproteins share a high degree of conservation of structural features with respect to their mouse counterparts. The primary sequences of the rat zonae proteins have been deduced from cDNA. Each zona protein undergoes extensive co- and post-translational modification prior to its secretion and incorporation into an extracellular zona matrix. Each has a predicted N-terminal signal peptide that is cleaved off once protein translation begins and an anchoring C-terminal transmembrane domain from which the mature protein is released. Mass spectrometric analysis with a limited amount of native material allowed determination of the mature N-termini of rat ZP1 and ZP3, both of which are characterized by cyclization of glutamine to pyroglutamate; the N-terminus of ZP2 was identified by Edman degradation. The mature C-termini of ZP1 and ZP3 end two amino acids upstream of a conserved dibasic residue that is part of, but distinct from, the consensus furin cleavage sequence, while the C-terminus of ZP2 was not determined. Each zona protein contains a "zona domain" with eight conserved cysteine residues that is thought to play a role in the polymerization of the zona proteins into matrix filaments. Partial disulfide bond assignment indicates that the intramolecular disulfide patterns in rat ZP1, ZP2, and ZP3 are identical to those of their corresponding mouse counterparts. Last, nearly all potential N-glycosylation sites are occupied in the rat zonae glycoproteins (three of three for ZP1, six or seven of seven for ZP2, and four or five of six for ZP3). In comparison, potential O-glycosylation sites are numerous (59-83 Ser/Thr residues), but only two regions were observed to carry O-glycans in rat ZP3.     

Cell migration through small gaps

Cell motility is a fundamental process associated with many phenomena in nature, such as immune response, wound healing, and cancer metastasis. In these processes, cells must squeeze through cell layers, and we characterize this ability to actively produce forces and simultaneously adapt their shapes. We have measured forward forces up to 15 nN that a migrating keratocyte was able to generate, in order to adjust its shape and successfully force its way under and past an obstacle. We also observed that 34 nN was capable of stalling the cell’s forward motion. Furthermore, we measured that under compression stresses up to 1,165 pN/μm² (1,165 Pa), cell morphology, and velocity remained unchanged. Additionally, we found that keratocytes were able to compress themselves up to 80% vertically in order to squeeze through a gap as small as 500 nm.Authors Claudia A. Brunner, Allen Ehrlicher, and Michael Goegler have contributed equally to this work.     

Mammalian sperm-egg interaction: Identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm

Sperm-egg interaction in mammals is initiated by binding of sperm to the zona pellucida, an acellular coat completely surrounding the plasma membrane of unfertilized eggs. Zonae pellucidae of mouse eggs are composed of three different glycoproteins, designated ZP1, ZP2 and ZP3, having apparent molecular weights of 200,000, 120,000 and 83,000, respectively Bleil and Wassarman, 1978, Bleil and Wassarman, 1980a, Bleil and Wassarman, 1980b. In this investigation, ZP1, ZP2 and ZP3 were purified from zonae pellucidae isolated individually from unfertilized mouse eggs and 2-cell embryos. Each of the glycoproteins was then tested for its ability to interfere with the binding of sperm to eggs in vitro. Solubilized zonae pellucidae isolated from unfertilized eggs, but not from 2-cell embryos, reduced binding of sperm to as little as 10% of control values. Similarly, ZP3 purified from zonae pellucidae of unfertilized eggs reduced the binding of sperm to eggs in vitro to an extent comparable to that observed with solubilized zonae pellucidae. On the other hand, ZP3 purified from zonae pellucidae of 2-cell embryos had no significant effect on the extent of sperm binding, consistent with the inability of solubilized zonae pellucidae from 2-cell embryos to affect sperm binding. In no case did purified ZP1 and ZP2 interfere significantly with the binding of sperm to eggs in vitro. These results suggest that ZP3 possesses the receptor activity responsible for the binding of sperm to zonae pellucidae of unfertilized mouse eggs. Fertilization apparently results in modification of ZP3 such that it can no longer serve as a receptor for sperm.     

Structural-functional relationships of the dynein,spokes, and central-pair projections predicted from an analysis of the forces acting within a flagellum

In the axoneme of eukaryotic flagella the dynein motor proteins form crossbridges between the outer doublet microtubules. These motor proteins generate force that accumulates as linear tension, or compression, on the doublets. When tension or compression is present on a curved microtubule, a force per unit length develops in the plane of bending and is transverse to the long axis of the microtubule. This transverse force (t-force) is evaluated here using available experimental evidence from sea urchin sperm and bull sperm. At or near the switch point for beat reversal, the t-force is in the range of 0.25-1.0 nN/ micro m, with 0.5 nN/ micro m the most likely value. This is the case in both beating and arrested bull sperm and in beating sea urchin sperm. The total force that can be generated (or resisted) by all the dyneins on one micron of outer doublet is also approximately 0.5 nN. The equivalence of the maximum dynein force/ micro m and t-force/ micro m at the switch point may have important consequences. Firstly, the t-force acting on the doublets near the switch point of the flagellar beat is sufficiently strong that it could terminate the action of the dyneins directly by strongly favoring the detached state and precipitating a cascade of detachment from the adjacent doublet. Secondly, after dynein release occurs, the radial spokes and central-pair apparatus are the structures that must carry the t-force. The spokes attached to the central-pair projections will bear most of the load. The central-pair projections are well-positioned for this role, and they are suitably configured to regulate the amount of axoneme distortion that occurs during switching. However, to fulfill this role without preventing flagellar bend formation, moveable attachments that behave like processive motor proteins must mediate the attachment between the spoke heads and the central-pair structure.     

Cardiomyocyte sensor responsive to changes in physical and chemical environments

Conventional cardiac physiology experiments investigate in vitro beat frequency using cells isolated from adult or neonatal rat hearts. In this study, we show that various cantilever shapes and drug treatments alter cardiomyocyte contraction force in vitro. Four types of cantilevers were used to compare the contractile forces: flat, peg patterned, grooved, and peg and grooved. Contraction force was represented as bending deflection of the cantilever end. The deflections of the flat, peg patterned, grooved, and peg and grooved cantilevers were 24.2 nN, 41.6 nN, 121 nN, and 134.2 nN, respectively. We quantified the effect of drug treatments on cardiomyocyte contractile forces on the grooved cantilever using Digoxin, Isoproterenol, and BayK8644, all of which increase contractile force, and Verapamil, which decreases contractile force. The cardiomyocyte contractile force without drugs decreased 8 days after culture initiation. Thus, we applied Digoxin, Isoproterenol, and BayK8644 at day 8, and Verapamil at day 5. Digoxin, Isoproterenol, and BayK8644 increased the cardiomyocyte contractile forces by 19.31%, 9.75%, and 23.81%, respectively. Verapamil decreased the contraction force by 48.06%. In summary, contraction force changes in response to adhesion surface topology and various types of drug treatments. We observed these changes by monitoring cell alignment, adhesion, morphology, and bending displacement with cantilever sensors.     

Is the mechanical activity of epithelial cells controlled by deformations or forces?

The traction forces developed by cells depend strongly on the substrate rigidity. In this letter, we characterize quantitatively this effect on MDCK epithelial cells by using a microfabricated force sensor consisting in a high-density array of soft pillars whose stiffness can be tailored by changing their height and radius to obtain a rigidity range from 2 nN/microm up to 130 nN/microm. We find that the forces exerted by the cells are proportional to the spring constant of the pillars meaning that, on average, the cells deform the pillars by the same amount whatever their rigidity. The relevant parameter may thus be a deformation rather than a force. These dynamic observations are correlated with the reinforcement of focal adhesions that increases with the substrate rigidity.     

Sperm-egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein

In this investigation, the interaction of mouse sperm with unfertilized eggs and embryos, solubilized zonae pellucidae isolated from eggs and embryos, and purified zona pellucida glycoproteins ZP1, 2, and 3 (J. D. Bleil, and P. M. Wassarman, (1980b) Dev. Biol. 76, 185-202) has been examined in vitro by light and electron microscopy. The experiments described were carried out in order to determine the temporal sequence of events during sperm-egg interaction in vitro and to identify the component(s) of zonae pellucidae responsible for inducing mouse sperm to undergo the acrosome reaction. "Pulse-chase" analysis of the sequence of sperm-egg interactions revealed that mouse sperm first "attach" loosely and then "bind" tightly to the unfertilized egg's zona pellucida. Binding of sperm to egg zonae pellucidae is followed by induction of the acrosome reaction. Induction of the acrosome reaction can be mediated by the zona pellucida, since solubilized zonae pellucidae isolated from unfertilized eggs were found to be just as effective as the calcium ionophore A23187 in inducing the reaction in vitro. Furthermore, ZP3 purified from zonae pellucidae isolated from unfertilized eggs, but not from two-cell embryos, was also just as effective as either solubilized zonae pellucidae from eggs or ionophore A23187 in inducing the acrosome reaction. ZP1 and 2 from both eggs and embryos, and ZP3 from embryos, had little effect on the extent of the acrosome reaction as compared to control samples. The results of these and other experiments (J. D. Bleil, and P. M. Wassarman, (1980b) Cell 20, 873-882) strongly suggest that, at least in vitro, mouse sperm recognize and bind to ZP3 of egg zonae pellucidae, and that such binding leads to the induction of the acrosome reaction. Modification of ZP3 following fertilization eliminates sperm binding to zonae pellucidae and, consequently, induction of the acrosome reaction is precluded.     

Keratocytes pull with similar forces on their dorsal and ventral surfaces

As cells move forward, they pull rearward against extracellular matrices (ECMs), exerting traction forces. However, no rearward forces have been seen in the fish keratocyte. To address this discrepancy, we have measured the propulsive forces generated by the keratocyte lamella on both the ventral and the dorsal surfaces. On the ventral surface, a micromachined device revealed that traction forces were small and rearward directed under the lamella, changed direction in front of the nucleus, and became larger under the cell body. On the dorsal surface of the lamella, an optical gradient trap measured rearward forces generated against fibronectin-coated beads. The retrograde force exerted by the cell on the bead increased in the thickened region of the lamella where myosin condensation has been observed (Svitkina, T.M., A.B. Verkhovsky, K.M. McQuade, and G. G. Borisy. 1997. J. Cell Biol. 139:397-415). Similar forces were generated on both the ventral (0.2 nN/microm(2)) and the dorsal (0.4 nN/microm(2)) surfaces of the lamella, suggesting that dorsal matrix contacts are as effectively linked to the force-generating cytoskeleton as ventral contacts. The correlation between the level of traction force and the density of myosin suggests a model for keratocyte movement in which myosin condensation in the perinuclear region generates rearward forces in the lamella and forward forces in the cell rear.     

Enhanced self-association of mucins possessing the T and Tn carbohydrate cancer antigens at the single-molecule level

Mucins are linear O-glycosylated glycoproteins involved in inflammation, cell adhesion, and tumorigenesis. Cancer-associated mucins often possess increased expression of the T (Galβ1,3GalNAcαThr/Ser) and Tn (GalNAcαThr/Ser) cancer antigens, which are diagnostic markers for several cancers, including colon cancer. We have used AFM based single-molecule forced unbinding under near physiological conditions to investigate the self-interactions between porcine submaxillary mucin (PSM) as well as between PSM analogs possessing various carbohydrates including the T- and Tn-antigen. Distributions of unbinding forces and corresponding force loading rates were determined for force loading rates from 0.18 nN/s to 39 nN/s, and processed to yield most probable unbinding forces f* and lifetimes of the interactions. Parameter f* varied in the range 27 to 50 pN at force loading rates of about 2 nN/s among the various mucins. All mucin samples investigated showed self-interaction, but the tendency was greatest for PSM displaying only the Tn-antigen (Tn-PSM) or a mixture of Tn-, T-antigen, and the trisaccharide Fucα1,2Galβ1,3GalNAc (Tri-PSM). Weaker self-interactions were observed for native PSM (Fd-PSM), which consists of a nearly equal mixture of the longer core 1 blood group A tetrasaccharide (GalNAcα1,3(Fucα1,2)Galβ1,3GalNAcαSer/Thr) and Tn-antigen. The data are consistent with the truncated Tn and T glycans enhancing self-interaction of the mucins. These carbohydrate cancer antigens may, thus, play an active role in the disease by constitutively activating mucin and mucin-type receptors by self-association on cells.     

Evidence for plasma membrane impermeability to small ions in acrosome-intact mouse spermatozoa bound to mouse zonae pellucidae, using an aminoacridine fluorescent pH probe: time course of the zona-induced acrosome reaction monitored by both chlortetracycline and pH probe fluorescence

Previous studies have shown that capacitated mouse spermatozoa bind to zonae pellucidae of mouse eggs with acrosomes apparently intact. The question addressed in this study was the following: are the membrane permeability barriers of the apparently acrosome-intact sperm still retained or is there a preliminary stage of the acrosome reaction in which these barriers are lost and the intracellular space becomes accessible to extracellular substrates? The experimental approach was to use the fluorescent pH probe 9-amino-3-chloro-7-methoxyacridine, which accumulates in intracellular spaces of lower pH than the suspending medium with the result that the cells become fluorescent. Freshly capacitated mouse spermatozoa bound to isolated zonae showed uniform fluorescence over the head and midpiece with this fluorescent probe at early times of binding. The fluorescence was abolished by NH4+ and nigericin, agents that equilibrate H+ across cell membranes. At these early times of binding, the acrosomes were fully intact as judged by chlortetracycline fluorescence pattern, which itself was unaffected by either N4+ or nigericin. The time course of the loss of this chlortetracycline pattern characteristic of acrosome-intactness was closely paralleled by loss of fluorescence of 9-amino-3-chloro-6-methoxyacridine over the first 90 min; thereafter, loss of the chlortetracycline pattern was somewhat more rapid. This result shows that acrosome-intact sperm bound to zonae pellucidae retain the permeability barriers of the plasma membrane to small cations; no evidence was found for an early stage of membrane "leakiness" preceding the acrosome reaction. The ionophore A23187 induced a very rapid acrosome reaction in sperm bound to isolated zonae, as judged with both fluorescence probes. This rapid reaction was partially inhibited by 3-quinuclidinyl benzilate, which, in the absence of ionophore, completely blocks the occurrence of the acrosome reaction in sperm bound to zonae. This suggests involvement of a specific calcium entry mechanism in the acrosome reaction of mouse sperm induced by mouse zonae pellucidae.     

Measurement of Subcellular Force Generation in Neurons

Forces are important for neuronal outgrowth during the initial wiring of the nervous system and after trauma, yet subcellular force generation over the microtubule-rich region at the rear of the growth cone and along the axon has never, to our knowledge, been directly measured. Because previous studies have indicated microtubule polymerization and the microtubule-associated proteins Kinesin-1 and dynein all generate forces that push microtubules forward, a major question is whether the net forces in these regions are contractile or expansive. A challenge in addressing this is that measuring local subcellular force generation is difficult. Here we develop an analytical mathematical model that describes the relationship between unequal subcellular forces arranged in series within the neuron and the net overall tension measured externally. Using force-calibrated towing needles to measure and apply forces, in combination with docked mitochondria to monitor subcellular strain, we then directly measure force generation over the rear of the growth cone and along the axon of chick sensory neurons. We find the rear of the growth cone generates 2.0 nN of contractile force, the axon generates 0.6 nN of contractile force, and that the net overall tension generated by the neuron is 1.3 nN. This work suggests that the forward bulk flow of the cytoskeletal framework that occurs during axonal elongation and growth-cone pauses arises because strong contractile forces in the rear of the growth cone pull material forward.     

Measuring the interaction forces between protein inclusion bodies and an air bubble using an atomic force microscope

Interaction forces between protein inclusion bodies and an air bubble have been quantified using an atomic force microscope (AFM). The inclusion bodies were attached to the AFM tip by covalent bonds. Interaction forces measured in various buffer concentrations varied from 9.7 nN to 25.3 nN (+/- 4-11%) depending on pH. Hydrophobic forces provide a stronger contribution to overall interaction force than electrostatic double layer forces. It also appears that the ionic strength affects the interaction force in a complex way that cannot be directly predicted by DLVO theory. The effects of pH are significantly stronger for the inclusion body compared to the air bubble. This study provides fundamental information that will subsequently facilitate the rational design of flotation recovery system for inclusion bodies. It has also demonstrated the potential of AFM to facilitate the design of such processes from a practical viewpoint.     

Mouse gamete interactions during fertilization in vitro. Chlortetracycline as a fluorescent probe for the mouse sperm acrosome reaction

We have developed an assay for detecting the acrosome reaction in mouse sperm using chlortetracycline (CTC) as a fluorescent probe. Sperm known to be intact with nonreacted acrosomes show CTC fluorescence in the presence of Ca2+ over the anterior portion of the sperm head on the plasma membrane covering the acrosome. Sperm which have undergone the acrosome reaction do not show fluorescence on the sperm head. Mouse sperm bind to zonae pellucidae of cumulus-free eggs in vitro in a Ca2+-dependent reaction; these sperm are intact by the CTC assay. Intact sperm bind to mechanically isolated zonae under the same conditions: the egg is apparently unnecessary for this inital reaction. Sperm suspensions, in which greater than 50% of the motile population had completed the acrosome reaction, were prepared by incubation in hyperosmolal medium followed by treatment with the divalent cation ionophore, A23187. Cumulus-free eggs challenged with such sperm suspensions preferentially bind intact sperm; acrosome-reacted sperm do not bind. We conclude that the plasma membrane of the mouse sperm is responsible for recognition of the egg's zona pellucida and that the obligatory sequence of reactions leading to fusion of mouse gametes is binding of the intact sperm to the zona pellucida, followed by the acrosome reaction at the zona surface, followed in turn by sperm penetration of the zona.