WO1995016916A1 - Methods and reagents for sperm assessment - Google Patents

Abstract

The present invention relates generally to methods of assessing spermatozoa morphology using an immunologically reactive molecule (IRM) and to reagents and kits used in such methods.

Description

METHODS AND REAGENTS FOR SPERM ASSESSMENT

The present invention relates generally to methods of assessing spermatozoa morphology and to reagents and kits used in such methods.

BACKGROUND OF THE INVENTION

Investigations into male fertility usually involve microscopic examination of spermatozoa in a sample. In such an examination a laboratory technician makes an assessment as to the normality of the. spermatozoa and likelihood of infertility. A high proportion of morphologically abnormal spermatozoa is an indicator of infertility. In addition, the proportion of sperm with intact acrosomes in a given sample is related to the likelihood of success of in vitro fertilization Taken together, acrosome status and sperm morphology provide very useful prognostic information for advising patients about their chances of achieving fertilization by standard in vitro fertilization procedures and for selection of patients likely to have poor results with assisted fertilization techniques such as intracytoplasmic sperm injection (Liu and Baker, 1992). Current methods of assessment are necessarily subjective and labour intensive requiring at least two sperm preparations and a specialised epifluorescence microscope for assessing the human acrosome (WHO 1992, Liu and Baker, 1992).

There is a need therefore to develop more reliable methods of investigating spermatozoa morphology.

Clusterin is an 80 kD glycoprotein dimer made of and β subunits. It is found at several sites in the human male reproductive tract (O'Brien et al, 1993). It has been suggested that clusterin has an array of functions ranging from inhibition of autologous complement attack (Murphy et al, 1989, Jenne and Tschopp, 1989), lipid transport (de Silva et al, 1990), programmed cell death (Buttyan et aL, 1989, Lee and Sensibar, 1987), cell maturation (Harding et al, 1991, O'Bryan et al, 1993A) and aggregation of cells in vitro (Fritz et al, 1983). Combined data from human and rat experiments suggest that clusterin is a major product of Sertoli cells, epididymal principal cells and seminal vesicle principal cells (O'Bryan et al, 1993, Sylvester et al 1984, Hermo et al, 1991, Aulitzky et al, 1992).

In work leading up to the present invention, the inventors discovered that morphologically normal spermatozoa and morphologically abnormal spermatozoa substantially have distinguishable forms of clusterin. Specifically the present inventors have discovered that morphologically abnormal spermatozoa display the 80 kD dimeric clusterin form over substantially the whole of the spermatozoa whereas morphologically normal spermatozoa have a different form of clusterin, "acrosomal clusterin", substantially associated with the acrosomal cap of the spermatozoa. Although not intending to be bound by theory at present this different form of clusterin is thought to be the α subunit or a portion or variant thereof.

SUMMARY OF THE INVENTION:

The object of the present invention is to provide a more reliable method for investigating spermatozoa morphology and, where required, acrosomal status of spermatozoa.

In a first aspect the present invention provides a method of assessing spermatozoa morphology in a spermatozoa sample comprising contacting said sample with an immunologically reactive molecule (IRM) capable of binding to at least one form of clusterin under conditions and for a time sufficient to allow binding of said IRM and clusterin, and detecting binding of said IRM to said clusterin, wherein the distribution of said IRM binding to abnormal spermatozoa is different to the distribution of IRM binding to normal spermatozoa.

In a second aspect the present invention provides a method of assessing acrosomal status of spermatozoa comprising contacting a sample of spermatozoa with an IRM reactive with the acrosomal form of clusterin as described herein under conditions and for time sufficient to allow binding of said IRM and clusterin, and detecting binding of" said spermatozoa and IRM, wherein morphologically normal spermatozoa which have not undergone an acrosomal reaction will display IRM binding to the acrosomal region.

In a third aspect the present invention provides a kit for assessing spermatozoa morphology and/ or acrosomal status comprising a first part or a multiplicity of parts adapted to receive a spermatozoa sample or samples, a second part or multiplicity of parts adapted to contain an IRM labelled with a reporter as herein described and capable of binding to at least one form of clusterin, and optionally a third part or parts adapted to contain substrate, buffers and other reagents necessary for detecting binding of said IRM to spermatozoa.

BRIEF DESCRIPTION OF THE FIGURES Figμre 1: A photographic representation of human spermatozoa stained for clusterin using avidin-biotin amplified immunoperoxidase technique and the G7 and E5 mAbs. All fields are magnified 1000X except Figure 1 E (X2,000).

A washed spermatozoa pellet stained for clusterin using the G7 mAb. Morphologically abnormal spermatozoa were intensely stained for clusterin. Spermatozoon 1. Has a thick tail and abnormal midpiece.

2. Has a thick tail, and

3. Has a coiled tail. Small spherical bodies (SB) were also intensely labelled for clusterin using the G7 mAb. Occasionally a band of equatorial staining (E) was seen a morphologically normal spermatozoa using the G7 mAb.

B. A washed preparation of human spermatozoa stained for clusterin using the G7 mAb. Aggregated spermatozoa, which also generally had abnormal morphology, tended to be intensely stained for clusterin using both th G7 and E5 mAbs.

C. Immunoperoxidase staining of a washed preparation of human spermatozoa using the G7 mAb showing the intense labelling of morphologically abnormal spermatozoa (Ab) and labelling of the equatorial region (E). D. Immunoperoxidase staining of a washed pellet of human spermatozoa stained for clusterin using the E5 mAb. Similar to the staining observed with the G7 mAb, abnormal spermatozoa were preferentially stained for clusterin. The majority of spermatozoa, however, were also stained in the acrosome region (A). Spermatozoon 1. has two heads and a thick tail, 2. has two tails and

3. coiled tail morphology. The small spherical bodies (SB) were also intensely stained for clusterin using the E5 Ab. E. A washed pellet preparation of human spermatozoa stained for clusterin using the E5 mAb and the immunoperoxidase technique. In addition to acrosomal staining of all spermatozoa, spermatozoa with immature tail morphology were intensely stained. F. The distribution of E5-reactive clusterin on spermatozoa prepared using the swim-up method. The majority of spermatozoa were stained in the acrosome region. The occasional spermatozoa which lacked acrosomal clusterin staining, however, maintained a region of E5-reactive clusterin around the equatorial region (E). Any remaining abnormal spermatozoa tended to be heavily stained (Ab).

G. The distribution of G7-reactive clusterin on spermatozoa prepared by the swim-up method. Only those remaining abnormal (Ab) spermatozoa were stained for clusterin using this mAb. Using spermatozoa prepared by the swim-up technique, spermatozoa with diffuse clusterin are infrequent. H. Swim-up preparation of human spermatozoa stained for clusterin using the E5 mAb after induction of the acrosome reaction with calcium ionophore. Spermatozoa which lack acrosomal clusterin staining (N) may have spontaneously undergone the acrosome reaction. Several of the spermatozoa without acrosome staining after induction of the acrosome reaction, have an equatorial band of clusterin staining (E). Some spermatozoa which have maintained acrosome clusterin staining (A) may be acrosome intact, whereas those with either no acrosomal clusterin or with equatorial clusterin banding, may be acrosome reacted. I. Spermatozoa treated with calcium ionophore and stained with clusterin using the G7 mAb. On several of these spermatozoa a region of equatorial banding (E) is seen.

Figure 2 A graphic representation which compares pelleted spermatozoa and swim- up spermatozoa in the distribution of G7-reactive and E5-reactive clusterin. Spermatozoa were prepared using each technique from halves of the same four seminal plasma samples.

Panels A and B show the reduction, after swim-up preparation, in the number of spermatozoa with diffuse clusterin staining using both antibodies. Panel C shows that spermatozoa with acrosome E5-reactive clusterin are more likely to be selected by the swim-up method. Panel D illustrates that the swim-up method selects for spermatozoa which do no-L have surface G-7 reactive clusterin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In one aspect the present invention provides a method of assessing spermatozoa morphology in a spermatozoa sample comprising contacting said sample with an immunologically reactive molecule (IRM) capable of binding to at least one form of clusterin under conditions and for a time sufficient to allow binding of said IRM and clusterin, and detecting binding of said IRM to said clusterin wherein the distribution of said IRM binding to abnormal spermatozoa is different to the distribution of IRM binding to normal spermatozoa.

The spermatozoa sample used in the method of the invention can be any spermatozoa sample in which the clusterin forms differ between morphologically normal and morphologically abnormal spermatozoa and where this difference is detectable with an IRM. Although this inventors' discovery has been specifically observed in humans, the spermatozoa of other animals including domestic animals such as cattle, sheep, goats, alpacas, llamas and horses may also be assessable in the method of the present invention.

The term "IRM" means any molecule capable of participating in an immunological reaction and displaying differential binding ability. The IRM is preferably an antibody and advantageously a monoclonal antibody (mAb). The term "antibody" includes naturally occurring antibodies, recombinant antibodies, synthetic antibodies including fusions or chimers of antibodies, fragments of any of the foregoing such as Fab and F(ab')₂ and genetically engineered scantibodies (See Skerra A. Bacterial expression of immunoglobulin fragments Curr. Opin. Immunol. 1993; 5:

256-262 which is a review of all the types of fragment available). Where the antibody IRM is a recombinant form, the molecule may be encoded by a naturally occurring or synthetic nucleotide sequence and expressed in any convenient expression system such as those disclosed in Sambrook et al (1989) and other Genetic Engineering Laboratory Manuals. Where the molecule is synthetic, it is conveniently prepared by a stepwise addition of single amino acid groups or amino acid fragments of, for example antibodies. With regard to the latter, the synthetic antibody may be a fusion or chimeric antibody comprising light or heavy chains derived from other antibodies.

The term "clusterin" used herein includes different forms of clusterin such as the 80 kD form, the α chain, the β chain, the acrosomal form of clusterin and mutants, variants, derivatives and parts thereof. The terms mutants and variants cover clusterin forms which display amino acid, glycosylation or other changes when compared to native clusterin, but that retain substantially the same immunological character as native clusterin or a part thereof. The term "part thereof means a portion or fragment of clusterin which retains at least some of the character of native clusterin. The term derivatives means a polypeptide molecule derived from clusterin which retains at least a part of the native clusterin character.

While the IRM must be specific for at least one form of clusterin the IRM may be capable of binding more than one form of clusterin. In addition the IRM may bind two forms of clusterin but have a higher affinity for one form than the other. When the IRM binds two forms of clusterin (viz the α subunit or acrosomal clusterin and the heterodimer) it will be understood that the differential clusterin distribution on abnormal and normal spermatozoa will enable these two categories of spermatozoa to be distinguished.

Antibodies and other IRM's of the present invention may be of any animal origin including mammals such as humans, live stock animals, companion animals, wild animals and laboratory test animals (eg. mice, rats, rabbits and guinea pigs). An "animal" also extends to non-mammalian species such as birds (eg. chickens and other poultry, emus and ostriches).

The IRM is labelled with a reporter providing, under suitable conditions, a detectable signal. Such reporters may include molecules such as chemiluminescent molecules, bioluminescent molecules, radio-nucleotides, fluorescent molecules or enzymes amongst others. Commonly used enzymes include peroxidises such as horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase, amongst others. Enzymes such as peroxidises maybe used in conjunction with avidin-biotin systems. Alternatively the reporter may consist of particles including colloidal gold, polyacrylamide or latex microbeads amongst others.

Those skilled in the art will be familiar with the conditions and time sufficient or suitable for achieving binding when the spermatozoa sample is contact with the IRM. Standard immunohistochemical techniques such as those described in the Examples may be used.

The phrase "distribution of antibody binding" refers to the physical distribution over the surface or exposed areas of the spermatozoa such as at the head, midpiece and tail and includes whether the binding is intense or diffuse.

Detecting of binding of said IRM can be achieved by any convenient detection means. Preferably the detection means is one that provides a good visual signal detectable upon microscopic examination. Preferably the IRM is labelled directly by a reporter as herein described, such as a reporter enzyme, or by reporter particles such as colloidal gold or biosensers. Alternatively the presence of antigen- antibody complex may be detected by an anti-immunoglobulin labelled with a label, reporter or other detector molecule capable of providing a detectable signal. In this embodiment the anti-immunoglobulin binds to the bound clusterin reactive antibody and then the label, reporter or detector molecule is read. Suitable reporters are as hereinbefore described.

Assessment of samples may be aided by automated means such as computer assisted methods for image analysis of IRM binding patterns on spermatozoa.

Preferably the IRM reacts with both the 80 kD form and the different form of clusterin ("acrosomal clusterin") described above so that morphologically abnormal spermatozoa can be distinguished from morphologically normal spermatozoa due to the binding pattern of the IRM to the spermatozoa. Specifically, substantially, in normal acrosome-intact spermatozoa, the acrosomal region is bound by the IRM whereas in abnormal spermatozoa there is a generalised binding over the whole spermatozoa. More preferably the IRM is an antibody, still more preferably a monoclonal antibody. In one of the most preferred forms of the present invention the IRM is mAbE5 or an antibody with the same reactivity or specificity (Murphy et al, J. Clin. Invest. SI: 1858-1864).

The advantage of using mAbE5, or an IRM with functionally similar characteristics is that the method of the invention provides an opportunity to assess the proportion of normal sperm with intact acrosomes as well as assess abnormal sperm status of a sample at one time. Thus the fact that one slide can be used to assess the two characteristics leads to substantial advantages such as reduced sample preparation time and lower costs. In addition the potential for use of automated computer assisted methods of image analysis reduces the subjectivity of present assessment methods.

The spermatozoa may be prepared prior to testing by an convenient means such as by centrifugation and washing or by the swim up technique (Baker e-L aL 1993).

In a further aspect the present invention provides a method of assessing acrosomal status of spermatozoa comprising contacting a sample of spermatozoa with an IRM reactive with the acrosomal form of clusterin under conditions and for a time sufficient to allow binding of said IRM and said clusterin, and detecting binding of said spermatozoa and IRM, wherein morphologically normal spermatozoa which have not undergone an acrosomal reaction will display IRM binding to the acrosomal region.

Preferably the spermatozoa sample is fixed or otherwise treated to expose the contents of the acrosome prior to contacting it with the IRM. Fixing may be carried out by standard methods known to those skilled in the art such as by acetone immersion. It will be understood that while the IRM must be reactive with at least the acrosomal form of clusterin it may bind other forms of clusterin. In the latter case the distribution pattern of clusterin on normal and abnormal spermatozoa will enable an assessment of the acrosomal status of the spermatozoa.

Preferably the IRM is an antibody, still more preferably a monoclonal antibody. In one of the most preferred forms of the present invention the IRM is mAbE5 or an antibody with equivalent reactivity (Murphy et al, J. Clin. Invest. 81: 1858-1864).

The spermatozoa may be prepared by an convenient technique such as those described above.

In still a further aspect the present invention provides a method of assessing spermatozoa morphology comprising contacting a spermatozoa sample with an IRM under conditions and for a time sufficient to allow binding of such IRM and clusterin wherein said IRM substantially binds with one form of clusterin but does not substantially bind with another form of clusterin, and detecting binding of said IRM to said spermatozoa.

Preferably the IRM is specific for the β-chain of clusterin and therefore binds substantially to morphologically abnormal spermatozoa but not substantially to morphologically normal spermatozoa. More preferably said IRM is an antibody, still more preferably a monoclonal antibody.

Similarly the spermatozoa may be prepared by any convenient technique such as those described above.

The invention also extends to an isolated preparation of IRM when used in any one of the methods of the invention. The IRM may be produced by standard methods. For example where the IRM is a monoclonal antibody the methods of Oi & Herzenberg 1980) may be used. Where the IRM is a natural antibody the methods of O'Bryan et al 1994 may be utilized. The following methods for making IRMs - u - may be used. These may be in the form of recombinant, synthetic antibodies and scantibodies according to Skerra A and Pluckthun A. Assembly of a functional immunoglobulin FV fragment in E. coli, Science 1988, 240; 1038-1041, Skerra et al

1993 (op cit), Bird R.E., Hardman K.D., Jacobson J.W., Johnson S., Kaufman B.M., Lee S., Lee T., Pope S.H., Riordan G.S., Witlou M. Single-chain antigen binding proteins. Science 1988, 242; 425-426, Carter P et al. High level E. coli expression and production of a bivalent humanized antibody fragment, Biotechnology 1992: 10;

163-167, Anthony J et al. Production of stable anti-digoxin FV in E. coli. Mol.

Immunol. 1992, 29:1237-1247. In addition to these methods the following methods may be used:

F^: Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual, pp 628-

631, Cold Spring Harbor Laboratory Press, Cold Spring Harbour.

Bacterial expression of F ^:

Buchner, J. and Rudolph, R. (1991) Renaturation, purification and characterization of recombinant F_ab-fragments produced in Escherichia coli. Biotechnology 9, 157-

162. dAbs:

Austin, P. (1989) Will dABs challenge mABs? Nature 341, 484-485; Ward, S.E.,

Gussow, D., Griffiths, A.D., Jones, P.T. and Winter, G. (1989) Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherischia coli. Nature 341, 544-546. dAbs & fAbs:

Hudson, P., (1990) DAbs and fAbs take on mAbs. Today's Life Science 2, 38-39.

SC Davis, G.T., Bedzyk, W.D., Voss, E.W. and Jacobs, T.W. (1991) Single chain antibody (SCA) encoding genes - one-step construction and expression in eukaryotic cells. Biotechnology 9, 165-169.

Miniantibodies/E scF-,:

Huston, J.S., Levinson, D., Mudgett-Hunter, M., Tai, M-S, Novotny, J., Margolies, M. N., Ridge, RJ. Bruccoleri, R.E., Haber, E., Crea, R. and Opperman, H. (1988(

Protein engineering of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherischia coli. Proc. Natl Acad. Sci. USA 85, 5879-5883; Glockshuber, R., Malia, M., Pfitzinger, I. and Pluckthun, A (1990) A comparison of strategies to stabilize immunoglobulin F_v- fragments. Biochemistry 29, 1362-1367; Pack, P. and Pluckthun, A. (1992) Miniantibodies - Use of Amphipathic Helices to Produce Functional, Flexibly Linked Dimeric Fv Fragments with High Avidity in Escherichia-Coli. Biochemistry 31, 1579-1584. Diabodies:

Holliger, P., Propero, T. and Winter, G. (1993) Diabodies - small bivalent and bispecific antibody fragments. Proc. Natl Acad. Sci. USA 90, 6444-6448. Camel heavy chain only antibodies:

Hamerscasterman, C, Atarhouch, T., Muyldermans, S., Robinson, G., Hamers, C, Songa, E.B., Bendahman, N. and Hamers, R. (1993) Naturally occurring antibodies devoid of light chains. Nature 363, 446-448. Chimaeric antibodies: Shitara, K., Nakamura, K., Tokuaketanaka, Y., Fukushima, M. and Hanai, N. (1994) A new vector for the high level expression of chimeric antibodies in myeloma cells. J.Immuno. Meth. 167, 271-278.

Other genetically engineered derivatives of antibodies: Alting-Mees, M., Amberg, J., Ardourel, D., Elgin, E., Greener, A., Gross, E.A., Kubitz, M., Mullinax, R.L., Short, J.M. and Sorge J.A. (1990) Monoclonal antibody expression libraries: A rapid alternative to hybridomas. Strategies in Molecular Biology (Sratagene technical publication). 3, 1,2,9; Clackson, T., Hoogenboom, H.R., Griffiths, A.D. and Winter, G. (1991) Making antibody fragments using phage display libraries. Nature 352, 624-628; Engelhardt, O., Grabherr, R., Himmler, G. and Ruker, F. (1994) Two-step cloning of antibody variable domains in a phage display vector. Biotechniques 17, 44-46; Owens, R.J. and Young, R.L. (1994) The genetic engineering of monoclonal antibodies. Journal of Immunological Methods 168, 149-165.

In addition, the invention extends to an isolated preparation of an IRM specific for one form of clusterin as hereinbefore defined. Preferably the IRM is an antibody more preferably a mAb labelled with a reporter as hereinbefore described. In another aspect the present invention provides a kit for assessing spermatozoa morphology comprising a first part or multiplicity of parts adapted to receive a spermatozoa sample or samples, a second part or multiplicity of parts adapted to contain an IRM labelled with a reporter and capable of binding to at least one form of clusterin and optionally a third part or parts adapted to contain substrate, buffers and other reagents necessary for detecting binding of said IRM to spermatozoa.

The reporter associated with the IRM provides, under suitable conditions, a detectable signal. Such reporters may include molecules such as chemiluminescent molecules, bioluminescent molecules, radio-nucleotides, fluorescent molecules or enzymes amongst others. Commonly used enzymes include horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase, amongst others. Alternatively the reporter may consist of particles including colloidal gold, polyacrylamide or latex microbeads amongst others.

Detecting of binding of said IRM can be achieved by any convenient detection means. Preferably the detection means is one that provides a good visual signal detectable upon microscopic examination such as those described above.

The kit may also be used for assessing acrosomal status of spermatozoa. In such a case the IRM must be at least reactive with the acrosomal form of clusterin. Preferably the IRM is monoclonal antibody E5 or a monoclonal antibody with eαuivalent activity.

The following invention will now be described with reference to the non-limiting Examples.

EXAMPLE 1

Materials and methods

Antibodies Clusterin was localised on human spermatozoa using the anti-human clusterin monoclonal antibodies E5 and G7 (Murphy et al, 1988). Western immunoblotting studies using native (heterodimeric) or separated clusterin α and β subunits indicate that the E5 mAb is directed against an epitope on the α-chain. E5 will therefore react with free α-chains and also with native clusterin heterodimer. The affinity of the E5 mAb however, is greater for free α-chains than for native heterodimeric clusterin.

The G7 mAb is strongly reactive with native heterodimeric clusterin but, does not react with either of the separated clusterin α and β subunits. The epitope for which G7 is specific must therefore encompass a portion of both the α-chain and β-chain, or be conformationally dependent on the heterodimeric structure.

Semen samples

Thirty eight semen specimens were obtained from diagnostic or donor samples provided by the Andrology laboratory, Royal Women's Hospital, Melbourne. Prior to isolation of spermatozoa, semen samples were analysed by standard procedures (WHO, 1992). The days of sexual abstinence prior to production of sample, volume, viscosity, spermatozoa concentration, percentage motile spermatozoa, the straight line velocity (Hamilton Thorn), the percentage normal spermatozoa morphology, the presence of spermatozoa aggregates and anti-spermatozoa antibodies (i-mmunobead test: Clarke, 1990) were all recorded. From 27 semen samples, spermatozoa were separated from seminal plasma by centrifugation and washing twice in Tyrode's solution (670g/10min) (Commonwealth Serum Laboratories, Melbourne, Australia). From the remaining 11 semen samples, motile spermatozoa were preferentially isolated using the swim- up technique (Baker et al, 1993). Spermatozoa were resuspended in 0.5 ml of Tyrode's solution and the concentrations determined in a counting chamber. Spermatozoa were diluted to a concentration of approximately 1 x 10^* spermatozoa/ml in 0.1M phosphate buffered saline, pH 7.4 (PBS). To control for intersample variation, 4 additional semen samples were obtained and divided in half immediately after liquefaction. Spermatozoa were prepared from one half of these samples by centrifugation and from the other half of the samples using the swim-up technique.

Immunohistochemistry

Fifty microlitres of each spermatozoa suspension were spotted onto glass microscope slides and allowed to air dry. Slides were then fixed by immersion in acetone for 10 min, stored at room temperature and used within 48 hrs of preparation. Prior to immunohistochemistry, smears were re-hydrated by immersion in PBS for 10 min. Clusterin was localised on spermatozoa using an avidin-biotin amplified immunoperoxidase technique. Non-specific antibody binding was blocked by pre- incubating slides in 10% non-immune rabbit serum (DAKOPATTS) for 20 min in a humid environment. Slides were then incubated with either G7 or E5 mAbs (1 μ/ml), followed by sequential incubations with biotinylated anti-mouse immunoglobulins (1:^-00: Amersham, E345) and streptavidin conjugated peroxidase solution (1:400: DAKOPATTS, 397). All antibodies were diluted in PBS/1.0% bovine serum albumin (BSA) and between each antibody application, slides were washed twice in PBS for 5 min. Bound peroxidase was detected using 0.05% diaminobenzidine tetrahydrochloride (Sigma) in PBS for 5 min. The reaction was stopped by transferring the slides into fresh tap water. Concurrent negative controls were performed using an irrelevant mAb of the same subclass (I_gG,). Preparations were counterstained using Harris's haematoxylin for 30 sees, dehydrated and coverslipped.

Analysis of the distribution of clusterin on human spermatozoa From each patient (n = 38), separate slides were prepared and stained for clusterin using both the G7 and E5 mAbs. A minimum of 200 spermatozoa were surveyed for each slide and samples were scored for the percentage and pattern of clusterin distribution. Clusterin distribution was classified into one of the following observed patterns: 1. Acrosomal.

The acrosomal region was positive, but the remainder of the spermatozoan was negative. 2. Equatorial.

The equatorial segment were positive, but the remainder of the spermatozoan was negative.

3. Diffuse.

There was intense staining over most or all of the entire spermatozoan surface. 4. Unstained.

No staining of spermatozoan.

Additionally, the presence and clusterin staining characteristics of any cellular debris and spermatozoa aggregates was noted.

Induction of the spermatozoa acrosome reaction using the calcium ionophore A23187.

To determine if the acrosome reaction altered spermatozoa clusterin localisation, the acrosome reaction was induced in three human spermatozoa preparations using a calcium ionophore method (Liu and Baker 1990). Spermatozoa (prepared by the swim-up method) were suspended at a concentration of 2 x 10⁶ motile spermatozoa/ml in human tubal fluid medium (HTF) (Quinn et al, 1985) containing 10% human serum (heat inactivated 56 °C, 30 min) and sterilised by filtration using an Acrodisc filter (0.2 μm: Gelman Sciences Inc., Ann Arbor, ML, USA). Spermatozoa were incubated with 10 μM A23187 (Sigma) in HTF for 1 hr at 37 °C in 5% C0₂ in air. After incubation, spermatozoa were pelleted, the supernatant removed, and resuspended in fresh HTF medium. Motility was assessed using phase-contrast microscopy. The percentage of acrosome reacted spermatozoa was determined using FITC conjugated pisum sativum agglutinin (FITC-PSA) (Sigma)(Liu and Baker, 1988) and slides were also prepared and stained for clusterin using the immunoperoxidase method outline above. As a negative control, two additional swim-up spermatozoa preparations, were treated in exactly the same manner with the exception that they were incubated in HTF without A23187. These control spermatozoa were also assessed for the percentage of acrosome reacted forms using the FITC-PSA, and the distribution of clusterin determined using the immunoperoxidase technique. It will be understood that in normal use of the present invention the acrosome reaction will not be artifically induced prior to treating the spermatozoa with the IRM.

Statistical analysis

The significance of the correlations by semen analysis and the immunohistological results was tested by non parametric Spearman tests.

Results

Clusterin localisation on washed spermatozoa

Table 1. lists the parameters of semen analysis and the clusterin staining characteristics of the 38 spermatozoa preparations labelled with both the E5 and G7 mAbs. Of the 38 semen samples, 17 were classified as normal using the WHO criteria (WHO 1992). None of the patients had anti-spermatozoa antibodies. Leucocytes, immature germ cells and small spherical bodies were found in variable numbers in many of the samples.

Abnormal spermatozoa were preferentially stained for clusterin using the G7 mAb (Table 2). G7 staining was usually intense, diffuse and was particularly noticeable on spermatozoa with residual cytoplasmic droplets, thick tails or abnormal head morphology (Fig. 1A and C). These data are similar to those previously observed with this antibody by immunofluorescence (O'Bryan et al, 1990). All spermatozoa aggregates were also intensely stained positive with G7 (Fig. IB). The aggregates were not however, caused by the G7 mAb itself, as spermatozoa from the same sample had similar rates of aggregate formation when incubated wither with G7, no mAb or with a negative control mAb. In -^*ome semen samples, a small percentage of spermatozoa ( < 5%) showed localised staining in the equatorial region (Fig. 1C).

Significantly different results were found using the E5 mAb (Table 2). Similar to the staining seen with the G7 mAb, abnormal spermatozoa and aggregated spermatozoa were intensely and diffusely stained for clusterin. In addition, however, the majority of morphologically normal spermatozoa were also stained in the acrosome region (Fig. ID, E, F). This acrosomal staining was less intense than the diffuse staining seen on abnormal spermatozoa (Fig ID). On a minority of apparently morphologically normal spermatozoa (15%, Table 2.), no E5-acrosome staining was seen; some of these spermatozoa had a region of E5-reactive clusterin in the equatorial region (Fig IF).

Using both the G7 and E5 mAbs, there was also staining, in the washed spermatozoa preparations, of small non nucleated, spherical structures (Fig. 1A & ID) and some immature germ cells. Non-specific (endogenous peroxidase) staining of leucocytes was also observed.

Immunoperoxidase staining of swim-up spermatozoa

In the 11 spermatozoa samples prepared by the swim-up technique, the differences between G7 and E5 mAb reactivity became more obvious as the percentage of normal, motile spermatozoa was increased by the swim-up technique. Swim-up spermatozoa, compared to pelleted and washed spermatozoa, contained fewer of the morphologically abnormal spermatozoa which were intensely reactive with both mAbs. Conversely, the higher proportion of morphologically normal spermatozoa was associated with E5 acrosome staining. Table 3. compares the clusterin distributions between swim-up and washed pellet preparations of spermatozoa.

The data shown in Figure 2. provide a direct comparison between centrifuged spermatozoa preparations and swim-up spermatozoa preparations derived from parts of the same semen sample. These data should therefore control for intersample variation and again they demonstrate that the swim-up method is associated with a loss of the diffuse stained abnormal spermatozoa and an increase in the E5 acrosomal stained, morphologically normal, spermatozoa.

Correlation analysis revealed that the proportion of spermatozoa, prepared with by wash or swim-up, with E5 equatorial staining was inversely related to the percentage abnormal morphology (Spearman rho = 0.418 p = 0.011). E5 diffuse staining was positively related with the percentage abnormal morphology (rho = 0.343, p = 0.37) and acrosome staining was inversely related to percentage abnormal morphology (rho = - 0.354, p = 0.031). G7 diffuse staining was also positively related to percentage abnormal morphology (rho = 0.326, p = 0.047). There was also an inverse relationship between the proportion of spermatozoa with no E5 staining and duration of abstinence before the sample was collected (rho = - 0.493, p = 0.003).

Clusterin staining of acrosome reacted spermatozoa After induction of the acrosome reaction using the calcium ionophore A23187, the acrosomal staining with the E5 mAb, disappeared from more than half of the morphologically normal spermatozoa leaving either no staining, or a narrow band of E5 staining around the equatorial regions (Table 4.) (Fig. 1H). The percentage of acrosome reacted spermatozoa in A23187 treated and control samples (as judged by PSA-immunofluorescence) was closely related to sum of the percentage of spermatozoa with either no E5 staining or only equatorial staining (Table 4.). This association suggests that induction of the acrosome reaction resulted in the release of E5-reactive acrosomal clusterin. E5-reactive acrosomal clusterin is likely to be an internal acrosomal component as no acrosomal region staining was seen in spermatozoa that were unfixed. The acetone fixation used in this study appears to allow access of the E5 monoclonal antibody to the acrosome contents.

An equatorial band of clusterin staining was also occasionally detected using the G7 mAb on acrosome reacted spermatozoa (Fig. II).

EXAMPLE 2

Material and Methods weie as in Example 1 with the exception that the E5 antibody only was used for immunohistology and only washed pellet sperm preparations were used.

A further 25 sperm preparations were made from washed pellets of semen samples obtained from 25 additional donors or patients (Andrology laboratory, the Royal Women's Hospital).

Preliminary analysis confirms the data described in Example 1; the proportion of morphologically normal sperm correlated with the presence of E5 acrosomal staining and the proportion of morphologically abnormal sperm with the presence of diffuse surface staining for clusterin.

EXAMPLE 3

Production of an antibody specific for the β-chain of clusterin.

An antibody against the clusterin β chain is produced to enhance the ability immunohistologically to detect abnormal sperm. The anti- β chain reacts with the surface heterodimeric clusterin on abnormal sperm but not the apparently α chain restricted form of clusterin in the acrosome of normal sperm.

Antibody production:

Heterodimeric clusterin is cleaved and the chains separated (Murphy 1988) and sheep immunized 3-4 times with β chain. Antiserum is harvested and tested for reactivity with β chain and any cross reactivity with α chain is absorbed using solid phase α chain. Alternatively mice are immunized with purified β chain and a series of murine monoclonal antibodies are produced against human clusterin β chain (Oi and Herzenberg 1980).

EXAMPLE 4

Production of an antibody specific for the α-chain of clusterin.

The same methods are used as in Example 3 except an isolated α-chain of clusterin is employed to -produce a α-chain specific antibody.

EXAMPLE 5 Spermatozoa morphology test kit

The kit contains a first part of parts in the form of a support for microscopic examination such as a microscope slide or slides for the spermatozoa sample. A second part or parts contains the IRM (preferably E5 or an equivalent antibody) coupled to a reporter (such as horseradish peroxidase) and is made available in a final working solution of buffer containing preservative and irrelevant protein for stablization (for example, Tris buffered saline containing 0.05% Na azide and 2% serum albumin). The kit also contains a substrate solution provided as a buffer to which supplied tablets are added prior to use. Wash buffer may be provided as powder sachets to be dissolved in water prior to use. Additional information in the kit includes detailed instructions, photographic examples, cover slips and mounting material.

Table 1

Semen characteristic mean range

Days abstinence 4.5 2 - 10

Volume (ml) 4.0 2.2 - 7.7

Sperm concentration (10⁶/ml) 112 0.2 - 402

% motility 51.5 2.0 - 89

% abnormal morphology 80 48 - 99

Table 1: Semi.. . analysis parameters of the 38 semen samples used in to prepare sperm for analysis of clusterin distribution.

Table 2

Pattern of antibody Mean percentage labelling of 200 sperm in the 38 samples reaction with sperm

Monoclonal antibody G7 Monoclonal antibody E5 (clusterin heterodimer) (clusterin α chain)

Not labelled 52% 15%

Most or all of 32% 24% sperm diffusely labelled

Acrosome 7% 62% labelling only

Equatorial labelling 2% 3% only

Table 2: The characteristics and range of sperm clusterin distributions in a group of 38 patients. These data include washed pelllet sperm preparations (n=27) and swim-up sperm preparations (n = 11).

Table 3. Anti-clυsterin monoclonal antibody reactivity - conipaiison between the swim-up speπn preparations and washed pellet sperm preparations

Pattern of antibody M ean percenta ge la belling of 200 spe rm pe r sa mple reaciion with sperm Monoclonal antibody G7 Monoclonal antibody E5

washe d swim-up washed swim-up

(n=27) (n=11) (n=27) (n=11)

Not labelled 56% 65% 17% 10%

Most or all of 35% 24% 25% 21% sperm diffusely labelled t

Acrosome 9% 1% 59% 68% labelling only

Equatorial labelling 1% 0.5% 0.5% 1% only

Table 4. E5 reactivity with acrosome reacted compared to control sperm

Ionophore treated samples % acrosome reacted (PSA) % with no E5 labelling % w^'rth equatorial Total with either no E5

E5 labelling labelling or with equatorial labelling

1. 50 35 16 51

2. , 59 41 9 50

3. 75 48 23 71

Control samples not exposed to ionophore

4. 7 1 7 8

5. 10 7 0 7

Table 4. Reactivity with the E5 (clusterin α chain) monoclonal antibody with swim-up sperm samples which had been treated with calcium ionophore to induce the acrosome reaction (n=3) compared to swim-up sperm not exposed to ionophore (n=2). Th acrosomal status of each sperm preparation was determined by PSΛ immunofluorescence. There is good concordance with th percentage of sperm that are acrosome reacted and the loss of E5 reactivity with the acrosome region. Equatorial staining is significantly increased following the acrosome reaction and the total percentage sperm with no acrosomal reactivity plus thos with equatorial reactivity is similar to the total percentage sperm which are acrosome reacted according to PSA labelling.

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Claims

CLAIMS:

1. A method of assessing spermatozoa morphology in a spermatozoa sample comprising contacting said sample with an immunologically reactive molecule (IRM) capable of binding to at least one form of clusterin under conditions and for a time sufficient to allow binding of said IRM and clusterin, and detecting binding of said IRM to said clusterin wherein the distribution of said IRM binding to abnormal spermatozoa is different to the distribution of IRM binding to normal spermatozoa.

2. A method of assessing acrosomal status of spermatozoa comprising contacting a sample of spermatozoa with an IRM reactive with the acrosomal form of clusterin as described herein under conditions and for a time sufficient to allow binding of said IRM and clusterin and detecting binding of said spermatozoa and IRM, wherein morphologically normal spermatozoa which have not undergone an acrosomal reaction will display IRM binding to the acrosomal region.

3. The method of claim 1 or claim 2 wherein the IRM is specific for more than one form of clusterin.

4. The method of any one of claims 1 to 3 wherein said IRM binds native clusterin and binds the a subunit of clusterin or acrosomal clusterin.

5. The method of claim 4 wherein said IRM has a higher affinity for the α subunit of clusterin or acrosomal clusterin than native clusterin.

6. The method of any one of claims 1 to 5 wherein said IRM is a natural or synthetic antibody.

7. The method of claim 6 wherein said antibody is a monoclonal antibody.

8. The method of claim 7 wherein said IRM is monoclonal antibody E5 or an antibody with equivalent specificity.

9. The method of any one of claims 1 to 8 wherein said spermatozoa sample is human spermatozoa.

10. The method of any one of claims 1 to 9 wherein said IRM is directly or indirectiy labelled with a reporter as described herein.

11. The method of any one of claims 1 to 10 wherein binding provides a visual signal and the detection of binding comprises microscopic examination.

12. A method of assessing spermatozoa morphology comprising contacting a spermatozoa sample with an IRM wherein said IRM substantially binds one form of clusterin but does not substantially bind another form of clusterin and detecting binding of said IRM to said spermatozoa.

13. A kit for assessing spermatozoa morphology comprising a first part or a multiplicity of parts adapted to receive a spermatozoa sample or samples, a second part or multiplicity of parts adapted to contain an IRM labelled with a reporter as herein described and capable of binding to at least one form of clusterin, and optionally a third part or parts adapted to contain substrate, buffers and od er reagents necessary for detecting binding of said IRM to spermatozoa.

14. The kit of claim 13 wherein said IRM is specific for more than form of clusterin.

15. The kit of claim 14 wherein said IRM is a natural or synthetic antibody.

16. The kit of claim 15 wherein said antibody is a monoclonal antibody.

17. The kit of claim 16 wherein said monoclonal antibody binds native clusterin and binds to the α subunit of clusterin or acrosomal clusterin.

18. A kit of claim 17 wherein said monoclonal antibody has a higher affinity for the subunit of clusterin or acrosomal clusterin than native clusterin.

19. The kit of claim 18 wherein said monoclonal antibody is E5 or an antibody with equivalent specificity.