EP0877757A1 - Method for evaluating and affecting male fertility - Google Patents

Abstract

A 22 kD sperm protein, SP-22, correlates with fertility and predicts fertility of males. The protein can be assayed to detect decreases in fertility resulting from exposure to toxicants and polluants which are known or suspected to decrease fertility. If an antibody is generated to this protein, the antibody recognition by sperm in an epididymal sperm sample or ejaculate would reflect the fertility of the sample. This antibody can be used as a contraceptive to inactivate sperm, screen for toxicity, select animals for artificial insemination, and select men for assisted reproductive technologies. The protein itself can be inactivated by gene knockout, which is another approach to contraception, or the protein can be added to sperm from infertile men to make fertility techniques more feasible.

Description

METHOD FOR EVALUATING AND AFFECTING MALE FERTILITY

FIELD OF THE INVENTION The present invention relates to a sperm protein which can be used for evaluating and/or inhibiting male fertility, as well as antibodies to the sperm protein. The present application is a continuation in part of application Serial No. 08/593,677, filed January 29, 1996, the entire contents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION Sperm production in the testis of human males is far less efficient than sperm production in other mammals, such as rat, rabbit and monkey (Amann, 1970) due to an increased rate of germ cell atresia. Together with this is the fact that a high incidence of sperm in the ejaculate of a fertile man is morphologically abnormal (Wyrobek et al . , 1982) . Thus, there is a heightened awareness of the possibility that the quantity and quality of sperm in the ejaculates of men are declining because of environmental influences (Sharpe, 1993) . A toxicant-induced alteration in the process of sperm maturation during sperm transit through the epididymis, the organ in which sperm acquire fertilizing ability, could render a man infertile. It has been hypothesized that specific proteins are added to sperm in the epididymis which confer fertility. Recently, Klinefelter et al., in Journal of mAndrology 25(4) , 318-327 (1994) demonstrated that an 18 kD epididymal sperm surface protein, presumably a plasma membrane protein, was well correlated with fertility, although it was not believed that this protein was predictive of fertility. There continues to be great interest in developing new and improved contraceptives. New contraceptives should be superior to existing products, e.g., oral contraceptives used by millions of women over the last 30 years are not only safe and effective, but even protect women against some cancers. However, other methods of contraception are still needed by many segments of the world's population, as many women do not have reliable access to oral contraceptives, or

RECTIFIED SHEET (RULE 91) ISA/EP may suffer adverse reactions to the hormones used in oral contraceptives.

Additionally, fertility testing is becoming more widespread as increasing numbers of apparently infertile couples seek medical assistance in conception. Because reproductive abnormalities of both sexes may affect fertility, assessing male fertility is common in fertility evaluations. While the most common starting point for evaluation of male fertility is an assessment of the sperm count in semen, also important to fertility is sperm motility. Therefore, in male fertility analyses, sperm motility has also been a factor.

Currently available techniques for measuring sperm count and sperm motility are microscopic in nature. A quantitative evaluation of sperm morphology and motility requires substantial experience on the part of the laboratory technician. The high level of experience required by laboratory technicians precludes general office evaluation of semen samples and generally requires referral to a specialized laboratory. Even with adequate resources, debris in semen samples can cause erroneous or inconsistent results.

Attempts to develop biochemical assays of semen have not resulted in simple procedures which may be performed in either the physician's office or a dedicated semen evaluation lab. Most biochemical markers have failed to demonstrate correlations with sperm number, motility, or fertility. Activity of fu arase, an enzyme present in semen, has been found to correlate to both sperm count and percentage motility, Crabbe, J. Reprod. Fert . 51 : 73-76 (1977) . Crabbe measured fumarase activity by spectrophotometric measurements. Unfortunately, spectrophotometric assays are not generally suitable for office assays because of the cost of these specialized devices as well as the training required for accurate and reproducible operation.

Dorian, in U.S. Patent No. 5,434,057, expanded on Crabbe's method by providing devices for assessing sperm ^"~ number and motility in semen samples comprising a solid support having a carrier matrix containing a fumarase substrate and malate dehydrogenase. The sample is applied to the carrier matrix and a visual signal is detected from the solid support resulting from metabolism of the fumarase substrate by fumarase in the sample. While this assay detects motile sperm in a semen sample, there is no method for inhibiting fertility nor of selecting out the most fertile sperm in a sample.

Feuchter et al . , in U.S. Patent No. 5,250,417, disclose a method for detecting the ability of sperm to undergo the acrosome reaction to permit determination of the fertility of male mammals. The acrosome reaction is a process by which sperm release hydrolytic enzymes that degrade the zona pellucida, which must be penetrated to enable the sperm and ovum to come into contact, fuse, and complete the fertilization process.

In recent years, other studies have targeted different proteins associated with sperm in an attempt to provide new contraceptive alternatives. Major research efforts involve immunological approaches to fertility control. The development of contraceptive vaccines is directed towards the immunoneutralization of reproductive processes or interfering with fertilization by inducing antibodies against oocytes and spermatozoa. Several sperm antigens shown to have high immunocontraceptive potential are human sperm membrane antigen (SP-10) and guinea pig sperm membrane protein (PH-20) . SP-10 is a sperm membrane specific antigen of 24-34 kD which was isolated using a monoclonal antibody

(MHS-10) that cross-reacts with the entire acrosomal region. It is associated with the outer aspect of the inner acrosomal membrane and the inner aspect of the outer acrosomal membrane of mature human sperm. It has been reproduced recombinantly in an Escherichia coli expression system.

PH-20, a guinea pig sperm protein of 64 kD, is present on both the plasma membrane and inner acrosomal membrane of sperm. It is essential for adhesion of sperm to the zona ^~ pellucida, the initial step in the fertilization process. Active immunization with PH-20 causes infertility in both male and female guinea pigs for a period ranging from six to fifteen months.

0' Rand et al., in U.S. Patent No. 5,175,148, disclose a sperm antigen corresponding to a sperm autoantigenic epitope which can be used as an immunocontraceptive agent aε well as for diagnosing autoimmune infertility. The synthetic peptide corresponds to an autoantigenic epitope of rabbit sperm membrane autoantigen.

Several other antigens with good immunocontraceptive potential have been identified and investigated in laboratory animals, including lactic dehydrogenase-x, an isoenzyme of lactic dehydrogenase confined to male germ cells. A synthetic peptide based upon a portion of this antigen has been shown to reduce fertility in laboratory animals. Unfortunately, most studies have found that the rate and duration of the immunocontraceptive effects are less than acceptable. A problem in immunological approaches to antifertility research is the need for a safe, effective adjuvant and suitable animal models for evaluating the efficacy and safety of methods.

Although most contraceptive research has been directed to use in females, there is an interest in male fertility control both from a scientific as well as a biological viewpoint. Many compounds have been identified as having male antifertility activity in various species, e.g., gossypol, 5-thio- D-glucose, and 6-chlorodeoxyglucose . Studies have also been conducted on the use of androgens to control male fertility. Unfortunately, most compounds identified as useful in controlling male fertility appear either to have irreversible antifertility effects, to be inherently toxic, or to affect libido. It has been demonstrated that sperm count could be depressed in men injected with large doses of androgens. However, there are still questions about the potential utility of androgens as male antifertility agents. The ideal male contraceptive would produce azoospermia without compromising libido or ^— sexual potency, and would be reversible.

SUMMARY OF THE INVENTION It is an object of the present invention to overcome the aforesaid deficiencies in the prior art, i.e., contraception.

It is another object of the present invention to provide a means for predicting male fertility in animals as well as in human males . It is another object of the present invention to screen for environmental endocrine disruptors, as endocrine disruption can lead to male-mediated infertility.

It is yet another object of the present invention to screen animals and humans exposed to known or suspected endocrine disruptors for fertility.

It is still another object of the present invention to select sires for artificial insemination who are good candidates for insemination.

It is a further object of the present invention to screen human semen for fertility prior to undertaking assisted reproductive technology techniques to improve the success of these techniques.

It is a further object of the present invention to improve fertility in males who fail to express a sufficient amount of SP-22, formerly known as SP-16, on sperm.

It is another object of the present invention to provide a reversible male contraceptive.

It has now been found that a 22 kD sperm protein, SP-22, formerly identified as SP-16, is very significantly correlated with fertility (p<0.0001; N=52) and predictive of fertility. If an antibody is generated to this protein, the degree of antibody recognition by sperm in an epididymal sperm sample or ejaculate would reflect the fertility of the sample. This antibody can be used as a contraceptive to block the expression of SP-22 to render sperm infertile, screen for toxicity, select superior sires for artificial insemination, and select men for assisted reproductive technologies. The protein itself can be used as a ^~ contraceptive vaccine, inactivated by gene knockout, which are other approaches to contraception, or the protein can be added to sperm from infertile men to make fertility techniques more feasible.

The protein SP-22, based upon initial electrophoretic runs, was originally thought to be a 16-kD protein, with a pi of 5.5, and was so identified in parent application Serial No. 08/593,677. However, after comparing several types of molecular weight standards, the molecular weight of this protein was found consistently to have an apparent molecular weight of 22 kD in 11% acrylamide gels. For purposes of the present invention, the protein will be identified as SP-22, although in the parent application USSN 08/593,677, the protein was identified as SP-16.

SP-22 has a partial amino acid sequence of Thr-Ser-Gly- Pro-Leu-Ala-Lys (SEQ ID NO:l) .

Thus, the present invention provides a convenient, reliable test for evaluating fertility, based upon changes to SP-22, a specific protein predictive of male fertility, as well as methods of calibrating changes in SP-22 in screening assays so as better to predict the likelihood of the sperm being fertile. Additionally, the present invention provides a method for blocking the expression of SP-22 so as to render the sperm infertile. The present invention provides a reliable test for evaluating fertility following exposure to reproductive toxicants, in candidate sires for artificial insemination, and for men considering assisted reproductive technology. Moreover, as indicated above, the invention offers a means to render men infertile through immunocontraceptive technology.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the results of a regression analysis correlating the amount of SP-22 to fertility. Figure 2 illustrates using an antibody to SP-22 to distinguish fertile sperm from infertile sperm.

Figure 3A and 3B illustrate immunolocalization of SP- 22 in a detergent extract of sperm using an antibody to SP 22.

Figure 4 illustrates immunolocalization of SP-22 on sperm using an antibody to SP-22.

DETAILED DESCRIPTION OF THE INVENTION

Purification of SP-22

SP-22 was purified by reverse-phase HPLC, 2D gel electrophoresis, and subsequent electroelution. Briefly, 50- 75 rats were killed and a detergent extract was prepared from the sperm recovered from cauda epididymal sperm. Next, 2 mg of concentrated, desalted extract was subjected to reversed-phase high pressure liquid chromatography (HPLC) . Aliquots of fractions were analyzed by mini-2D SDS gel electrophoresis, and the remaining volume of 5 fractions containing SP-22 were pooled and the protein (approximately 100 micrograms) was resolved by mini-2-D SDS gel electrophoresis. The Coomassie-stained SP-22 then was punched from the gels, electroeluted and either used for amino acid sequencing or mixed with Freund's adjuvant for polyclonal antibody generation. For amino acid sequencing, SP-22 as digested with TRCK- trypsin in 4 M urea, pH 8.0, and peptides were generated with a Microbore C8 HPLC column with 0.1% TFA/acetonitrile. Sequence analysis then was performed on an ABI 473 A sequenator. Since a characteristic fragment of SP-22 is known, i.e., Thr-Ser-Gly-Pro-Leu-Ala-Lys (SEQ ID N0:1) , it is possible to prepare functional derivatives of SP-22 as well. By "functional derivative" is meant a fragment,variant, analog, or chemical derivative of SP-22, which terms are defined below. A "functional derivative" retains at least a portion of the amino acid sequence of SP-22, which permits its utility in accordance with the present invention, namely, determining or affecting male fertility. A "fragment" of SP-22 refers to any subset of the SP-22 molecule, that is, a shorter peptide. Fragments of interest are those which can be used to determine or affect male fertility. A "variant" of SP-22 refers to a molecule which is ^~ substantially similar to either the entire SP-22 protein or a fragment thereof. Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide, using methods well known in the art.

Alternatively, amino acid sequence variants of SP-22 can be prepared by mutations in the DNAs which encode the synthesized SP-22. Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity. Obviously, the mutations that will be made in the DNA encoding the variant peptide must not alter the reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (cf . European Patent Publication No. EP 75,444) .

At the genetic level, these variants ordinarily are prepared by site-directed mutagenesis, as exemplified by

Adel an et al . , DNA 2 :183, 1983, of nucleotides in the DΝA encoding the peptide molecule, thereby producing DΝA encoding the variant, and thereafter expressing the DΝA in recombinant cell culture. The variants typically exhibit the same qualitative biological activity as the nonvariant peptide.

An "analog" of SP-22 refers to a molecule which is substantially similar to either the entire molecule or a fragment thereof . The analog may be prepared by chemical synthesis.

A "chemical derivative" of SP-22 contains additional chemical moieties not normally part of the SP-22 amino acid sequence. Covalent modifications of the amino acid sequence are included within the scope of this invention. Such modifications may be introduced into the SP-22 by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Amino terminal residues can be reacted with succinic or other carboxyric acid anhydrides. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4- pentanedione; and transaminase-catalyzed reaction with glyoxylate .

Specific modifications of tyrosyl residues per se have been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl side groups such as aspartyl or glutamyl are selectively modified by reaction with carbodiimides (R'N-C-N-R*) such as l-cyclohexyl-3- [2-morpholinyl- (4-ethyl)] carbodiimide or 1- ethyl-3- (4-azonia-4, 4 dimethylpentyl) carbodiimide . Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions .

As used herein, the term "muteins" or "variants" refers to analogs of SP-22 in which one or more of the amino acid residues of the natural SP-22, preferably 1-10, and more preferably 1-5, residues, or even only a single residue, are replace by different amino acid residues or are deleted, or one or more amino acid residues, such as 1-10, 1-5, or only one residue are added to the natural sequence of SP-22. These muteins are prepared by known synthesis techniques and/or site-directed mutagenesis techniques, or by any other known technique suitable therefor. The substitutions are preferably conservative, see, e.g., Schulz, G.E. et al . , Principles of Protein Structure, Springer-Verlag, New York, 1978; and Creighton, T.E., Proteins : Structure and Mol ecular Properties, W. H, . Freeman & Co., San Francisco, 1983; both of which are hereby incorporated by reference in their entireties. The types of such substitutions which may be made in ^~ the protein or peptide molecules of the present invention may be based on analysis of the frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schulz et al . , op . ci t . , and Figure 3-9 of Creighton, op . ci t . Based upon such analysis conservative substitutions may be defined herein as exchanges within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues:

Ala, Ser, Thr, Pro, Gly

II. Polar, negatively charged residues and their amides :

Asp, Asn, Glu, Gin III. Polar, positively charged residues: His, Arg, Lys

IV. Large, aliphatic nonpolar residues: Met, Leu, Ile, Val, Cys

V. Large aromatic residues: Phe, Try, Trp

Within the foregoing groups the following 5 substitutions are considered "highly conservative":

Asp/Glu

His/Arg/Lys Phe/Tyr/Trp

Met/Leu/Ile/Val

Semi-conservative substitutions are defined to be exchanges between two of groups (I)-(V) above which are limited to supergroup (A) , comprising (I) , (II) and (III) above, or to supergroup (B) , comprising (IV) and (V) above. Substitutions are not limited to the genetically encoded, or even the naturally occurring amino acids. When the epitope is prepared by peptide synthesis, the desired amino acid may^¬ be used directly. Alternatively, a genetically encoded amino acid may be modified by reacting it with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Screening with SP-22 Antibody It is apparent from the above description of SP-22 ^— antibodies that a wide variety of diagnostic tests is possible using the antibodies of the invention. In attempting to diagnose causes of infertility, an immunoassay to detect decreased levels of SP-22 on sperm is a useful adjunct to known hormone assays. Further uses for the antibodies include testing livestock for candidates for artificial insemination: the higher the levels of SP-22 in the potential donor, the more likely artificial insemination is to be successful. Isolation of SP-22 allows production of antisera containing antibody to SP-22 for possible crossreaction with other species, including human SP-22. This antibody enables preparation of an enzyme-linked immunosorbent assay (ELISA) . For example, to evaluate antibody binding, polystyrene microwells are precoated with extract of a particular epididymal sperm (rat) or ejaculate (horse, bull, human) sample containing an unknown amount of SP-22. Next, SP-22 antibody is added, followed by the addition of avidin- biotin-peroxidase complex. A precipitate is formed when a substrate such as DAB is oxidized by peroxidase in the presence of hydrogen peroxide. A standard curve for SP-22 is generated using increasing known amounts of SP-22. The amount of SP-22 in a sample is then determined by the optical density of the colored precipitate in the sample and the linear regression obtained from the set of SP-22 standards .

Aside from ELISA, the amount of SP-22 present on the surface of sperm in a sample (epididymal or ejaculate, animal or human) can be determined using quantitative fluorescence spectroscopy or fluorescent light microscopy. For this, sperm will be incubated with SP-22 antibody and then with labelled Rhodamine or FITC-conjugated second antibody. It is first necessary to determine the relationship between fluorescence of a sample in a fluorometer or a microscopic image, and the optical density of SP-22 separated by 2D gel electrophoresis. Once this is established, fluorescence can be related to fertility. It is also important to determine the relationship between the number of sperm in a sample which express SP-22 (i.e., fluoresce) , the degree of the expression or fluorescence, and fertility. This is particularly true for men considering assisted reproductive technologies. For example, if only a critical number (x) of sperm is needed to express a threshold amount (y) of SP-22 for a successful attempt at fertility, it is possible to selectively remove those sperm not expressing SP-22 in the ejaculate and use only those sperm that do express a sufficient amount of

SP-22, for assisted reproductive technologies such as intra uterine transfer or IVF following dissociation of SP-22 expressing sperm from the SP-22 antibody.

To determine if there is a relationship between the number of sperm expressing SP-22 and the extent to which they express it, sperm binding SP-22 antibody are evaluated by quantitative indirect fluorescence microscopy. For this, Rhodamine or FITC immunolabeling is performed on an aliquot of sperm equivalent to that used for in utero insemination, and the number of sperm that fluoresce is determined along with the relative degree of fluorescence of individual sperm in a sample. The resulting fluorescence histograms are related to fertility assessed by artificial (in utero) insemination. To determine whether a critical number of SP-22 expressing sperm are requisite to fertility, an aliquot equivalent to that used for in utero insemination is subjected to immunoabsorption. Polystyrene microwells are precoated with SP-22 antibody and sperm in the ejaculate are allowed to bind. Those sperm not binding are washed away, antibody-bound sperm are recovered following dissociation of the antibody with incubation in 0.1 M lithium diiodosalicylate, and increasing numbers of these SP-22 expressing sperm are inseminated in utero.

Conservative amino acid substitutions according to the present invention, e.g., as presented above, are known in the art and would be expected to maintain the biological and structural properties of the polypeptide after such amino acid substitutions. Most deletions, insertions and substitutions according to the present invention are those which do not produce radical changes in the characteristics of the protein or peptide molecules. One skilled in the art will appreciate that the effect of substitutions can be evaluated by routine screening assays, either immunoassays or bioassays. For example, a mutant typically is made by site-specific mutagenesis of the peptide molecule-encoding nucleic acid, expression of the mutant nucleic acid in recombinant cell culture, and, optionally, purification from the cell culture, or a biological sample containing SP-22, for example, by immunoaffinity chromatography using a specific antibody on a column to absorb the mutant by binding to at least one epitope. Antibodies to SP-22

Antibodies to SP-22 can be prepared by any conventional mans, e.g., by coincubating purified SP-22 with hybrid mouse cells in vi tro to produce monoclonal antibodies, or by generating polyclonal antibodies in mice or rabbits in vivo . The antibody produced by the hybrid cultures is collected by chromatography for subsequent use. Preparation of Antibodies to SP-22

Antibodies (both polyclonal and monoclonal) to SP-22 may be prepared for diagnostic and therapeutic uses including but not limited to fertility control (contraception) and fertility assessment (screening) . "Antibody" in this context refers to a synthetic protein which binds SP-22 and negates its biological function. Antibodies to SP-22 are prepared by either polyclonal or monoclonal techniques:

A. Polyclonal Antibody Production For polyclonal antibody product, adult mice or rabbits are immunized with 25 or 100 mg of SP-22 suspended in Freund's complete adjuvant. This preparation is injected subcutaneously and is followed by booster injections of SP-22 mixed with incomplete adjuvant. Sera obtained after the final booster injection are checked for titer, affinity, and specificity.

Specifically, for rabbits, 100 micrograms of SP-22 protein as obtained above, or peptides which are analogs of SP-22, are solubilized in 0.5 mL physiological saline and emulsified with an equal volume of Freund's adjuvant to prepare inoculum sites in the back. Two New Zealand White female rabbits, weight 2.5-3.5 kg, are bled via the marginal ear vein for pre-immune serum. Approximately 50 microliters of inoculum is injected into 20 sites within the shaved area. The rabbits are boosted in similar fashion four weeks later. Two weeks later, the rabbits are boosted a final time. Two weeks later the rabbits are bled again via the marginal ear vein, and sera containing the polyclonal antibodies is obtained.

Figure 3A shows, in the upper photograph, a silver- stained, two-dimensional gel profile of detergent-extracted cauda epididymal rat sperm. SP-22 is indicated by a circle. The acidic lower molecular weight protein appears to be a product of SP-22 degradation.

Figure 3B is an immunoblot of a sample that is identical to the one used in Figure 3A using mouse anti-rat SP-22 antiserum. Both authentic SP-22 and its degradation products are localized with antiserum.

To obtain the results shown in Figures 3A and 3B, antiserum to SP-22 was produced as follows. A detergent extract of cauda epididymal sperm was chromatographed by reversed-phase HPLC and fractions enriched in SP-22 were run an analytical 2D gels. Coomassie-stained SP-22 punches were subsequently subjected to electroelution and the electroeluted material was desalted, concentrated, and assayed for protein. After verifying that the concentrated electroeluted material was SP-22, 25 micrograms was mixed with Freund's complete adjuvant and injected subcutaneously into each of six mice. Four other mice received only adjuvant .

After four weeks, each mouse was boosted with 12.5 micrograms of SP-22 mixed with Freund's incomplete adjuvant. After another ten days, a final similar boost was given.

The mice were euthanized ten days after the final boost, and the serum was collected.

B. Monoclonal Antibody Production Monoclonal BALB/c mice are immunized with SP-22 protein^" or substantially similarly active fragments or analogs thereof by intraperitoneal injection with 50 micrograms of immunogen. Thereafter, the spleens are collected and cell suspensions are prepared by perfusion with DMEM. The BALB/c spleen cells are fused with SP 2/0-Ag 14 mouse myeloma cells by PEG and the resultant hybridomas grown in HAT selected tissue culture media plus 20% fetal calf serum. The surviving cells are allowed to grow to confluence. The spent culture medium is checked for antibody titer, specificity, and affinity.

Specifically, the mice are immunized with SP-22 adjuvant emulsion described above. Each mouse first receives 0.2 mL of this emulsion intraperitoneally, and then is reinjected in similar fashion with 0.1 mL six weeks later.

Mouse serum is obtained ten days after the second injection and then tested for anti-FRP activity via ELISA. The mouse exhibiting the highest absolute anti- FRP activity is chosen for cell fusion. Spleen cell suspension containing B-lymphocytes and macrophages is prepared by perfusion of the spleen. The cell suspension is washed and collected by centrifugation. Myeloma cells are also washed in this manner. Live cells are counted and the cells placed into a 37"C water bath. One L of 50% polyethylene glycol in DMEM is added slowly.

The cells are incubated in the PEG for one to 1.5 minutes at 37^"C, after which the PEG is diluted by the slow addition of media. The cells are pelleted and 35 to 40 L of DMEM containing 10% fetal bovine serum is added. The cells are then dispensed into tissue culture plates and incubated overnight in a 37^'C, 5% C0₂, humidified incubator.

The next day, DMEM-FCS containing hypoxanthine, thymidine, and aminopterin (HAT medium) are added to each well. The concentration of HAT in the medium to be added was twice the final concentrations required, i.e., H_fmal = 1 x lO^M, A^ = 4.0 x 10⁷M, and T_fιna, = 1.6 x 10-⁵M.

Subsequently, the plates are incubated with HAT medium every three to four days for two weeks. Fused cells are thereafter grown in DMEM-FCS containing hypoxanthine and thymidine. As cell growth becomes 1/2 to 3/4 confluent on the bottom of the wells, supernatant tissue culture fluid is taken and tested for SP-22 specific antibody by ELISA. Positive wells are cloned by limiting dilution over macrophage or thymocyte feeder plates, and cultured in DMEM-FCS. Cloned wells are tested and recloned three times before a statistically significant monoclonal antibody is obtained. Spent culture media from the chosen clone contains antibody which binds SP-22 in all dilutions tested. C. Antibody to SP-22 Peptides

SP-22 is identified by its biological functions and activities set forth herein, as well as by its size of approximately 22 kD and isoelectric point of 5.25. However, changes in form and the substitution of fragments or equivalents are contemplated as circumstances may suggest or render expedient. For instance, it may be necessary to generate polyclonal antibodies to peptide fragments of SP-22 if sufficient amounts of purified SP-22 cannot be obtained relatively easily.

In addition to the antibodies which are identical to the naturally-occurring SP-22 peptide antibody, the present invention embraces epitopes which are substantially homologous with such antibodies. The term "substantially homologous", when used in connection with amino acid sequences, refers to sequences which are substantially identical to or similar in sequence with each other, giving rise to a homology of conformation and thus to retention, to a useful degree, of one or more biological (including immunological) activities. The term is not intended to imply a common evolution of the sequences.

Substantially homologous peptide epitopes may be identified by a variety of techniques. It is known in the art that one may synthesize all possible single substitution mutants of a know peptide epitope. Geysen et al . , Proc . Na t . Acad. Sci . (USA) 81 : 3998-4002, 1984. While the effects of different substitutions are not always additive, it is reasonable to expect that two favorable or neutral single substitutions at different residue positions in the epitope- can safely be combined in most cases.

One may also synthesize a family of related single or multiple substitution mutants, present the mixture to a cell line capable of presenting the desired epitopes, and expose the eels to suitable restricted antigens. If the cells are lysed, effective epitopes may be identified either by direct recovery from the cells or by a progressive process of testing subsets of the effective peptide mixtures. Methods for the preparation of degenerate peptides are described in Rutter, U.S. Patent No. 5,010,175; Haughter et al., Proc . Na t . Acad . Sci . (USA) 52:5131-5135 (1985) ; Geysen et al . , op. ci t . ; W086/06487; W086/00991.

In devising a multiple mutagenesis strategy, a person of ordinary skill would of course give weight to the single substitution mutant data in determining both which residues to vary and which amino acids or classes of amino acids are suitable replacements.

It is also possible to predict substantially homologous epitopes by taking into account studies of sequence variations in families of naturally occurring homologous proteins. Certain amino acid substitutions are more often tolerated than others, and these are often correlated with similarities in size, charge, etc., between the original amino acid and its replacement. Insertions or deletions of amino acids may also be made, as described above.

Clones

Once an antibody to, or portion sequence of, SP-22 is available, it will be necessary to screen cDNA libraries to identify the cDNA clones that produce SP-22. SP-22 may be produced by methods other than recovery from male animals. In particular, a cDNA probe is prepared against a partial sequence of SP-22 and used to identify the SP-22 genome in cells from any mammalian species. The identified genome is then inserted into a plasmid which is then employed to produce recombinant SP-22 in proliferating bacteria or other hosts according to methods known in the art. This will be useful in the methodologies, e.g., immunocontraception, ^_ addition to sperm, described herein. However, not only will these utilities require large amounts of SP-22, they will also require large quantities of the SP-22 antibody. This is accomplished in batch hybridoma cell culture using proven methods .

Contraception

Antibodies to SP-22 can be used for contraception as well as for assaying fertility. A reversible contraceptive vaccine is provided by administering to an animal subject SP-22 as described above in an amount effective to reduce the fertility of that subject via generation of antibodies to SP-22. Partial reduction in fertility, i.e., effects which are reflected as a reduction in fertility in a population of subjects, are intended as within the scope of the present invention.

Any animal which expresses sperm surface SP-22 may be treated by the immunocontraceptive method of the present invention, including both birds and mammals. Exemplary mammals include mice, rabbits, dogs, cats, cows, pigs, sheep, horses, and humans. Mammalian subjects are preferred. The vaccine can be administered to either females or males by any suitable means, including by intramuscular injection, by intravenous injection, or by intraperitoneal injection. The antibody can be administered topically, as by vaginal foam or by any convenient topical method in an appropriate carrier, e.g., by nasal spray.

The term "protection", as used herein, is intended to include prevention or suppression of production of fertile sperm.

It will be understood that in medicine, it is not always possible to distinguish between preventing and suppressing, since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events. The term "protection", as used herein, is meant to include prophylaxis. The form of administration may be systemic or topical. For example, administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, vaginal or buccal routes. Alternatively, or concurrently, administration may be by the oral route. Parenteral administration can be by bolus injection or by gradual release over time.

It is understood that the suitable dose of a composition according to the present invention will depend upon the age, health and weight of the recipient. However, the most preferred dosage can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. This typically involves adjustment of a standard dose, e.g., reduction of the dose if the patient has a low body weight.

Prior to use in humans, a drug is first evaluated for safety and efficacy in laboratory animals. In human clinical trials, one begins with a dose expected to be safe in humans, based on the preclinical data for the drug in question, and on customary doses for analogous drugs, if any. If this dose is effective, the dosage may be decreased to determine the minimum effective dose, if desired. If this dose is ineffective, it will be cautiously increased, with the patients monitored for signs of side effects. See, e.g., Berkow et al . , eds., The Merck Manual , 15th edi tion, Merck and Co., Rahway, N.J., 1987; Goodman et al . , eds, Goodman and Gilman ' s The Pharmacological Basis of Therapeutics , 8th edi tion, Pergamon Press, Inc., Elmsford, N.Y. (1990) ; Avery ' s Drug Trea tment : Principles and Practice of Clinical

Pharmacology and Therapeutics, 3rd edi tion, ADIS Press, LTD., Williams and Wilkins, Baltimore, MD (1987); Ebadi, Pharmacology, Little, Brown and Co., Boston (1985) , which references and references cited therein are entirely incorporated herein by reference.

The appropriate dosage form depends on the composition administered, i.e., the carriers used for the antibody, as well as the mode of administration. Modes of administration include tablets, capsules, lozenges, dental pastes, suppositories, inhalants, solutions, ointments, and parenteral depots. See, e.g., Berker, supra , Goodman, supra , Avery, supra and Ebadi, supra, which are entirely incorporated herein by reference, including all references cited therein.

In addition to the antigen of the invention, a pharmaceutical vaccine composition may contain suitable pharmaceutically acceptable carriers, such as excipients, carriers, and/or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The amount of antigen administered depends upon factors such as route of administration, species, and the use of booster administrations. In general, a dose of about 0.1 to about 100 micrograms per kg of body weight may be used. The antigen to SP-22 may be prepared as both human and veterinary vaccine formulations. Vaccine formulations of the present invention comprise the antigen in a pharmaceutically acceptable carrier. The antigen is included in the carrier in an amount which is effective to reduce the fertility of the subject being treated. Pharmaceutically acceptable carriers are preferably liquid, particularly aqueous carriers, such as sodium phosphate buffered saline. The vaccine formulations may be stored in a sterile glass container sealed with a rubber stopper through which liquids may be injected and formulations withdrawn by syringe. Vaccine formulations of the present invention may optionally contain one or more adjuvants. Any suitable adjuvant can be used, such as aluminum hydroxide, aluminum phosphate, plant and animal oils, and the like, with the amount of adjuvant depending on the nature of the particular adjuvant employed. In addition, the vaccine formulations may also contain at least one stabilizer, such as carbohydrates such as sorbitol, mannitol, starch, sucrose, dextrin, and glucose, as well as proteins such as albumin or casein, and buffers such as alkaline metal phosphates and the like.

In addition to the active ingredient, i.e., the antigen or antibody to SP-22, pharmaceutical compositions according to the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active ingredients into preparations for pharmaceutical use. Preferable, the preparations contain from about 0.1 to about 99 percent, preferably from about 25 to 85 percent of active ingredient, together with the excipient. The excipient may be any pharmaceutically acceptable excipient or carrier which can be used with the antigen or antibody. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol and sorbitol, cellulose preparations and derivatives and/or calcium phosphates. Also useful as excipients are binders such as starch, gelatin, gums, methyl cellulose, hydroxyproplymethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. Lubricants such as silica, talc, stearic acid, or salts thereof, and/or polyethylene glycol can also be used.

For vaginal application, suppositories, lotions, creams, sprays, or foams may be used to incorporate the active ingredient. Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanols. Foam formulations may include oily suspensions or aqueous solutions of the active ingredient with suitable foaming agents. Other topical carriers for vaginal application include pharmaceutically acceptable liquids in which the active ingredient is suspended or dissolved.

For administration by nasal spray, the active ingredient is incorporated into a pharmaceutically acceptable liquid that can be sprayed into the nose.

SP-22 can also be used to identify male animals who are good candidates for supplying sperm for artificial insemination. Since many livestock animals are reproduced by artificial insemination or embryo transfer, it is important to be able to identify males who are fertile as well as possessing desirable characteristics to pass on to the next generation. Techniques for reproducing animals by embryo transfer are described in U.S. Patents 3,854,479; 4, 816, 257f and 4,326,505, the entire contents of each of which are hereby incorporated by reference. By determining the amount of SP-22 in the sperm of a subject animal, the fertility of the animal can be predicted.

Toxicity Testing

It is known that sperm proteins are affected by toxicants and pollutants. According to the present invention, the changes in SP-22 level are calibrated to predict the likelihood of the sperm having been rendered infertile because of exposure to the toxicant.

In Klinefelter et al . , op . ci t . , it was demonstrated that endocrine-disruptive chemicals decreased the fertilizing ability of cauda epididymal sperm in four days. Tests were conducted to determine if this infertility was related to decrease of SP-22 associated with the sperm.

In order to evaluate the effects of exposure to toxicants that perturb the androgen status of the animal, such as EDS, sperm are subjected to analysis for SP-22. Adult (90 to 120 day old) male Sprague-Dawley rats are housed two to three per cage with laboratory-grade pine shavings as bedding. The rats are maintained under controlled temperature (22 °C) and humidity (40-50%) conditions, and are given Purina laboratory rat chow and tap water ad libi tum. Males are maintained in a 14-hour light, 10-hour dark schedule. Each male is numbered and randomly assigned to a treatment group. The test toxicant is administered either as a single intraperitoneal injection or as four daily injections. After four days the rats are killed in the caudal epididymides of each male is placed in a 35-mm culture dish containing 2 mL of Medium 199. Detergent extracts representing 10-40 x 10⁶ mL sperm, depending on treatment, are prepared and aliquots equivalent to 30 micrograms are electrophoresed in a mini, two-dimensional electrophoresis system (BioRad) for quantitative analysis of SP-22. Specifically, sperm are transferred to a microcentrifuge tube and washed twice by centrifugation (3000 x g, five minutes) in Dulbecco's ^— phosphate buffered saline, pH 7.2, with freshly added 0.2 M phenylmethylsulfonyl fluoride (PMSF) . After the final wash the sperm are extracted for one hour at room temperature with 1 mL of 40 mM n-octyl- -glucopyranoside in 10 mM Tris, pH 7.2, containing freshly added PMSF. Following a final centrifugation at 3000 x g, the supernatant is removed and frozen at -70^°C.

Upon thawing, each extract is concentrated in 1 M Tris buffer by two centrifugations (3000 x g for 45 minutes at 4^'C) in Centricon-10 units (Amicon) . Protein concentration is determined using a Pierce protein assay kit . Sample volumes containing 30 μg protein are lyophilized, and protein is solubilized for 30 minutes at room temperature in 45 μL of sample buffer consisting of 5.7 g urea, 4 mL 10% NP-40, 0.5 mL ampholytes (70% 3-10, 30% 5-7) and 0.1 g dithiothreitol per 10 mL. Isoelectric focusing (750 V, 3.5 hours) is carried out in gels consisting of 6.24 g urea, 1.5 g acrylamide (30% acrylamide, 1.2% bisacrylamide) , 2.25 mL 10% NP-40, and 0.65 mL ampholytes (60% 3-10, 40% 5-7) per 10 mL. Molecular weight separation is carried out in 11% acrylamide gels (200 V, 45 minutes) . Gels are soaked in 50% methanol and silver stained. A Kepler 2D gel analysis system (Large Scale Biology Corp., Rockville, MD) was used for background correction, spot matching, and spot area quantitation. Images were acquired by transmittance at 80 μm spatial resolution and 4096 gray levels on an Ektron 1412 scanner and converted to 256 gray levels. Quantitation was done by fitting two-dimensional gaussian distributions to the density distribution of the spot area following background subtraction. Of the 124 proteins (spots) that were identified in the 50 gel data set, 22 were common to gels representative of sperm extracts of vehicle-treated animals. Of these 22 proteins, only SP-22 was affected by all test chemicals in a dose-related fashion. In fact, SP- 22 was the only one, of the 124 that were identified, that changed in either a dose or treatment-related fashion.

It has been determined that insemination (in utero) of 5 x IO⁶ epididymal sperm from a control rat results in ^~" approximately 75% fertility, thereby providing relatively greater sensitivity than insemination of a number of sperm that would result in 100% fertility. The various data (fertility and SP-22, as well as other endpoints such as motility parameters and testosterone concentrations) are collected and analyzed using two-way analysis of variance for both block and treatment effects. An initial analysis is performed to determine whether experimental block differences influenced the parameters measured. Where overall block effects are significant (P<0.05), the least-square means are compared for significant (P<0.05) treatment differences. A correlation analysis as performed to determine whether significant (P<0.01) correlations exist between each of the measured endpoints, and fertilizing ability and Pearson correlation coefficients (R) are calculated.

In a study reported in Klinefelter et al . , in Journal of Andrology 15(4) , 318-327 (1994) , the authors used in utero insemination of epididymal sperm and exposure to a chemical which disrupts androgen status of the epididymis, ethane dimethanesulfonate, or EDS, to investigate the hypothesis that EDS comprises the fertilizing ability of sperm by affecting epididymal function directly. Fertilizing ability, sperm motility, serum testosterone, and tissue testosterone were evaluated. In addition, sperm proteins were extracted and analyzed by quantitative two dimensional gel electrophoresis. An 18 kD protein was well correlated with fertility. However, the authors felt that changes in this protein were not specific either to EDS itself or the dose that was tested.

In a subsequent study, the insemination procedure was modified to permit assessment of fertility (implants/corpora lutea) rather than fertilizing ability (percentage of eggs fertilized) . In this study, multiple chemicals that disrupt endocrine status were tested. Adult males were exposed either to ethane dimethanesulfonate (EDS; 25 and 50 mg/kg) , epichlorohydrin (EPI; 3 and 6 mg/kg) , or hydroxyflutamide (Hflut; 12.5 and 25.0 mg/kg) or chloroethyl ethanesulfonate (CEMS; 12.5 and 18.75 mg/kg) . Each of these compounds perturbs the endocrine balance of the male reproductive system. The animals exposed to the known antiandrogen hydroxyflutamide were castrated and implanted with testosterone implants just prior to the first injection. The vehicle controls for all treatments except hydroxyflutamide received daily injections of 30% DMSO in water. The vehicle controls for the hydroxyflutamide animals were castrated, implanted with testosterone implants, and given daily injections of 15% ethanol. Four days after the onset of dosing, the males were killed and the epididymides were removed. The caput/corpus was frozen on dry ice for subsequent steroid extraction and testosterone assay. Sperm were released from the epididymal tubule into insemination medium and held in a C0₂ incubator at 34 ^*C for no more than 15 minutes until insemination. Adult, estrus-synchronized female rats were monitored for lordosis behavior just after lights out on the day of insemination. Females displaying mating behavior were cervically stimulated either with vasectomized teaser males at least 15 minutes prior to insemination. A volume equal to 5 x IO⁶ sperm was inseminated into each uterine horn at day 0. On day 9, the females were killed and fertility was assessed. A kepler 2D gel analysis system (Large Scale Biology Corp.,

Rockville, MD) was used for background correlation, spot matching, and spot area quantitation. Images were acquired by transmittance at 80 um spatial resolution and 4096 gray levels on an Ektron 1412 scanner and converted to 256 gray levels. Quantitation was done by fitting two-dimensional gaussian distributions to the density distribution of the spot area following background subtraction. Of the 124 proteins (spots) that were identified in the 50 gel data set, 22 were common to gels representative of sperm extracts of vehicle-treated animals. Of these 22 proteins, only SP- 22 was affected by all test chemicals in a dose-related fashion. In fact, SP-22 was the only one, of the 124 that were identified, that changed in either a dose or treatment- related fashion.

Measurements of sperm motion and sperm morphology were not significantly affected by any of the treatments. Based on a scatter plot of the data relating the amount of SP-22 to fertility (frequent) , fertility classes greater and less than 50% were chosen. Variables were then entered into the discriminant analyst to predict fertility by class, as shown in Table l. Since in this study, fertility for the control animals was targeted at 68% ± a standard deviation of 18%, 50% represented a reasonable cutoff for the fertile class.

TABLE 1 Discriminant Analysis Based on SP-22

PERCENTAGE CORRECTLY

CLASS PREDICTED

Fertile 90 (17/19) (>50%)

Subfertile 94 (29/31) (<50%)

The regression analysis shown as Figure 1 shows that the amount of SP-22 is significantly correlated to fertility (p<0.0001; r2=0.83) . A nonlinear fit of the data was indicated, since a threshold of 10,000 integrated optical density units (I.O.D.) of SP-22 was necessary to achieve greater than 50% fertility.

In Figure 1, data (N=50) for fertility and the integrated, background-corrected area (I.O.D.) of SP=22 were fitted to the following equation:

SP22 ^X (F P

in which F_SP.22 is the fertility at protein concentration SP-22, F₀ is the fertility at 0 protein concentration. A and B are constants (A is the initial increase in fertility and B is the rate of exponential decay of the increase in ^~ fertility) . The dotted lines represent the 95% confidence limits around the fitted line. The fitted parameters are F₀=2.810-5 + 3.567; A=4.283-3 ± 3.965-5; and B=2.984-4 +_. 1.140-ln with r₂=0.83.

Thus, by entering the level of SP-22 of a sperm sample into an appropriate mathematical model, it is possible to predict the fertility of the sperm sample with a reasonably high degree (i.e., >90%) of success. An antibody to SP-22 can be used to evaluate the fertility of sperm in an epididymal sperm sample or an ejaculate. Since the antibody to SP-22 recognizes a single protein on immunoblots of gels of both human and stallion sperm extracts (not shown) , this antibody will most likely be applicable to evaluation of animals in which maximum fertility is important, e.g., cattle, horses, dogs, and humans, among other animals. Additional endpoints may be included to predict fertility.

The toxicants tested above do perturb the endocrine balance of the male reproductive system. Other environmentally relevant "endocrine disruptors", such TCDD (dioxin) , could also compromise the expression of SP- 22. The present application and preliminary data indicating that TCDD does indeed decrease SP-22 expression (not shown) thus prompts development of a screening kit to test such chemicals.

Antibodies to SP-22

Antibodies against SP-22 can be prepared by any conventional means, and can be either polyclonal or monoclonal. They may be raised in rabbits, mice, or other animals or tissue culture cells derived therefrom, or can be products or cells of human origin. They may also be produced by recombinant DNA technology either in a form identical to that of the native antibody or as chimeric molecules, constructed by recombination of antibody molecules of human or animal origin or in other forms chosen to make the antibodies most suitable for use in therapy.

For preparation of the antibodies, either purified SP-22 or a peptide identical to the known sequence or a fragment thereof, e.g., to the N-terminal protein sequence, may be used to immunize animals. A further possibility is to fuse one of the possible nucleotide sequences coding a fragment of SP-22 to the gene coding for Protein A, to express the antibody. The antibody is then purified by affinity chromatography on a Sepharose column and used to immunize animals.

The monoclonal antibodies according to the present invention are prepared using conventional hybridoma techniques (Kohler et al . , (1975) Na ture 256 : 495; Kohler et al (1976) Eur. J. Immunol . 6 : 511) . After immunization, spleen cells alone or together with lymph node cells of the immunized animals are isolated and fused with a suitable myeloma cell line. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium and then cloned. The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding SP-22. After identification, the desired clones are grown in bulk, either in suspension culture or in ascitic fluid, by injecting the cells into the peritoneum of suitable host mice. The monoclonal antibodies produced by the hybridomas are then isolated and purified. The monoclonal antibodies may also be immunized and used for the purification of SP- 22 in affinity purification procedures using an immunoadsorbent column.

The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, anti- idiotypic antibodies to antibodies that can be labeled in soluble or bound form, as well as active fractions thereof provided by any known technique, such as, but not limited to, enzymatic cleavage, peptide synthesis, and recombinant techniques.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen. A monoclonal antibody contains a substantially homogeneous population of antibodies specific to antigens, which population contains substantially similar epitope binding sites.

Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having the variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Chimeric antibodies are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine monoclonal antibodies have high yields from hybridomas but higher immunogenicity in humans, such that human murine chimeric monoclonal antibodies are used.

Chimeric antibodies and methods for their production are known in the art [Cabilly et al . , Proc . Na tl . Acad. Sci . USA 51:6851-6855 (1984) ; Boulilanne et al . , Na ture 312 : 643646 (1984) ; Cabilly et al . , European Patent Application 125023, published November 14, 1984; Neiberger et al . , Na ture 314 : 268-270 (1985) ; Taniguchi et al . , European Patent Application 171496, published February 19, 1985 ; Morrison et al . , European Patent Application 173494, published March 5, 1986; Neuberger et al . , PCT Patent

Application WO 8601533, published March 13, 1986; Kudo et al . , European Patent Application 184187,published June 11, 1986; Sahagan et al . , J^". Immunol . 137 : 1066-1074; Robinson et al., International Patent Publication WO 8702671, published May 7, 1987; Liu et al . , Proc . Na tl . Acad . Sci . USA 84:3439-3443, 1987; Sun et al . , Proc . Natl . Acad. Sci . USA 84:214-218, 1987; Better et al . , Science 240 : 10411043 (1988) ; and Harlow and Lane, Antibodies, a Labora tory Manual . Each of these references is hereby incorporated herein by reference in its entirety.

An anti-idiotypic antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. An anti-idiotypic antibody can be prepared by immunizing an animal of the same species and genetic type (e.g., a mouse strain) as the source of the monoclonal antibody with the monoclonal antibody to which an anti-idiotypic antibody is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants, i.e., the antiidiotypic activity. See, for example, U.S. Patent No. 4,699,880, the entire contents of which are hereby incorporated by reference.

The anti-idiotypic antibody may also be used as an immunogen to produce an immune response in yet another animal, producing a so-called anti-anti-idiotypic antibody. The anti- anti- idiotypic antibody may be epitopically identical to the original monoclonal antibody which induces the anti-idiotypic antibody. Thus, by using antibodies to the idiotypic determinants of a monoclonal antibody, it is possible to identify other clones expressing antibodies of identical specificity. Accordingly, monoclonal antibodies generated against

SP-22, and related proteins of the present invention, may be used to induce anti-idiotypic antibodies in suitable animals, such as BALB/c mice. Spleen cells from such immunized mice are used to produce anti-idiotypic hybridomas secreting antiidiotypic monoclonal antibodies. Further, the anti-idiotypic monoclonal antibodies can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional BALB/c mice. Sera from these mice will contain anti-anti idiotypic antibodies that have the binding properties of the original monoclonal antibodies specific for SP- 22 or epitopes thereof.

The term "antibody" is also meant to include both intact molecules as well as active fractions thereof, such as, for example, those which are capable of binding antigen. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non specific tissue binding than an intact antibody, cf. Wahl et al . , J. Nucl . Med. 24 : 316-325, 1983. Compositions according to the present invention are prepared for administration by mixing the SP-22 antibody or its derivatives with physiologically acceptable carriers, stabilizers, and excipients, and prepared in dosage form, e.g., by lyophilization in dosage vials, foam, generating compositions, creams, jellies, lotions, suppositories, etc. ^~ The amount of antibody to be administered will depend on the route of administration, the size of the patient, etc.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various application such specific embodiments without departing from the generic concept, and therefore such adaptations and modifications are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation.

All references cited in this specification are hereby incorporated by reference.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: U.S. ENVRONMENTAL PROTECTION AGENCY (B) STREET: 401 M Street S.W.

(C) CITY: Washington

(D) STATE: D.C.

(E) COUNTRY: U.S.A.

(F) POSTAL CODE (ZIP) : 20024 (A) NAME: Gary KLINEFELTER

(B) STREET: 302 Cross Lake Drive

(C) CITY: Fuq ay-Varina

(D) STATE: North Carolina

(E) COUNTRY: U.S.A. (F) POSTAL CODE (ZIP) : 27526

(ii) TITLE OF INVENTION: METHOD FOR EVALUATING AND AFFECTING MALE

FERTILITY

(iii) NUMBER OF SEQUENCES: 1 (iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/593,677

(B) FILING DATE: 29-JAN-1996

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Thr Ser Gly Pro Leu Ala Lys 1 5

Claims

WHAT IS CLAIMED IS:

1. A purified protein SP-22.

2. A purified protein according to claim 1 which has the partial amino acid sequence Thr-Ser-Gly-Pro-Leu-Ala-Lys (SEQ ID N0:1) .

3. A method for predicting fertility of a male animal comprising assaying sperm from said male animal for levels of protein SP-22 and relating the amount of SP-22 in the sperm to fertility.

4. The method according to claim 3 wherein the male animal is a human.

5. A method according to claim 3 wherein the male animal is a farm animal to be selected as a sire for artificial insemination.

6. A method for screening for environmental endocrine disruptors comprising assaying for protein SP-22 in sperm from a male animal and correlating the amount of SP-22 on sperm to the degree of environmental endocrine disruption caused by said disruptor.

7. Antibodies to SP-22.

8. An antibody according to claim 7 wherein said antibody is a monoclonal antibody.

9. A method for temporarily rendering an animal infertile comprising administering vaginally to said animal an effective amount of antibody to SP-22.

10. The method according to claim 9 wherein the animal is a human.

11. The method according to claim 10 wherein the human is a female.

12. The method according to claim 11 wherein the antibody to SP-22 is administered vaginally.

13. A vaccine for rendering an animal infertile comprising administering to said animal an effective amount of SP-22 in a physiologically acceptable carrier.

14. A method according to claim 13 wherein said animal is a human.

15. A method for improving fertility in a male animal^-' comprising adding an effective amount of SP-22 to a semen sample or a subset of sperm of said male animal .

16. A method according to claim 15 wherein said male animal is a human.

17. A method for improving fertility in a male animal comprising: testing sperm from said male animal for expression of SP-22; harvesting only sperm which expresses SP-22; and fertilizing a female with said sperm which expresses SP-22.

18. A method according to claim 17 wherein said male animal is a human.