The EMBO Journal Vol.2 No. I I pp.2053 - 2058, 1983

A thermodynamic study of sperm-egg interaction

V.Elia, F.Rosatil.2, G.Barone, A.Monroyi* andA.M.Liquori3.4Istituto Chimico, Universita, Napoli, 'Stazione Zoologica, Napoli, and3Centro Interdisciplinare dell'Accademia Nazionale dei Lincei, Roma, Italy

Communicated by A.M.Liquori and A.MonroyReceived on 18 July 1983

We have studied the binding of spermatozoa to the receptorsites on the vitelline coat (VC) of glycerol-treated eggs (ghosteggs) of the Ascidian, Ciona intestinalis (Protochordate).Glycerol treatment cytolyses the egg without affecting theability of the VC to bind spermatozoa in a species-specificmanner; however, in this system binding is not followed bythe acrosome reaction. The ghost eggs are metabolically inert.As a base line for our analysis, we have studied theconcentration-dependent heat evolved and oxygen consump-tion of spermatozoa when diluted in sea water. The processhas been analyzed on the basis of equations derived by Li-quori and Tripiciano to describe cell growth. Upon binding tothe ghost eggs, the spermatozoa produce an explosive heatevolution (excess heat) which is not accompanied by oxygenconsumption. The excess heat produced plotted againstsperm concentration (at constant egg concentrations) gives anasymmetric bell-shaped curve. This is interpreted as being dueto the competitive effect of sperm agglutination at a highsperm concentration. It is concluded that only spermatozoathat attach singly (monomeric spermatozoa) to the eggundergo metabolic activation.Key words: sperm-egg binding/heat evolution/sperm activa-tion.

IntroductionThe primary event of fertilization is the species-specific

recognition and binding between spermatozoa and eggs. Thisprocess occurs at the level of specific sites, called sperm recep-tors, located on the vitelline coat (VC) of the glycoprotein eggenvelope (Figure la). In the Ascidians (Protochordates) onlya small fraction of the spermatozoa bound to the VC under-goes the acrosome reaction (Rosati and De Santis, 1978; DeSantis et al., 1980), which is a prerequisite for the subsequentstep of sperm-egg fusion. Treatment of these eggs withglycerol results in the removal of the follicle cells, lysis of thetest cells (which are interspersed between the VC and theoocyte) and of the oocyte (Rosati and De Santis, 1978). TheVC is apparently unaffected by this treatment and in fact itretains its ability to bind the spermatozoa in a species-specificmanner. However, the bound spermatozoa fail to undergothe acrosome reaction (De Santis et al., 1980). These 'ghosteggs' (Figure lb) thus offer an excellent tool to dissect out theevents of sperm binding from those of the acrosome reaction,sperm-egg fusion and egg activation. The key components ofthe sperm receptors on the VC are fucosyl-containing glyco-proteins (Rosati and De Santis, 1980; Pinto et al., 1981; De

20n leave from the University of Siena.40n leave from the University of Rome.*To whom reprint requests should be sent.

() IRL Press Limited, Oxford, England.

Santis et al., 1983). When extracted from the VC of ghosteggs these glycoproteins did in fact inhibit sperm attachmentand elicit acrosome reaction (De Santis et al., 1983).To investigate the complex processes of the sperm-egg

interaction independently of the acrosome reaction andgamete fusion, an extensive thermochemical study has beenundertaken using a highly sensitive microcalorimeter (Beezer,1980). Among the advantages of this method is the possibilityof quantitatively comparing the thermochemical events oc-curring upon interaction of the spermatozoa with the ghosteggs and those taking place in a sea water suspension of sper-matozoa. Both the temporal evolution of the heat evolvedand the relationship between total heat and sperm concentra-tion were analyzed on the basis of a theoretical formulationfirst introduced by Liquori and Tripiciano (1980).The results obtained, together with preliminary results on

oxygen consumption, lead to the conclusion that interactionwith the egg triggers in the spermatozoa an explosive heatevolution not accompanied by oxygen consumption. Anasymmetric bell-shaped plot of the excess heat as a functionof the sperm concentration at the optimal egg concentrationwas obtained. A theoretical analysis of this plot combinedwith direct observations suggests two concomitant processes(A and B). Process A corresponds to binding of individualspermatozoa ('monomers') to the available sites on the sur-face of VC. Process B corresponds to self-binding (agglutina-tion) of spermatozoa, which results in the formation ofclumps. Therefore, only monomeric spermatozoa bound tothe eggs are probably biochemically activated. The com-petitive binding and self-binding processes suggest that thereceptor glycoproteins on the surface of the two gametes aresimilar.

ResultsSpermatozoa in sea water

Temporal evolution. Figure 2 shows a typical power time(TP) diagram obtained at a given sperm concentration (15.0 x107 sperm/ml). Preliminary experiments indicate that dif-ferent TP diagrams are obtained at the same sperm concen-tration by performing the experiments in air and in nitrogen.The TP diagram corresponds to the flux Ju(t) = du of thermal

dtenergy as a function of time.The total heat evolved Qexp may therefore be obtained asfollows:

00

Qexp= Ju(t)dt (1)

By integrating the TP diagram within increasing intervals, thevalues of function Q(t) are obtained as follows:

,. tQ(t) = Ju(t) d t (2)

A plot of Q(t) as a function of times give a typical sigmoidalcurve which is characterized by an inflection and by a trendtoward an asymptotic value Q = limQ(t). The following re-

duced function: t- 0

2053

V.Elia et al.

Fig. 1. (a) Spermatozoa attached to the VC (arrow) of an egg of Ciona fertilized after removal of the follicle cells. x 1500. (b) Spermatozoa attached to aglycerol-treated egg (ghost egg). x 1500.

.50

Ju(t)(mW)

.25

20 40 60 80t (minutes)

Fig. 2. Typical TP plot obtained from a suspension of spermatozoa at aconcentration of 15 x 107 sperm/ml.

y(t) Q(t) (3)Q

may therefore be constructed, where the asymptotic value Qmay be obtained by suitable graphic extrapolation. (For in-stance the inverse plot [Q(t)] - 1 may be extrapolated to t- 0.)Plots of y(t) as a function of t show the typical features of theprocess displaying kinetic cooperativity (or autocatalyticbehaviour). Cell growth is a typical example.

Several empirical equations have been adopted to describesigmoidal cell growth curves. A novel equation has been

derived by Liquori and Tripiciano (1980) as a relaxation auto-catalytic function by linking in a master equation the solu-tions of two rate equations typical of linear non-equilibriumthermodynamics. These two linear differential equations cor-respond to a 'slow' and to a 'fast' growth and may be writtenas:

d_t -1 l(~t)-)

d__ - 1(It)- 2)d t Q2

(4a)

(4b)

(1(t) and t2(t) are the extents of the two kinetics processes, iand t2 are the corresponding asymptotic values (homeostaticvalues), and r1 and T2 are time constants (or relaxation times).The solutions of (4a) and (4b) are:

41() = 4i (1 - exp - t/r1) (5a)42() = 42 (1 - exp - t/r2) (5b)The reduced function y1(t) = _1(0obtained:yl(t) = 1 - exp - t/rly2(t) = 1 - exp - t/r2

Y2 = 4t) may be~2

(6a)(6b)

Notice that y1(t) and y2(t) are dimensionless quantities varyingbetween 0 and 1.The autocatalytic transition from the slow to the fast

growth is expressed by the autocatalytic master equation:y(t) = y1(tO(t) + y2(t) [1 - y(t)] (7)By rearranging equation (7) and replacing y1(t) and y2(t) ac-cording to 6a and 6b the reduced growth equation was finallyobtained:

y(t)= 1 - exp - t/T11 - exp - tITI + exp - t/r2 (8)

In order to apply equation 8 to cell multiplication, experimen-tal plots may be constructed with values y(t) = N() where N(t)is the number of cells at time t and N is the correspondingextrapolated homeostatic value. Y(t) plots may also be ob-

2054

0.5

V(Cs)

1.0

0 40 80t (minutes)

Fig. 3. Plot of the function y(t) fitted to the experimental data correspon-ding to Figure 2. (r, and r2 are 41 min and 15 min, respectively.)

tained from calorimetric growth curves using values ofy(t) = Q-). The fitting of equation (8) to experimental re-

duced growth curves was found to be most satisfactory forbacterial and yeast cells.

It is remarkable that equation (8) also satisfactorily fits ex-perimental plots of y(t) = Q{t) at given sperm concentrations.

QFigure 3 shows a typical example. The experimental y(t)values were obtained from the TP diagram shown in Figure 2according to the transformations (2) and (3), whereas the con-tinuous curve was calculated according to the theoreticalequation (8) by a fitting procedure.

The total heat evolved as a function of sperm concentra-tion. When the total heat Qex obtained from the TPdiagram according to (1) is plotteS against sperm concentra-tion (Cs), a sigmoidal plot is obtained. A reduced plot may beconstructed using values y(Cs) = Qexp(Cs) where Qexp(Cs) is

Q(exp)the total heat evolved at the sperm concentration Cs, andQexp is the asymptotic value defined as Qexp =lim Qexp(Figure 4). (The value of Qexp may be obtained by a suitableCs- X0 graphic extrapolation procedure.)A theoretical sigmoidal function describing the dependence

of y(t) on Cs may be obtained using a model mathematicallyanalogous to that introduced by Liquori and Tripiciano forkinetic cooperativity (1980). It may be formulated as follows.Let us consider the following two functions, mathematicallyanalogous to (5a) and (5b), corresponding to two distinct pro-cesses (1 and 2):Q1exp(CS) = Q exp (1 - exp - Cs/-yl) (9a)Q2exp(CS) = Q exp (1 - exp - Cs/72) (9b)QIexp(Cs) and Q2exp(Cs) are the total heats evolved, Q1exp andQ exp are their saturation (asymptotic values) and -y and 72

Spenn-egg interaction

QaepC3)Qexp 0

0

0

7.0

0O~~< o Cs

7.5

Fig. 4. Semilogarithmic plot of Qexp as a function of sperm concentra-Qex

tion. The continuous curve corresponds to the function -(Cs) according toequation 12 with the following value of the parameters: -Yi = 1.5 x 1010sp/ml and 72 = 1.3 x 107 sp/ml.

are two constants having the same dimension as Cs. (9a) and(9b) may be transformed into the following reduced dimen-sionless functions:y-l(Cs) = 1 - exp - Cs/'y1 (lOa)Y2(Cs) = 1 - exp - Cs/y2 (lOb)The function -,(Cs) and -2(Cs) may be linked by the follow-ing master equation:y-(Cs) = y1(Cs)W2(Cs) + Y2(Cs) [1 - y(Cs)] (11)

where -,(Cs) = Qlexn(Cs) and y2(Cs) = Q2exn(CS)QIexp Q2exp

which is the mathematical analogue of equation (7). Rear-ranging (11) and replacing y-,(Cs) and y-2(Cs) according to(lOa) and (lOb) yields the following sigmoidal function:

y-(Cs)= 1 - exp - Cs/lyl1 - exp - Cs/,1 + exp - CS172

(12)

Considering the large degree of (unavoidable) scattering ofthe experimental results as mentioned under Materials andmethods, the plot obtained from the above equation satisfac-torily reproduces the experimental plots of y-(Cs) as a functionof Cs (see Figure 4).Spermatozoa and eggs in sea water

Excess total heat. When increasing volumes of semen(dry sperm) are mixed with a constant volume of sea watercontaining a given concentration of ghost eggs (glycerol-treated eggs), the heat evolved is - 50% greater than thatevolved in a reference cell containing only spermatozoa in seawater at the same concentration: this is the 'excess heat'Q*exp. When the latter quantity is plotted against sperm con-centration Cs, a peculiar plot Q*ex (Cs) is obtained. At a suf-ficiently high egg concentration, 6*exp(Cs) increases steeplyto a maximum value and then decreases.The shape of the plot is that of an asymmetric bell. If each

value of Q*exp(Cs) is divided by the maximum value, name-ly (Q*exp)max, the adimensional plot of QQep(a). shown in

(Q*exp)maxFigure 5 is obtained. Parallel experiments on oxygen con-sumption show that the excess heat (described above) is notassociated with oxygen consumption by the spermatozoa.The negative results of experiments where the supernatant ofa suspension of eggs in sea water was added to dry sperm ex-clude the hypothesis that the excess heat is caused by the eggsreleasing some factors activating anaerobic metabolism.Therefore, this activation must be connected with the binding

2055

V.Elia et al.

y

1.0

0.5

log Cs 8.0

Fig. 5. Semilogarithmic plot of Q as a function of sperm concentra-Q*expmax

tion at an egg concentration of 5 x 103 eggs/ml. (Q*ex,)max is the value of

Q*exp at the maximum of the plot. The values of bound sperm are alsoalso shown as full circles (see text). (boud sperm)max

A

Fig. 6. The photograph shows spermatozoa agglutinated head-to-head athigh sperm concentration (15 x 107/ml). Arrow: a group of five sper-matozoa; small arrow: a group of three spermatozoa; arrow heads:doublets. x 1200.

of the spermatozoa to the VC of the eggs. However, theasymmetric bell-shape of the Q* (Cs) plot means that sper-

Q exd)maxmatozoa binding to the VC must be modulated by more thanone concomitant process.Two possibilities may be envisaged either by assuming an

inhibitory binding of spermatozoa to a different class of sites(sites B) on the VC, or a process involving aggregation ofspermatozoa which again is competitive with their binding as

A'monomeric' spermatozoa + eggs = bound spermatozoa

lBt

agglutinated spermatozoa (polymeric)Scheme I

single units to the VC. The latter phenomenon has beenobserved under the microscope (Figure 6) when sperm con-centration exceeds the range beyond the maximum of theQ*exp(Cs) plot, namely (Q*exp)max(The scheme describes competitive coupling between the

binding (process A) of spermatozoa to the available sites onthe VC, and process B corresponding to self-binding, orsperm agglutination.

According to the scheme, the probability <y> that asperm cell bound to an available site on the VC surface is bio-chemically activated may be expressed by the 'joint pro-bability':<Y> = YA(l - YB) (13)where YA is the 'a priori probability' that the spermatozoon isbound to an available site on the VC, and 1 - YB is the 'apriori probability' that the spermatozoon is not bound toother sperm cells to form a clump.The metabolic heat fired by the spermatozoa should then begiven by:

Q*exp(Cs) = <y> Qexp (14)where Q*exp(Cs) is the excess heat at a sperm concentrationCs, and Qexp is a normalization factor having the dimensionof heat.The following semi-empirical functions may be used to

describe binding and self-binding of spermatozoa accordingto the above scheme:

Y 1= KA(Cs)A (ISa)KA(C'n

YB = KB(CS)B (15b)1 + KB(Cs)nBKA and KB are binding constants, nA and nB are 'cooperativi-ty parameters' analogous to Hill numbers. It should be notic-ed that for nA = 1 and nB = 1 (15a) and (15b) reduce to thefamiliar form of Langmuir and Michaelis-Menten equations.

According to (15b) the fraction of free, non-aggregatedspermatozoa is:

1 + KB(CS)nB (16)replacing (15a) and (16) into (13) the latter becomes

< y> - KA(CS)A O3bs[1 + KA(CS)nA][l + KB(CS)nB] (13 bis)

A plot of <y> as a function of Cs, using a set of appropriatenumerical values of KA, KB, nA and nB is shown in Figure 7where the construction of the function (13 bis) from its com-ponent functions (15a) and (15b) is also shown.

(13 bis) satisfactorily reproduces the asymmetrical bell-shaped plot of Q*exp(Cs). The use of (13 bis) is also supportedby the fact that the numbers of egg-bound spermatozoa atincreasing-concentrations divided by the maximumnumber bound fits the normalized plot Q*exp (see Figure5). (Q*exp)maxThe concentration of the eggs was kept constant in both ex-periments (5 x 103 eggs/ml).

2056

Sperm-egg interaction

attached to one another by their heads above a critical rangeof sperm concentration (similar to 'inverted micelles' of longamphyllic molecules); (iii) the deviation from the linearity ofthe light scattered by suspensions of spermatozoa in sea waterabove the same critical sperm concentration range. The com-petition between egg binding and self-binding of sper-matozoa, that is adhesion between the sperm head and theVC and self-adhesion between the sperm heads, suggests thepresence of at least similar receptor sites at the surface of bothgametes. This possibility is currently being investigated.

log CS

7.0 75 8.0

Fig. 7. Semilogarithmic plot of the function YA (1 - YB) calculated accor-ding to 13 bis as a function of the sperm concentrations. Plots Of YA and 1- YB are shown in the same figure. The values of the parameters appear-ing in 13 bis are -logKA = 24.52, -logKB = 30.38, nA = 3.27, nB =3.71.

DiscussionSperm binding to the VC is the first step of the sperm-egg

interaction. The analysis of the time sequence of the events ofsperm-egg binding leads to the conclusion that for a suc-cessful interaction (i.e., one leading to egg activation) thespermatozoon itself needs to be activated. The expression'sperm activation' means the alterations the spermatozoonundergoes upon interacting with the egg. In ascidians, spermbinding to the VC brings about morphological changesculminating in a few spermatozoa undergoing the acrosomereaction. The present results which were obtained using'ghost eggs' that are unable to induce the acrosome reaction,add to our knowledge on the molecular processes controllingsperm activation. In fact, when spermatozoa become attach-ed to the ghost eggs they fire an excess heat not associatedwith oxygen consumption and possibly corresponding to analteration of their metabolism. This may be related to thechanges the sperm mitochondrion undergoes in the activatedascidian spermatozoa (swelling and sliding in the tail)(Lambert and Epel, 1979; De Santis et al., 1983). Uncouplingof respiration has been found to occur in sea urchin sper-matozoa activated by egg jelly (Christen et al., 1983). Alter-natively, a shift of ATP utilization may occur as spermatozoaare bound to the VC as a consequence of the diversion of theATP breakdown to generate heat rather than to providepower for motility. These alternatives are presently being in-vestigated.

It is interesting that, unexpectedly, the experimental bell-shaped curve describing the dependence of the firedmetabolic heat on sperm concentration at an optimal egg con-centration can be analyzed in terms of a joint probability,namely:<Y> = YA( - YB)where <y> corresponds to the probability that a spermato-zoon is activated, YA is the 'a priori probability' that anavailable site is occupied on the VC and 1 - YB correspondsto the 'a priori probability' that the spermatozoon is notbound to other spermatozoa, thus forming clumps.The above model is fully consistent with: (i) determinations

of the number of spermatozoa bound to the eggs at increasingsperm concentrations; (ii) direct microscopic observationsshowing the formation of clumps consisting of spermatozoa

Materials and methodsThe experiments were performed on the ascidian Ciona intestinalis. Eggs

and sperm were collected from the gonoducts. The semen was stored as 'drysperm' i.e., undiluted semen; in these conditions the spermatozoa remainviable and fertile for several hours. Glycerol treatment of the eggs was per-formed as previously described (Rosati and De Santis, 1978).To minimize individual variability, the experiments were carried out with

sperm pooled from several animals. However, seasonal variability, which con-cerns the animal's reproductive cycle, and which influences both the thermalresponse and the rate of oxygen consumption, could not be avoided. Hencecaution should be taken when comparing experiments carried out at differenttimes of the year.CalorimetryThe heat flux was estimated using a standard batch LKB 2107-112 micro-

calorimeter. This instrument is equipped with two separate gold reactionvessels 10 ml in volume) in thermal contact with a pair of thermocouples anda heat sink. The heat produced in the reaction vessels causes a heat flow fromthe vessels to the heat sink and an electromotive force proportional to thetemperature difference. The output voltage is amplified and fed to a recorder.The calorimeter was calibrated using an electric heater. Each reaction vesselhas two compartments into which the reactants are introduced. Duringmeasurements, the calorimeters are kept in rotatory motion to ensure constantmixing of the reactants in the two compartments. The thermocouples in con-tact with the cells are connected in such a way that the electromotive forcecaused by the heat flow from the two cells to the heat sink, are balanced out.In this manner the amplified output voltage from thermopiles is proportionalto the difference between the heats produced into the two vessels.Two different systems have been investigated: (i) dry sperm diluted with sea

water; (ii) dry sperm mixed with eggs in sea water. In experiments (i) one ofthe two compartments of the reaction vessel contained 4 ml of sea water, andthe other compartment contained different measured amounts (from 5 to60 d1) of 'dry sperm'. The second reaction vessel was left empty. The experi-ment started when a stable base-line without drifts, indicating thermalequilibrium, was obtained (-2 h). In experiments (ii) one of the reactionvessels contained dry sperm and 4 ml of a suspension of glycerol-treated eggsin sea water. The other vessel contained the same quantity of dry sperm and4 ml of sea water. In series (ii) the experimentally measured heat flow wasautomatically subtracted from the heat evolved by the spermatozoa alone,thereby giving the amount of excess heat due to the sperm-egg interaction. Allexperiments were conducted at 25°C and the measurements were continueduntil the initial stable base-line was reached (2 h). The sperm concentrationwas routinely checked by measuring the absorbance of a fixed suspension ofthe initial dry sperm according to Rosati and De Santis (1980).Oxygen consumption

In parallel experiments, the oxygen consumption of the same sperm con-centrations in the presence or absence of glycerol-treated eggs (ghost eggs) wasdetermined with an oxygen electrode using a Gilson oxygraph. Most of theoxygen was absorbed - 20 min after dilution. Under the same conditions, andat all sperm concentrations tested, no changes in the oxygen consumption as aresult of spermatozoa binding to the ghost eggs were detected. The curves ofoxygen consumption by the spermatozoa in the presence of eggs is similar tothat in the absence of eggs.Sperm motility and the binding reaction of the sperm used in these ex-

periments was always checked in the light microscope under dark field il-lumination.Quantitative estimate of sperm bound to ghost eggsThe binding reaction of different sperm and egg concentrations was quan-

titatively estimated according to the subtraction method of Vacquier andPayne (1973) and has been described in a previous paper (Rosati and De San-tis, 1980).

With a constant egg concentration (5 x 103/ml) and increasing the sperm

2057

V.EJia et al.

concentration, it was confirmed that the optimum conditions for sperm bin-ding are reached at a sperm concentration of 4-5 x 107/ml with a sperm/eggratio of 0.8-I x 104. Under these conditions, depending on the seasonal andindividual variability of the animals, 30-50% of the spermatozoa in thesuspension become bound to the eggs within 6 min. When the sperm concen-tration is increased to 15 x 107/ml while also increasing the egg number, thepercentage of sperm bound drops to 25%o and reaches very low values atsperm concentrations of 30 x 107/ml. Increasing the sperm concentration alsoresults in an increasing formation of clumps of spermatozoa agglutinated bytheir heads.

In the reverse experiment in which the sperm concentration is kept constant(4-5 x 10 -7/ml) and the egg concentration is increased to >5-8 x 103/ml,the percentage of spermatozoa that become bound to the eggs, does not in-crease. Indeed, egg concentrations > 10 x 103/ml appear to cause a decrease inthe percentage of bound spermatozoa.

AcknowledgementsWe thank Drs.R.De Santis, R.Pinto, A.De Santis for their help in some ex-periments, Drs.A.Tripiciano and C.A.Mattia for their help in some of the fit-ting procedures, and G.Princivalli for the painstaking typing of themanuscript. We also thank Dr.D.Epel and Professor H.Kornberg for theircritical reading of the manuscript and for their suggestions. This work hasbeen supported by CNR grants of Progetto Finalizzato Chimica Fine e Secon-daria to A.M. and to G.B. The calorimeter was purchased with a special grantfrom the University of Naples.

ReferencesBeezer,A.E., ed. (1980) Biological Microcalorimetry, published by Academic

Press, New York.Christen,R., Schackmann,R.W. and Shapiro,B.M. (1983) Dev. Biol., 98,

1-14.De Santis,R., Jamunno,G. and Rosati,F. (1980) Dev. Biol., 74, 490-499.De Santis,R., Pinto,M.R., Cotelli,F., Rosati,F., Monroy,A. and D'Alessio,G. (1983) Exp. Cell Res., in press.

Lambert,C.C. and Epel,D. (1979) Dev. Biol., 69, 296-304.Liquori,A.M. and Tripiciano,A. (1980) in Barigozzi,C. (ed.), Lecture Notes

in Biomathematics, Proceedings of Vito Volterra Symposium on math-ematical models in biology, Roma 1979, Springer, New York.

Pinto,M.R., De Santis,R., D'Alessio,G. and Rosati,F. (1981) Exp. Cell Res.,132, 289-295.

Rosati,F. and De Santis,R. (1978) Exp. Cell Res., 112, 111-119.Rosati,F. and De Santis,R. (1980) Nature, 283, 762-764.Vacquier,V.D. and Payne,J.E. (1973) Exp. Cell Res., 82, 227-295.

2058