EP0380536A1

EP0380536A1 - In vitro method for the induction of the spermatozoal acrosome reaction and application of said method to the assessment of spermatozoa and the treatment of male-related infertility

Info

Publication number: EP0380536A1
Application number: EP88908291A
Authority: EP
Inventors: Paul Theodore Tomkins
Original assignee: HOUGHTON James Anthony
Current assignee: HOUGHTON James Anthony
Priority date: 1987-09-23
Filing date: 1988-09-23
Publication date: 1990-08-08
Also published as: AU2489188A; IE872567L; JPH03501203A; WO1989002743A1

Abstract

Procédé permettant d'induire la réaction de l'acrosome dans des spermatozoïdes, consistant à soumettre les spermatozoïdes in vitro à l'électroperméabilisation mettant en oeuvre l'application d'un champ électrique suffisant pour élever le potentiel de la membrane du plasma spermatique à partir d'environ -70 mV jusqu'à +1 V, afin de permettre un influx d'ions Ca++. Grâce à ce procédé on peut atteindre jusqu'à 75 % de pénétration dans ledit dosage de pénétration de sperme (SPA) avec des échantillons oligospermiques. Le procédé est applicable dans l'évaluation des spermatozoïdes, dans la préparation de spermatozoïdes pour la fertilisation in vitro et dans le traitement de la stérilité chez l'homme.Method for inducing the acrosome reaction in sperm, comprising subjecting the sperm in vitro to electropermeabilization using the application of an electric field sufficient to raise the potential of the membrane of the sperm plasma from from about -70 mV to +1 V, to allow an influx of Ca ++ ions. Thanks to this process, up to 75% penetration in said sperm penetration assay (SPA) can be achieved with oligosperm samples. The method is applicable in the evaluation of spermatozoa, in the preparation of spermatozoa for in vitro fertilization and in the treatment of infertility in humans.

Description

IN VITRO METHOD FOR THE INDUCTION OF THE SPERMATOZOAL ACROSOME REACTION AND APPLICATION OF SAID METHOD TO THE ASSESSMENT OF SPERMATOZOA AND THE TREATMENT OF MALE-RELATE INFERTILITY

This invention relates to an in vitro method for the induction of the spermatozoal acrosome reaction and to the application of said method in artificial insemination and in vitro fertilisation (IVF) .

The vast majority of ejaculated spermatozoa (sperm) are not immediately capable of achieving fusion with an ovum. In the normal course of events, the spermatozoa attain this competence while ascending the female reproductive tract, in particular, in the uterus and the fallopian tubes. The spermatozoa first of all undergo a. reversible capacitation step followed by an irreversible acrosome reaction. In in vitro fertilisation techniques, the acrosome reaction is obtained by a number of routes.

The true sequence of events in the in vivo acrosome reaction has not been fully elucidated and it cannot be assumed that any in vitro method is mediated by a similar sequence of events. The in vivo process is multifactorial and temporal and this doubtless explains why the acrosome reaction is attainable in vitro by a variety of routes.

A spermatozoan is a highly differentiated cell whose role is the protection, transport and delivery into the ooplasm of the male genome. The structure of the mammalian spermatozoan varies widely from species to species, however a general mammalian structure is apparent:

It is oval shaped in rabbit, boar and bull, resembles a long cylinder in fowl and is curved in mouse, hamster and rat. Size variations also exist. The human sperm head is about 5.0 μM long, 3.5 μM wide and 2.0 μM thick, whereas that of the bull is approximately 8.5 μM long, 4.0 μM wide and 0.3-0.5 μM thick. These size variations could be important in selective sperm penetration through cervical mucus and the layers surrounding the ooσyte.

The spermatozoan comprises a head portion linked to a tail portion by a fragile neck portion. The tail portion is responsible for the motility of the spermatozoan. The spermatozoan head can be divided into two parts, the nucleus and the surrounding membrane structures. The membrane structures include the plasma membrane, that covers the entire structure of the head, the acrosome, a bag like structure that surrounds the anterior portion of the nucleus, a postnuclear cap that covers the posterior portion of the nucleus and the equatorial segment which represents the area of overlap between the postnuclear cap and the acrosome. The acrosome consists of an inner acrosomal membrane and an outer acrosomal membrane which is in close contact with the plasma membrane.

The reversible capacitation step enables sperm to penetrate the acellular glycoprotein layer, the zona pellucida, of the ovum (Bedford, J.M. , 1970, Biol.

Reprod. (Suppl.) 2 , 128-158). The exact mechanism is still unclear. Recent models suggest membrane alteration in surface charge, cholesterol depletion and motile behaviour.

The acrosome reaction is a morphological event where the plasma and the outer acrosomal membranes undergo multiple point fusion (Green, D.P.L. , 1978, The mechanism of the acrosome reaction, in Development in mammals, Ed., Johnson, M.H., Vol. 3, 65-81, Nth. Holland, Amsterdam) , resulting in the release of acrosomal hydrolases (Akruk, S.R. et al., 1979 Gamete Res., 2., 1-3). A basic assumption is that elevation of Ca ions in the cytoplasm between the plasma and outer acrosomal membranes is the key event just prior to initiation of the acrosome reaction. Capacitation involves the preparation of the sperm for the elevation

_ ++ . in Ca ions.

A viable acrosome reaction is an essential prerequisite for sperm fusion with the egg oolemma in mammalian fertilisation.

Since the original observations of sperm functional development began in the 1950's, a number of techniques have been developed to try and mimic the events leading to a viable acrosome reaction under in vitro conditions, taking into account species, sample state and quality. These techniques include:

(1) Pre-incubation in ligated rodent or rabbit uteri, Austin, C.R., 1951, Aust. J. Sσi. Res. 4_., 581-596;

(2) Pre-incubation in simple or complex culture !_ media supplemented with albumin or serum, Miyamoto, H. & Chang, M.C., 1973, J. Reprod. Fert. , 32. 193-205;

(3) Exposure to high ionic media, Brackett, B.G. and Oliphant, G. , 1972, Biol. Reprod., 12., 260-274;

(4) Treatment of sperm with Ca ++ lonophore,

Aitken, R.J. et al., 1984, J. Androl. 5., 321-329;

(5) Direct microinjection of sperm inter alia. Lassalle, B. et al., 1987, Gamete Res., 16., 69-78;

(6) Exposure to phosphatidylcholine liposomes,

Graham, J.K. et al., 1986, Biol. Reprod., 35, 413-424;

(7) Pre-incubation in simple medium supplemented with defined synthetic polymers and high Ca , Tomkins, P. T. et al., 1988; Hum. Reprod. 3,

367-376;

(8) Pre-incubation in the presence of glycosaminoglycans, Lee, C.N. et al., 1986, J. Anim. Sci., .63., 861-867; and

(9) Pre-incubation for 48 hours at + 4°C in TEST - yolk buffer, Bolanos, J.R. , 1983, Fert. Steril., 39. 536-540.

A research tool that is becoming increasingly used by clinicians is the so-called sperm penetration assay (SPA) (Yanagimachi, R. et al., 1976, Biol. Reprod. 15.

471-476) . This assay assesses the ability of capacitated human sperm to penetrate surrogate zona-free hamster eggs; quantitative estimates of % penetration, polyspermy and attachment are produced. The technique is not standardised, but does nevertheless give good predictive ability of samples' in vivo fertilizing potential, particularly if supported by motility data. Using this system, along with other tests, some categories of patients attending an infertility clinic can be ranked with some accuracy according to fertility potential.

The SPA has essentially been confined to assessment of infertility in humans to date, although there is a demand at present for extension of the assay for use in assessment of infertility in a number of domestic animals. Apart from fusion with human sperm, fusion of the zona-free hamster egg has been observed with sperm from the following species: budgerigar, bat, dolphin, mouse, deer mouse, rat, guinea pig, rabbit, dog, pig, goat, bull, horse and marmoset monkey.

Using an SPA system and specific stains, the Applicants have shown that traditional sperm capacitation regimes based on albumin or serum (2) are only capable of 'activating' ≤ 20% of the cell population. High ionic treatment (3) and TEST-yolk (9) are capable of elevating in vitro penetration levels of some samples, but are unsuitable for oligospermic samples. Exposure to Ca ++ ionophore (4) results in a dramatic and rapid increase in the in vitro penetration of fertile control samples, but has no effect on oligospermic and many other categories of infertile samples. Ionophore is also hard to control and due to its toxicity is unlikely to be countenanced for use in IVF or similar programs. Direct micro-injection (5) of sperm into egg ooplasm or the periviteline space is impractical as a routine technique, except perhaps in large research/clinical centres. The Applicants have achieved some success using media based upon synthetic polymers (7) . However, whereas this approach ensures uniformity and rapidity of response, it again appears to have little benefit for apparent sub-fertile donors.

Effectively, there is currently no simple 100% effective method for preparing sperm of any species for fertilization.

Electropermeabilisation or eleσtroporation is a technique involving use of an electric field pulse of high intensity to generate transient pores in cellular membranes. This technique has been used inter alia to introduce biologically active foreign genes into primary rat hepatoσytes. Ran Tur-Kaspa et al., 1987, Molecular and Cellular Biology jβ, 2, 716-718 and in cell-to-cell fusion, Zimmermann, U. and Vienken, J. 1982, J. Membrane, Biol. 62, 165-182.

The Applicants are not aware of any previous application of electropermeabilisation to spermatozoa per se of any species or in sperm-to-egg fusion.

It is an object of the present invention to provide an improved method for preparing spermatozoa of any species for fertilization.

It is a further object of the present invention to provide a method for assessing sperm and, thereby, male fertility.

It is a still further object of the present invention to provide a method for the treatment of male-related infertility. Accordingly, the invention provides a method of inducing the acrosome reaction in spermatozoa, which comprises subjecting spermatozoa in vitro to electro¬ permeabilisation involving application of an electric field sufficient to raise the spermatozoal plasma membrane potential from about -70 mV to +1 V to allow an influx of ca⁺⁺ ions, such that up to 100% penetration is observed in the so- called sperm penetration assay (SPA) with normal sperm and up to 75% penetration observed in said assay in oligospermic samples.

The invention also provides a method of assessing the developmental potential of spermatozoa, which comprises subjecting spermatozoa in vitro to electro¬ permeabilisation involving application of an electric field sufficient to raise the spermatozoal plasma membrane potential from about -70 V to +1 V to allow an influx of Ca ions, and determining the ability of the spermatozoa so-treated to penetrate surrogate zona-freβ hamster eggs, thereby indicating the developmental potential of said spermatozoa.

The invention further provides a method of preparing spermatozoa for in vitro fertilisation, which comprises inducing the acrosome reaction in said spermatozoa in vitro by subjecting the spermatozoa to electro¬ permeabilisation involving application of an electric field sufficient to raise the spermatozoal plasma membrane potential from about -70 mV to +1 V to allow an influx of Ca ions.

The invention further provides a method of treating male infertility, which comprises converting otherwise non- viable spermatozoa to viable spermatozoa by subjecting said spermatozoa in vitro to electropermeabilisation involving application of an electric field sufficient to raise the spermatozoal plasma membrane potential from about -70 mV to +1 V to allow an influx of Ca ions, thereby inducing the acrosome reaction in said spermatozoa.

The invention further provides a method of diagnosing male-related infertility which comprises subjecting a sample of spermatozoa in vitro to electro¬ permeabilisation involving application of an electric field sufficient to raise chβ spermatozoal plasma membrane potential from about -70 V to +1 V to allow an influx of Ca⁺⁺ ions and determining the ability of the spermatozoa so treated to penetrate surrogate zona-free hamster eggs, thereby indicating the developmental potential of the spermatozoa.

The methods according to the invention effectively side step all the preparatory capacitation stages by directly permitting calcium ion entrance to a large number of cells inducing rapid and synchronised acrosome reactions. When cells are subjected to a large and transient DC field such that the plasma membrane resting potential is raised from about -70 V to +1 V, localised regions of the membrane break down and pores are realised. The number, size and duration of these pores is a function of the applied voltage, duration of pulse and pulse medium, temperature, as well as, other factors such as cell size and the cholesterol/phospholipid ratio in the membrane.

The induction of the acrosome reaction in accordance with the invention, which brings about direct and rapid influx of Ca⁺⁺ ions into sperm, probably bypasses all or some of the membrane changes associated with the strict capacitation phase, i.e., it creates a large and relatively instant population of sperm in various stages of acrosome induction, some of which retain otility. The end result of ionophore(4) exposure is similar to electropermeabilisation, but the mechanism is different. Ionophore effectively transports calcium ions through the membrane. Electropermeabilisation would seem to be more effective, yet much less deleterious than ionophore, which usually kills more than half the cells, is difficult to control and does not appear to benefit the majority of sub-fertile samples. All other previous pre-treatment systems generally only induce acrosome reaction in a small percentage of the sperm.

The electric field is applied in the methods according to the invention as a pulse at a voltage of 250-1000 V corresponding to a field strength of 625-2000 V cm^" , especially 400-800 V corresponding to a field strength of 1000-2000 V cm^"1.

The pulse preferably either emanates (i) from the discharge of a capacitor, (ii) from the variable switching of a static power-supply or (iii) by means of a high voltage pulse generator. The former approach gives rise to a pulse which is exponential in character, the latter two approaches will both give relatively clean square pulses of defined shape and duration. Additionally, one could amplify the output from a standard pulse - generator or stimulator.

A Bio-Rad (Trade Mark) Gene Pulsar may be used to generate a pulse which is exponential in character.

Suitable operating conditions for human spermatozoa have been found to be 500 V at 25 μF with a time constant of approximately 2.5 mS. An inter-electrode distance of 4 mm is used, such that the application of 500 V implies 1250 V/cm. In the case of the Gene Pulsar, penetration remains at 100% for normal samples at 750 V, but polyspermy is decreased, while voltages below 200 V are sub-threshold. With the Bio-Rad machine, application of a single pulse has proved effective. A Hoefer Instruments Progenetor may be used to generate a square wave pulse. Appropriate operation conditions for the Hoefer Progenetor are 450 V and 10-50 S pulses. Electropermeabilisation is usually conducted at +4 C, since the putative pores tend to stay open longer at lower temperatures before resealing. At lower temperatures the threshold of the breakdown potential is raised. Accordingly, in the methods according to the invention, electropermeabilisation is normally carried out at a temperature in the range 20-25°C. However, the basic method is extremely rapid, taking only minutes, while the time from sample arrival to insemination is only approximately one hour, thereby obviating the need to rigidly regulate temperature. We have found that sperm incorporation in the SPA decreased by 34% when samples were pulsed at +4°C and 49% when pulsed at 37°C.

The pulse medium includes a source of Ca ions at a concentration in the range 2.0-20 mM, most especially 10 mM.

The pulse medium may be an ionic, partially ionic or an essentially non-ionic medium. A suitable non-ionic medium for use in the method according to the invention is one based on a polyol, especially a monosaccharide or disaccharide, especially sucrose. Advantages of using non-ionic sucrose is that local current heating effects are minimised and pulse strength is increased in capacitance discharge systems. Mannitol is also suitable for such systems and has the added advantage of scavenging hydroxyl radicals. However, Mannitol has been found to be about 20% less effective than sucrose. A suitable ionic-based pulse medium has been found with the following composition: 137 mM NaCl, 20 mM HEPES, 5 mM C1, 0.7 mM Na₂HP0 , 6 mM glucose and 5 mM CaCl₂. A very successful partial ionic medium has been developed with the following formulation: 210 mM inositol, 30 mM KC1, 5 mM HEPES, 100 μM inositol triphosphate, 2 mM CaCl_ and 1 mg ml BSA. With both of these media the need for a post-pulse centrifugal wash is eliminated and the sample can simply be diluted to the desired concentration, provided this involves a dilution factor of at least X5. The advantages of ionic pulse media are best exploited using square wave pulse generators.

The pulse medium should also include an agent which prevents sticking of spermatozoa to reaction chambers containing it and maintains motility thereof. A suitable such agent is a polymer such as polyvinyl alcohol. Serum albumin can also be employed, though its chelation properties will ensure that the concentration of available ionized Ca is reduced up to a third.

Preferably, post-pulse gamete interaction is conducted in protein containing media such as albumin - based media.

In the accompanying drawings:

Fig. 1 illustrates the relationship between calcium concentration in a sucrose based pulse medium and SPA score parameters, mean percentage penetration, and sperm/egg ration measured as the average number of decondensed sperm heads detected in each zona-free hamster egg. Plot a represents mean percentage penetration and plot b represents regression of sperm/egg ratio on CaCl_;

Fig. 2 illustrates the relationship between applied pulse voltage (Gene-Pulsar) , mean total detected acrosome reactions and the SPA score parameters, mean percentage penetrations and mean sperm/egg ratio. Curve a. represents mean percentage penetration, curve b represents mean total percentage and partially acrosome reacted sperm, and plot σ represents mean sperm/egg ratio; and

Fig. 3 illustrates the relationship between postpulse incubation time, percentage penetration, and sperm incorporation. Plot a indicates mean percentage penetration and plot b represents mean sperm/egg ratio.

When penetration in the SPA is near or at 100%, the s/e ratio is a more sensitive measure of 'fertility' than the other parameters measured in the SPA.

The invention will be further illustrated by the following Example.

Example

Semen samples were obtained from patients referred from the Infertility Clinic of Galway Regional Hospital. The patients were a fairly homogenous group with only 20% exhibiting apparent secondary infertility. In 15% of couples, the spouse also demonstrated a reproductive problem. The mean age of the group was 33.5 ± 0.7 years while the mean duration of infertility was 4.3 ± 0.54 years. Approximately 25% showed an abnormal spermiogram on first referral, with the bulk of the remainder being- classified as unexplained infertility. The control group was smaller (N = 5) with a mean age of 37.2 ± 3.2 years. All had fathered a child within the previous ten years, had a normal spermiogram and no clinical indications of ill health.

Semen samples were obtained by masturbation into a sterile Sterilin (Sterilin is a Trade Mark) 60 ml container and kept warm until delivered to the laboratory.

Fresh semen samples were received within minutes of collection and allowed to liquify for a maximum of 30 minutes. A preferentially motile population of sperm was isolated by layering 1 ml of the sample over a simple discontinuous two-step Percoll gradient. The lower layer consisted of 95% Percoll in 20 X HEPES buffer containing 2.6 M NaCl, 0.08 M KC1, 20 mM

CaCl₂.2H₂0, 0.28 M glucose and 200 mM HEPES, pH 7.3-7.4 (osmolality 360 mOsm, density 1.10 g ml- ) . HEPES was prepared as a 1 M stock solution by mixing tissue culture grade acid and base solutions at 37°C to a stable pH of 7.3-7.4. The final osmolality was 312 mOsm. The HEPES stock solution was diluted as necessary • to give the desired molarity. The upper layer was composed of 47.5% Percoll, 2.5% HEPES buffered saline in complete medium (osmolality 335 mOsm, density 1.05 g ml^" ) . The gradient was slightly hyperos otic for the reasons described by Vincent, R. and Nadeau D. , (1984) Anal. Biochem. 141. 322-328. The loaded gradient was centrifuged at 350 g for 25 minutes in an angle head rotor at room temperature. The diffuse sperm pellet at -360 mOsm was then carefully removed, resuspended in 10 ml of fresh medium (freshly prepared medium of the same formulation) as above and 1 ml of Lipiodol (Lipiodol is a Trade Mark for a radio-opaque oil of May & Baker Limited) and twice centrifuged in a swing-out rotor at 350 g at room temperature. The sperm were then diluted with BWW/DM medium as hereinafter defined and subjected to a further centrifugal wash. BWW (herein referred to as BWW/n) is a modified Tyrodes medium, the Biggers, Whitten & Whittinghams medium (Biggers et al., 1971). The invariant constituents were: 94.8 mM NaCl, 4.77 mM KC1, 1.2 mM KH₂P0₄, 1.2 mM MgS0₄.7H₂0, 5.5 mM glucose, 25 mM NaHCO,, 0.27 mM sodium pyruvate, 19.5 mM sodium lactate, 0.001% penicillin-streptomycin and 20 mM HEPES. BWW/DM is the defined medium containing a combination of polyvinyl alcohol (PVA) and dextran (Tomkins, P.T. et al., 1988 supra). The complete formulation for BWW/DM is given in Table 1. The sperm were suspended at a concentration of 0.5 x 10 8 ml—1 in a pulse medium consisting of 0.26M Analar sucrose, 10 mM Analar

CaCl₂.2H₂0, 5 mM HEPES pH 7.3 - 7.4 and 50 _μg ml^"1 polyvinyl alcohol (M.W. 125,000). 800 μl aliquots of the sperm suspension were subjected to high voltage pulses in a 'Bio-Rad Gene Pulsar' of 500 V at 25 μF with a time constant of « 2.5 mS (1250 V/cm) . After pulsing, the suspension was diluted with BWW/DM medium, washed once, resuspended at a concentration of 0.5 x 10 7 ml—1 and immediately processed through the SPA.

Subsequent comparative experiments carried out have demonstrated that SPA results for all samples are higher and more consistent if post-pulse gamete interaction is conducted in protein containing media. Thus while

BWW/DM has proved superior to BWW/n containing 0.3 - 0.5% human serum albumin for inducing acrosome reactions and fusiogenic sperm by means of pre-incubation, it appears to be inhibitory after sperm have been pulsed. One possible explanation is that BWW/DM increases cell leakiness and the post-pulse situation does not adequately preserve cell integrity; motility certainly declines more in BWW/DM than in BWW/n under post-pulse conditions. The complete formulation for BWW/n is given in Table 1.

The total time that elapsed from sample arrival to insemination of the egg drops was approximately one hour. The results of the SPA in terms of attachment, % penetration and s/e ratio for the "pulsed" samples according to the invention relative to normal controls and oligospermic samples are depicted in Table 2.

The group designated *5 hr cap/HSA' in Table 2 was the result of pre-incubating sperm in BWW/n containing 0.5% human serum albumin (HSA) , prior to running in the SPA as usual. The group designating '24 hr cap/HSA' in Table 2 was the result of pre-incubating the sperm overnight for 18-24 hours in BWW/n containing 0.3% HSA. Control sperm concentrations ranged from 8 x 10 7 ml-1 to 2.5 x 10 8 ml—1, while oligospermic sample concentrations ranged from 1 x 10⁶ ml^"1 to 1.5 x 10⁷ ml^-1.

Experimental details are described by Tomkins, P.T. et . al., 1988 supra. Apart from the use of BWW/DM medium, the only other modification of the SPA is the use of hyaluronidase and trypsin at lower concentrations viz 0.05% relative to the standard SPA of Yanagimachi, R. et al. , 1976 supra.

A number of experiments were carried out to determine mean penetration and sperm/egg ratios in the SPA for four classes of sperm samples, namely (i) those of known fertility, (ii) oligospermic, (iii) asthenospermic and (iv) those with unexplained fertility, using the electropermeabilisation method according to the invention. The results are depicted in Table 3. TABLE 2

Technique Sample Attachment % Penetration s/e Ratio

5 hr cap/ control < 20 50 0.65

HSA

24 hr cap/ control ≤ 40 72 0.9

HSA

5 hr cap/ control < 100 90 1.2

BWW/DM

PULSE control < 500 100 10.25

5 hr cap/ oligo- < 5 <10 -

HSA

5 hr cap/ oligo- < 10 <10

BWW/DM

PULSE oligo- 50-500 75 1.2

Cap =capacitation HSA =human serum albumin DM=defined medium Oligo- =oligospermic

NOTE: long term incubation of oligospermic samples in albumin media was not employed because such material does not survive well in culture. TABLE 3 Mean Penetration and Sperm/Egg Ratio Measurements in the SPA for Four Classes of Sperm Samples

Method of Sperm Preparation Control Electropermeabilisation

No. of Mean Mean Mean Mean

Class of Sample expts. % penetration sperm/egg % penetration sperm/egg

Known fertility 12 35 15 0.41 ± 0.1 100 8.9 + 3.8 (4)

Oligospermia 3.0 ± 2.0 0.03 0.03 57 ± 10.1 0.8 ± 0.09 (3)

Asthenosper ia 15.7 ± 4.5 0.21 0.05 61 ± 12.4 2.47 ± 0.9 (2)

Unexplained infertility 27.0 ± 9.7 0.32 ± 0.08 100 4.3 ± 1.9

* The numbers in parentheses refer to the number of donors

Fig. 1 demonstrates the effect of Ca ion concentration of the medium on the electropermeabilisation of sperm in accordance with the invention as measured in terms of s/e ratio and % penetration according to the SPA. Some of these results have been published, Tomkins, P.T. and Houghton, J.A. Fertil. Steril. 1988, 50, 329-336.

The results of manipulating pulse voltage are graphically displayed in Fig. 2. Standard errors were within ± 10% for penetration and acrosome reaction responses and two units for sperm/egg ratio. The level of acrosome induction was directly quantified using a PSA-FITC fluorescent stain (Cross, N.L. et al. 1986, Gamete Res. 15, 213) . Mean penetration and sperm incorporation both approximate to normal bell-shaped curves. The proportionate increase in acrosome induction with voltage saturates beyond 1250 V cm^" at least with a fixed concentration of 5 mM Ca present.

Membrane vesicular fusion events and the release of hydrolytiσ enzymes during the acrosome reaction are not instantaneous processes. While rapid Ca influx may synchronise acrosome induction in the majority of cells, it is possible that the kinetics differ among subpopulations of sperm. Manipulation of postpulse incubation time was used to investigate this possibility. The mean results of two experiments are shown in Fig. 3. Penetration responses markedly declined if post-pulse incubation at 37 C was continued beyond 1 hour. This may reflect impending senescence of an active acrosome reacted population. Under tube incubation conditions, percentage otility was 72 ± 8% after the

3-hour culture period, but this had declined to only 18 ± 6% after a further 3-hour gamete interaction period. The electropermeabilised induction of the acrosome reaction according to the invention is significantly more efficient, as evidenced by the results depicted in Tables 2 and 3, rapid, and less harmful than any of the known in vitro techniques. The assumption that the basic method according to the invention is only inducing acrosome reaction in the sperm can be inferred from the SPA results. It is postulated that the electropermeabilisation method according to the invention alters the membrane of intact sperm in a manner permitting fusion. This is one explanation for the good results achieved with sub-fertile (oligospermic) samples.

Because normally only acrosome reacted sperm can fuse with an egg oolemma, the SPA is usually held to measure a sperm's ability (and hence the sample) to undergo capacitation, the acrosome reaction, fuse with and be incorporated into an egg and permit DNA decondensation. The results of Tables 2 and 3 indicate the methods according to the invention elevate penetration in the SPA. Accordingly, it is expected the basic method according to the invention will prove useful for use as an adjunct to this in vitro test by enabling grouping and class separation on the basis of distinct s/e data, as well as, establishing the ultimate fusiogenic potential of a sample. The SPA is now routinely conducted in a great many medical centres but interpretation and cross-evaluation has been hampered by the lack of standardisation in methodology and a relatively high associated level of false-negative responses in the text system (see Tomkins, P.T. et al.. supra 1988) for a discussion of the problem. Application of the present invention to the SPA results in a simple and efficient methodology that enables each sperm sample to achieve its maximal and optimised response in the test assay. This increases the resolution of the assay's discriminative powers and can potentially isolate a genuine population of males that do demonstrate pathological failings of the fusiogenic apparatus in all or a proportion of their sperm cells. Conversely it would be of benefit as a pre-screen to isolate couples that may profit from the use of interventionest reproductive technology such as IU-AIH (intra-uterine artificial insemination by husband) , GIFT (gamete intrafallopian transfer) or IVF, provided sperm are not rendered incapable of passage through the zona-pellucida by premature acrosome induction.

The basic method according to the invention can be used for all in vitro capacitation procedures prior to gamete interaction. The basic method according to the invention can also be used in the extension of the SPA known as the sperm chromosome assay (SCA) which establishes the level of normal sperm karyotypes.

There is as yet little animal IVF, one of the reasons being that capacitation of important domestic species such as bull and horse are difficult to achieve. It is anticipated the basic method according to the invention, which is applicable to the sperm of all mammalian species, should overcome these difficulties. The general applicability of the methods according to the invention to sperm of mammalian species is based upon the electrically mediated induction of a universal Ca dependent and triggered event. The specific conditions of pulse shape or amplitude, media employed and gamete handling conditions will differ from species to species.

The electropermeabilisation method according to the invention can also be used to achieve benign electropermeabilised entry of compounds, in which some sub-fertile sperm may be deficient such as cAMP, taurine and superoxide dismutase, into sperm. Unlike the sperm from a number of mammalian species, human sperm from known fertile individuals do not appear to exhibit high levels of acrosome induction following σapacitative pre-incubation treatments (C.E. Stock & L.R. Fraser 1987, Hum. Reprod. 2, 109-119). Since ≤ 0.001% of sperm in-vivo normally obtain the chance of interaction with the ovum, a combination of low sperm cell synchrony, temporal as well as physical factors may limit the size of the available pool of cells that are capable of an acrosome reaction at the appropriate time. Application of electropermeabilisation to such a sample may increase this available pool when coupled to other appropriate reproductive technology.

Human polyzoospermic samples and those with a high incidence of morphologically round headed sperm appear to demonstrate a high level of acrosomal aberrations (W.B. Schill et al. 1988, Hum. Reprod. 3, 139-145). These aberrations effectively preclude the probability of a normal acrosome reaction occurring in a proportion of the sperm population. However both such groups are likely to benefit from the efficiency of electropermeabilised acrosome induction when coupled to appropriate selection mechanisms.

Accordingly, the advantages and potential of the methods according to the invention will be appreciated.

Claims

CLAIMS :

1. A method of inducing the acrosome reaction in spermatozoa, which comprises subjecting spermatozoa in vitro to electropermeabilisation involving application of an electric field sufficient to raise the spermatozoal plasma membrane potential from about -70 V to +1 V to allow an influx of Ca⁺⁺ ions, such that up to 100% penetration is observed in the so called sperm penetration assay (SPA) with normal sperm and up to 75% penetration is observed in said assay in oligospermic samples.

2. A method of assessing the developmental potential of spermatozoa, which comprises subjecting spermatozoa in vitro to electropermeabilisation involving application of an electric field sufficient to raise the spermatozoal plasma membrane potential from about -70 mV to +1 V to allow an influx of Ca⁺⁺ ions, and determining the ability of the spermatozoa so-treated to penetrate surrogate zona-free hamster eggs, thereby indicating the developmental potential of said spermatozoa.

3. A method of preparing spermatozoa for in vitro fertilisation, which comprises inducing the acrosome reaction in said spermatozoa in vitro by subjecting the spermatozoa to electropermeabilisation involving application of an electric field sufficient to raise the spermatozoal plasma membrane potential from about -70 mV to +1 V to allow an influx of Ca⁺⁺ ions.

4. A method of treating male infertility, which comprises converting otherwise non-viable spermatozoa to viable spermatozoa by subjecting said spermatozoa i vitro to electropermeabilisation involving application of an electric field sufficient to raise the spermatozoal plasma membrane potential from about -70 V to +1 V to allow an influx of Ca⁺⁺ ions, thereby inducing the acrosome reaction in said spermatozoa.

5. A method of diagnosing male-related infertility, which comprises subjecting a sample of spermatozoa i _ vitro to electropermeabilisation involving application of an electric field sufficient to raise the spermatozoal plasma membrane potential from about -70 V to +1 V to allow an influx of Ca⁺⁺ ions, and determining the ability of the spermatozoa so treated to penetrate surrogate zona-free hamster eggs, thereby indicating the developmental potential of the spermatozoa.

6. A method according to any one of Claims 1-5, wherein the electric field is applied as a pulse at a voltage of 250-1000 V, corresponding to a field strength of 625-2000 V cm^"1.

7. A method according to any preceding claim, wherein the pulse is exponential in character.

8. A method according to any one of Claims 1-6, wherein the pulse is a square wave pulse of defined shape and duration.

9. A method according to Claim 7, wherein the pulse applied is approximately 500 V at 25 μF with a time constant of approximately 2.5 mS.

10. A method according to Claim 8, wherein 10-50 mS pulses are applied at 450 V.

11. A method according to any preceding claim, which is carried out a temperature in the range 20-25 C.

12. A method according to any preceding claim wherein the pulse medium includes a source of Ca ++ i•ons at a concentration in the range 2.0-20 mM.

13. A method according to any preceding claim, wherein the medium is selected from an ionic, partially ionic or an essentially non-ionic medium.

14. A method according to any preceding claim, wherein the pulse medium includes an agent which prevents sticking of spermatozoa to reaction chambers containing it and maintaining motility thereof.

15. A method according to any preceding claim, wherein the post-pulse gamete interaction is carried out in a protein-containing medium.

16. A method according to any preceding claim, wherein the spermatozoa is human spermatozoa.