CA2568512A1 - Gender selection with the use of antibodies - Google Patents

Abstract

Epitope polypeptides, corresponding to antigenic regions of hydrophilic sequences selected from within mammalian gene Y-chromosome protein sequences, have been prepared and used to generate antibodies in vivo. The antibodies have been found to bind preferentially to the Y-chromosomal sperm component in semen.
The binding has been found sufficient to decrease motility of such Y-chromosomal sperm component leading to substantial increases in female offspring when using the treated semen in artificial insemination.

Description

DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.

NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.

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THAN ONE VOLUME.

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GENDER SELECTION WITH THE USE OF ANTIBODIES
BACKGROUND OF THE INVENTION

Gender selection of husbandry animals has been a goal of veterinarians for the last 40 years'. The selection of male and female offspring of farm animals would greatly help the competitive edged of producers. Several approaches have been tried to separate X and Y spermatozoids. This includes the separation of sperm (X
and Y) by the amount of DNA within X and Y containing spermatozoids2"5. This approach employs Hoechst stain that intercalates DNA/RNA. A cell with increasing amount of DNA/RNA

such as spermatozoids with X chromosome will intercalate more dye and fluoresce more.6 A flow cytometer is used to distinguish between the levels of fluorescence.
Some flow cytometers are able to separate the two types of sperm due to the amount of fluorescence that the cells emit. However, the distinction between Y and Y
spermatozoid using this technology is poor, time consuming, costly, and the yield poor7.
Viability of the cells is another concern.

Immunological approaches to gender selection have been contemplated since the discovery of male antigens8, which consists of a family of molecules (H-Y
antigens) found only on the surface of male cells9,10. The H-Y antigens are able to elicit an immune reaction when cells or tissues from a male donor are grafted on a female acceptor. Putative anti H-Y have been be used to treat whole sperm to modify the X/Y
ratio of spermatozoa to select for female gender in the offspring' 1-14 Antibodies to the male specific antigens have been used to discriminate between male and female embryos in swine and cattle.

Females can form antibodies (Abs) to spermatozoa. These Abs induce infertility in human females15 by inhibiting the motility of spermatozoa. The immune response of females to male antigens has also been described and the antigens are defined as a family of gene products (called H-Y antigens)16. The family of H-Y antigen structures is ill defined. The H-Y antigens induce a humoral (antibody) and cellular (CD4 and CD8 T
cells) responses'o"1-21 However, since the H-Y antigen family is composed of multiple gene products on the Y chromosome or controlled by genes on the Y chromosome that induces the expression of gene products found on other chromosomes of the genome, it is not known how many H-Y gene products there are and if all of the H-Y gene products induce an Ab and/or cellular responses.

Antibody (Ab) production to H-Y antigens has been induced in females with the use of whole male cells or cell lysates from males with or without the use of adjuvants22-24. The H-Y antigen family gene products across species are either identical or homologous25-2'. Many of the Y chromosome H-Y gene products may have a homologous gene on the X chromosome but the gene product are not necessarily identical28,29. Thus these antigens have low immunogenicity due to the similarity or near identity with self proteins.

Ab production is usually performed by immunizing in a different species from which the antigen was derived. Ab production is facilitated when the antigen is foreign to the host i.e. no shared sequences. As the protein sequences of an antigen are homologous to self protein, the immunological response becomes less vibrant or non-existent. The degree of homology and identity between X and Y homologous gene products (as discussed above) is high and thus the immune response to the differences is poor. It can be visualized that the host is being challenged with many self-antigens.
This approach of employing whole cells and cell extract has yielded Ab to the H-Y antigens and to self structures. The Abs are of the IgM isotypes and of low affinity and of multiple specificity 10. The failure to produce high affinity specific reagents was due probably due to the use of whole cells or fractions of membrane extracts used in the immunization. These extracts and membrane preparations from male cells are antigen preparations that are predominantly self-antigens expressed in male and female cells.

Attempts to employ these Abs failed to select female gender offspring since the Ab failed to discriminate between spermatozoa containing X and Y-chromosomes.
A
different approach to the generation of anti-H-Y antigen is necessary. Our approach was to obtain an immunizing antigen that would generate high affinity specific Ab. Many protein sequences of the H-Y proteins of various species are known since the complete (human) and partial genome sequences of livestock animals have been published.
A
protein adopts a specific three-dimensional structure. Globular proteins bury their hydrophobic groups in their core and expose on their surface hydrophilic moieties. The same holds for portions of proteins that are located at the cell surface. The three-dimensional structure determines which areas of the molecule will protrude more and most likely be accessed first and recognized by Ab. i.e. hydrophilic areas of the protein.
One recent example of sex determination involving separation of sperm determinative of each sex using supported antibody is US Patent No. 6,489,098, December 3, 2002 to T.L. Benjamin et al. A reference mentioned in this patent (J.

Reproductive Immunology, 1984, Vol. 5, pp. 109, Hoppe & Koo) described tests in which the sex ratio of eggs fertilized in vitro was not influenced by reacting sperm with certain monoclonal H-Y antibodies (evidently the selection criteria for the antigen used were not appropriate).

SUMMARY OF THE PRESENT INVENTION

With the present invention, the applicants have utilized a novel approach based on a technique to identify H-Y proteins that may be associated with the outer membrane of spermatozoa (Table 1). In general, the protein sequences from H-Y antigens were analyzed with the e.g. Bio Edit software to determine the hydrophilic areas of each sequence. Based on these areas, epitopes were synthesized, and e.g. biotin was added to the N terminal end of the peptide. Each peptide was added to a carrier protein, e.g. streptavidin and selected mammals e.g. rabbits, mice were immunized employing e.g. RIBI as the adjuvant.

While peak hydrophilicity was found for gene fragments of about 4-7 amino acids, significant hydrophilicity (and Ab reactivity) was observed for fragments of up to about 22 amino acids.

The peptide Elisa, anti-motility, flow cytometry results showed that pre-immune serum does not react with swine spermatozoa whereas antiserum to some of the peptides reacted with a subset of swine spermatozoa. One of the anti-sera also reacted with bovine spermatozoa but not the pre-immune serum nor the pre-immune or immune sera to the other peptides. Employing androgen motility and sedimentation chambers, motile versus non-motile spermatozoa were separated and a significant depletion of Y
chromosome containing spermatozoa was achieved in the motile group (as detected by fluorescent in situ hybridization (FISH) technology). We conclude that the antiserum even though produced in rabbits (or mice) is comprised of multiple monoclonal antibodies with a single specificity but multiple affinities to the single peptide. A total of 5 peptiditic antigenic determinants from four different proteins have been identified that 5 were used to inhibit motility of swine or bovine spermatozoa bearing the Y
chromosome.
Accordingly, as disclosed herein, one aspect of the present invention relates to a novel epitope polypeptide corresponding to antigenic regions of hydrophilic protein sequences, selected from within mammalian gene Y-chromosome protein sequences;
and preferably wherein the Y-chromosomal sequences are selected from genes from the group having accession numbers D30811, AB027133, BC074923 and G49470 (NCI). More preferably the genes are selected to be common to more than one mammalian species (Table 1, Table 2); e.g. wherein the species are homo sapiens, porcine and bovine; and the genes most preferably having an amino acid sequence selected from the group in the Sequence Listing.

In another aspect of the present invention, there is disclosed an antibody to the epitope polypeptide, preferably which comprises at least one monoclonal antibody.
There is further provided a mixture of monoclonal antibodies having a single specificity.
There is also provided an antibody selected to react with Y-chromosomal sperm from more than one species.

In a still further aspect of the present invention, there is disclosed a method of selecting epitope polypeptides from Y-chromosome genes comprising obtaining an H-Y
protein sequence from a male chromosome of a mammalian species of interest, determining the hydrophilic regions of the sequence, and preparing epitope polypeptides corresponding to the hydrophilic regions; followed by immunizing at least one mammalian species with the prepared epitope polypeptides to generate corresponding antibodies and recovering the antibodies. A still further preferred embodiment of the method of the present invention includes the additional step of selecting an antibody able to react with sperm of at least two species. Yet another embodiment of the method of the present invention provides a method of treating sperm to increase the incidence of female offspring therefrom, comprising contacting the sperm with at least one antibody recovered according to the invention. A still further embodiment provides a method of decreasing motility of Y-chromosomal sperm comprising treating the sperm with antibody as described.

In addition, the present invention also embraces a composition for artificial insemination comprising sperm and antibody as described herein selected to bind to the surface of only the Y-chromosomal sperm component.

In addition, the present invention also embraces a pharmaceutical composition comprising as the active ingredient the antibody as described herein, together with a carrier therefor.

EXAMPLES AND DESCRIPTION OF PREFERRED EMBODIMENTS

Having generally described the invention, reference will now be made to preferred embodiments, including reproduction of the photographs designated as Figures herein, in which Figure 1 is a photograph of a slide showing the results of anti-sperm activity using peptide 1, in Table 2;

Figures 2A and 2B are graphs illustrating the anti peptide activity on swine spermatozoa as measured by flow cytometry;

Figures 3A and 3B are graphs illustrating the anti-peptide activity on bovine spermatozoa as measured by flow cytometry; and Figure 4 is an enlarged electron photograph showing the detection of the "Y"
chromosome by FISH assay.

The following description outlines various examples of the present invention relating to the preparation of the various aspects of the invention described herein.
Materials and Methods Peptide selection The sequence of the selected chromosomal 4 proteins was obtained from Pub Med (Tablel). Hydrophilic peptide sequences within these were selected employing the BioEditT"' program 30 (Ibis Therapeutics, a division of Isis Pharmaceuticals, Inc) (Table 2). The peptides were synthesized by Sheldon Biopharmaceutical (McGill University Montreal QC). The amino terminus of the peptide was biotin labelled. Tables 1 and 2 are as follows:

Table 1 Y Chromosome genes.

r5ecies Gene Accession number Box taurus MEA D30811 Homo sapiens SRY BC074923 Homo sapiens Mea-2 AB027133 Homo sapiens DBY G49470 able 2: Summary of peptide sequence and reactivity of Abs to swine or bovine spermatozoa.

Antigen Peptide sequence Peptide synthesized Ab Reactivity Swine Bovine Mea 1 Ab Biot 1 PTEGTGDWSSEEPEEEQEETG YES YES YES
--;
SRY Ab Biot 4 RDQRRKMALENPRMRNSEISKQ YES YES NO
DBY Ab Biot 5 EMESHSVTQAGVQWPDLGSLEV YES YES NO
Mea 2 Ab Biot 6 LQRRLEEFEGERERLQRMADSAA YES YES NO
Mea 2 Ab Biot 9 RKWLEEQLKQYRVKRQQERSSQ YES YES NO
The hydrophilicity screening tests were done with selected gene regions ranging from about 5 to about 20 amino acids in length. Hydrophilicity peaks were found for four sequences of five amino acids as underlined in the gene sequences in Table 2.

Significant hydrophilicity remains in sequences of up to about 22 amino acids (e.g. as shown in Table 2). Sequences of various lengths (e.g. 4-22 amino acids) may be used for the immunization protocol as long as the hydrophilicity peak sequence is included therein. We have found that sequences of about 20-22 amino acids that retain significant hydrophilicity (and comprise at least one of the hydrophilicity peaks) are very 5 suitable for the immunization protocol. For immunization purposes it is desirable to build a "platform" of amino acids around the peak hydrophilicity sequence. These surrounding amino acids need not be the exact sequences occurring in the gene but should act similarly. Suitable criteria for choosing such surrounding sequences are to include amino acids with polar groups.

10 Antigen preparation and immunization of rabbits A 1 mg per ml of each peptide was prepared in Hanks Buffered solution pH 7.2.
The preparation of the immunizing antigen was performed by incubating 4 times the molar ratio of peptide to streptavidin (Sigma St-Louis Mo) for 30 minutes at room temperature. The immunizing antigen consisted of 40 ug of streptavidin-peptide complex with an equal volume of RIBI as the adjuvant 31 (Cedarlane Laboratories Toronto ON). (The concentration of streptavidin was taken into consideration only. The contribution of the peptide mass was not used in the calculation). RIBI
adjuvants are oil-in-water emulsions where antigens are mixed with small volumes of a metabolizable oil (squalene) which are then emulsified with saline containing the surfactant Tween 80.

This system also contains refined mycobacterial products (cord factor, cell wall skeleton) as immunostimulants and bacterial monophosphoryl lipid A.

Animals were pre-bled before the first immunization and animals were immunized at monthly intervals for four months. Serum samples were obtained from each animal 2 weeks after the first immunization. One additional serum sample was obtained 7 days after each subsequent immunization. After the four immunizations, all animals were exsanguinated. The animal protocol was approved by the University of Ottawa (ON) Animal Ethics committee.

Swine and Bovine semen preparation Swine sperm was obtained from Dr Daniel Hurnik Atlantic Veterinary College Charlettown PEI, Mr Gingerich, Ontario Swine improvement Corporation, Innerkip ON or Dr A Afshar, (Canadian Food Inspection Agency, Ottawa ON). Bovine semen was obtained from Dr S Scott (Perth Veterinary Clinic, Perth ON).

Semen was used either "as is" or diluted in semen extender. Swine and bovine extender was prepared as directed by the manufacturers, IVO Zeist Semen extender (ISTI Inc Princeton ON) and Triladyl (MiniTube Ingersoll ON).

Semen (swine or bovine) was washed in extender, diluted in extender and placed on glass slides enumerated by microscopy to determine viability and motility.
Directional movement, vibration and tail movement were enumerated. Agglutination, head to head or tail to tail was also quantitated.

Inhibition of Spermatozoa Motility The quantization of the motility and inhibition of motility of spermatozoa was performed by two assays. The first assay (swim up assay32) was performed in conical tubes by under laying 1 million spermatozoa (90% motile) in 100 pl under an equal volume of control serum or Ab diluted in swine extender. The sample was incubated at 37 C for 30 minutes and the top 25 pl containing spermatozoa were placed on a pre-warmed slide. The motility and agglutination was assessed by light microscopy.

The second assay that measure motility and the inhibition of motility employed migration and sedimentation chambers from Zander Medical supplies33. This was performed since the actual separation of motile and non-motile spermatozoa could be achieved. Fresh sperm (swine or bovine) with over 90% motility was placed in the outer chamber. Dilutions of the Ab in saline were layered above the sperm and into the capture well (inner chamber). The unit was incubated for 30 minutes at 37 C.
Spermatozoa were harvested from the capture well, enumerated by microscopy to determine % motility. The percentage of Y chromosome positive spermatozoa was determined FISH technology as described below. The Ab used for swine was a pool of Ab 1, 4, 5, 6, 9 that had been fractionated with saturated ammonium sulfate to remove the albumins and desalted using column chromatography employing swine extender3a The Ab for bovine experiment was Ab to peptide one only that had been prepared as described above. The FISH for the bovine spermatozoa was not performed since we do not have the probes for the bovine species.

Microscopic and Flow cytometric analysis of spermatozoa treated with Ab Flow cytometric analysis was performed with spermatozoa preparations from the anti-motility experiments. The additional step of adding goat anti rabbit Ig labeled was performed. This included washing the spermatozoa after the incubation of the primary Ab with 5 ml of extender. The pellet was re-suspended with goat anti-rabbit Ig-Alexa 647 (Molecular Probes, In VitroGen Toronto ON) (Microscopic analysis) or with goat anti-rabbit Ig Fitc Flow (Cytometric analysis) (Tago Diagnostics, Cedarlane Laboratories Toronto ON) that had been diluted in extender. The tubes were incubated for an additional 30 minutes at RT and washed. For fluorescent microcopy, the spermatozoa were mounted on a glass slide and viewed on a Leica microscope equipped with a Photometrics PXL 1400 CCD camera to capture images. Images were imported into PowerPointT" and viewed. An EPICS XL MCL Flow Cytometer (Beckman Coulter Electronics) was employed to acquire the data from samples and the data was saved as listmode files. Samples of spermatozoa without primary or secondary antibodies (auto control) and spermatozoa with secondary antibody but no primary (secondary control) were also acquired. Both pre-immune and Ab preparations taken after each immunization were tested.

Anti-peptide and anti-streptavidin Elisa assay:

The Elisa assay were performed by binding 1 pg of peptide or streptavidin in PBS
in nuclon type II Elisa plates35. After overnight incubation at 4 C, the plates were washed 3 times employing a Biorad plate washer (Biorad Inc Toronto, ON) using PBS
pH 7.2 (0.01 M) 0.005% Tween 20 as the washing solution. The pre-immune serum and serum samples were diluted in PBS pH 7.2 (0.01 M) Tween 20 0.1 % and 0.1 %
FCS

(dilution buffer) and plated in the Elisa plates. After 1 hr incubation at RT, the plates were washed three times with washing buffer above, and goat anti-rabbit Ig HRP
(Tago Diagnostics) in dilution buffer was added to the plates. After 1 hour the plates were washed and OPD in a sodium citrate buffer ph 4.0 was added to the plates.
After 30 minutes, the reaction was stopped by the addition of 0.1 M H2SO4. The optical density of each well in the plate was measured in a Packard Spectra Count Elisa plate reader using 450 filter.

Preparation of swine chromosome Y DNA probes for FISH analysis Preparation of DNA direct probes for FISH analysis for Y-chromosome specific genes was performed as follows. The sequence for swine Y chromosome (X12696, 3832 nucleotides in length with no marked internal repetitions36 was chosen as the gene to detect in the FISH analysis. The chromosome Y-specific primers were designed according to oligonucleotide sequences described by Rubes et al. 37.

PCR primers for the Y chromosome that resulted in a 377 bp probe are as follows:
Forward: 5-AAT CCA CCA TAC CTC ATG GAC C-3 Reverse: 5-TTT CTC CTG TAT CCT CCT GC-3 The probe was obtained by employing a pUC57 plasmid that had the probe inserted into it (BioBasic Inc., Markham ON). The plasmid was transfected into E Coli and isolated as described by Colligan et al (chapter 10)38. Sufficient amounts of plasmid were produced but the generation of the fragment by cutting the probe from the plasmid was not sufficiently specific and many other products were produced. The plasmid was isolated and used in a PCR amplification system that resulted in a pure product of 377 bp for the Y specific fragment.

The PCR reaction was performed as follows. All reagents were thawed in advance and kept on ice before use in the PCR reaction. The master mix was prepared by mixing 0.4pl of a mixture of 4 dNTPs (dTTP, dATP, dGTP and dCTP each at 2 mmol 1-1), 2.5 pl (from a 100 pmol pl-1 stock solution) of primers for chromosomes Y, 5 pl of purified plasmid y and 1, 5 pl of 1 OxPCR buffer (100 mmol Tris-HCI 1-1, pH
8.3 at 25 C;
500 mmol KCI I-'; 15 mmol MgC12 1-1) and 1 pl of 5 U Taq DNA polymerase pl-'.
The volume reaction was made up to 50ul with water. Amplification cycles were performed in a programmable thermal controller (PTC-100, MJ Research Inc) and consisted of a first denaturation step before the first cycle at 95 C for 5 min, followed by 35 cycles of the following program: denaturation at 95 C for 1 min, annealing at 48 C for 1 min extension at 72 C for 1 min and a final elongation step for 10 min at 72 C.The PCR
product were 5 subjected to electrophoresis in 1%(w/v) agarose gels. A portion of the gel was stained with ethidium bromide, visualized and then photographed under UV light. The product in the non-stained gel was located, by using the stained gel as a template, cut and the product extracted from the gel and resin purified (Promega Technical Bulletin).
Labeling of Y chromosome probes with Alexa 594 UTP:

10 The isolated Y chromosome DNA probe was labelled using the modified deoxyuridine triphosphates (dUTP), Alexa 594-dUTP (Molecular Probes Eugene OR).
The Alexa 594 emits in the red region of the spectra. The labeling was performed as recommended by the manufacturer (Nick Translation kit N 5500 from Amersham Pharmacia Biotech Europe GmbH (Barcelona)).

15 Fluorescence in situ hybridization of swine spermatozoa:
Preparation of spermatozoa.

Spermatozoa were centrifuged for 5 min at 200 g and washed three times with 6 ml of KCI hypotonic solution (75 mmol/1). The supernatant was discarded and the pellet was re-suspended in fresh, cold fixative (methanol:glacial acetic acid 3:1), bringing the sperm suspension to a volume of 4 ml. The fixed spermatozoa suspension was spread on a clean glass slide and air-dried. The slides were stored in the dark at 4 C until 3' hybridization'39 Hybridization and detection The slides were prepared for hybridization as follows. The slides were washed in 2xsaline-sodium citrate buffer (SSC) to remove excess fixative, dehydrated by passing through a series of ethanol (70%; 85%; 100%) and air-dried. The slides were incubated for 30min at 37 C in a 10 mmol dithiothreitol (DTT) solution (pH 7.4) to reduce the protamine disulphide bonds and, to decondensate the spermatozoon nucleus. The slides were incubated for 1-3 hours in the dark at room temperature in a 10 mmol lithium 3,5 diiodosalicylicacid. (Sigma St Louis MO). The slides were washed in 2xSSC, dehydrated in ethanol (70%; 85%; 100%) and air-dried. The spermatozoa were denatured in 70% (v/v) formamide/2xSSC solution at 75 C for 5min. The spermatozoa were dehydrated for a third time as described above and dried at room temperatureao,47 The detection of swine Y chromosome of spermatozoa was performed by using the above slides and adding 1 ng/10p1 of denatured (75 C for 5 min) Y-chromosome Alexa 594 labelled probe. The slides were covered with a coverslip and sealed with rubber cement. They were placed in a dark moist chamber at 37 C for 24 h. The slides were washed sequentially with 0.4xSSC at 75 C for 2min, 2xSSC/0.1 %(v/v) Tween at room temperature for 2 min followed by ethanol dehydration (70%; 85%; 100%) and air-dried.

The slides were viewed by fluorescent microscopy on a Leica fluorescent microscope equipped with a Photometrics PXL 1400 CCD camera to capture images.
Images were imported into PowerPointT"' and viewed.

Results Anti-peptide and anti-streptavidin activity of rabbit sera.

The anti-peptide and anti-streptavidin activity of the pre-immune and post-immunization of the serum from the rabbits were assessed by an Elisa assay.
All pre-immune sera did not have any Ab activity to streptavidin (Table 3) whereas antibody was detected and increased after each immunization. Table 3 is as follows:

Table 3: Anti-streptavidin activity of rabbits immunized with peptide-streptavidin complex.

Streptavidin Pre immunization 1st immunization 4t immunization activity Optical density Optical density Peptide Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit 1 .052 .063 .262 .192 3.43 .619 4 .05 .053 .243 .241 2.69 2.44 5 .06 .067 .211 .186 2.41 .634 6 .058 .056 .056 .344 .467 3.11 9 .063 .064 .236 .057 2.81 .559 The antibody response to the peptide could not be observed by Elisa therefore a flow cytometric assay was performed using swine and bovine spermatozoa.

Detection of anti-peptide antibody on swine and bovine spermatozoa employing fluorescent microscopy and flow cytometry.

Swine spermatozoa was obtained, enumerated on a microscope and 1 million cells were placed stained with a 1:100 dilution of pre-immune or anti-peptide Ab diluted in swine extender. The cells were washed after 30 minutes, and goat anti-rabbit Ig-Alexa 647 was added to each of the tubes. None of the pre-immune serum reacted with the swine spermatozoa as observed by fluorescent microscopy. Serum from animals immunized to peptide 1, 4, 5, 6 and 9 reacted with swine spermatozoa. Figure 1 shows a representative figure. We overexposed the slides to determine if low level binding could be observed and binding was not observed. We confirmed this microscopic data by performing a flow cytometric experiment using goat anti rabbit Ig Fitc as the detection antibody. Figure 2 is a representative figure showing two populations of swine spermatozoa were observed. Pre-immune serum did not react with the spermatozoa.
A similar flow cytometric experiment with bovine spermatozoa was performed and we demonstrated that only anti-peptide 1 but not its pre-immune serum reacted with the cells (Figure 3).

Anti-motility response of antibodies to peptides.

Antibodies to spermatozoa have been shown to have anti-motility activity and it is known that such an activity can inhibit fertilization. Thus we performed experiments to determine the anti-motility activity of the antibody preparations. The data in Table 4 shows that the pre-immune serum had marginal or no anti-motility activity and that the anti-motility activity was observed in serum from animals immunized with peptides 1, 4, 5, 6 and 9. Table 4 is as follows:

Table 4: Motility and Agglutination of swine spermatozoa incubated with pre-immune and immune serum I~Swim up method for sperm preparation Concentration=20 M/mI and Mot=95%) Pre-immune % ype of Immune % ype of serum Motility gglutination serum Motility gglutination Antigen ~dilution dilution ~C1(1/10 85 None 1 1/10 10 H-H & T-T
C1(1/100) 87 None 1 1/100 0 H-H & T-T
01(1/1000) 88 None 1 1/1000 78 None C1 1/10000 85 None 1 1/10000 78 None C3(1/10) 86 None 3 1/10 78 H-H & T-T
,C3(1/100 70 None 3 1/100 70 H-H & T-T
03(1/1000) 80 None 31/1000 62 None 03(1/10000 61 None 31/10000 65 None - - -a C4 1/10 80 None 4 1/10 15 H-H & T-T & shakin C4(1/100) 85 None 4 1/100 9 H-H & T-T & shaking C4 1/1000 88 None 4 1/1000 80 H-H & T-T & shaking C4(1/10000 80 None 4 1/10000 78 H-H & T-T & shaking 6 _ C5(1/10) 75 H-H & T-T 5 1/10 51 H-H & T-T
C5 1/100 75 H-H & T-T 5 1/100 53 H-H & T-T
C51/1000 70 None 51/1000 76 None 051/10000 70 None 51/10000 76 None ~- ---;
9 C7(1/10) 65 None 71/10 3 None 07(1/100 70 None 71/100 55 None _ C71/1000 87 None 71/1000 78 None 071/10000 85 None 71/10000 85 None Confirmation of selective reactivity of anti-peptide serum to spermatozoa with Y
chromosome:

The selective reactivity of anti-peptide antibodies to spermatozoa with Y

5 chromosome was performed with magnetic beads or with the anti-motility assay using the androgen motility chambers. Spermatozoa that had migrated were tested for the Y
chromosome by employing a specific fluorescing genetic probe for swine Y
chromosome (Figure 4). The initial experiments were performed with the use of magnetic beads coated with anti-rabbit Ig that would react with spermatozoa coated 10 witti immune serum. However, the complete removal of the beads was not possible and the results of the FISH assay were inconclusive. However, with the use of the androgen migration and sedimentation chambers we were able to separate X and Y
containing spermatozoa. These flasks contain two chambers - an outer and inner chamber.
The outside wall of the outer chamber was higher than the outside wall of the inner chamber 15 and thus after placing semen in the outer chamber, the antibody solution was placed in the inner chamber and overlay the semen in the outer chamber. The flask was incubated at 37 C and the spermatozoa migrated through the Ab solutions to the innner chamber and accumulated in the inner well. We demonstrated a dose dependent inhibition of migration of swine and bovine spermatozoa using anti-peptide antibodies.

20 In case of swine semen, a drastic inhibition of Y containing spermatozoa was observed (Table 5). The X and Y FISH technology for bovine has not been established as yet.
Table 5 is as follows:

Table 5: Androgen anti motility activity and Y chromosome FISH detection Swine Experiment Ab dilution Spermatozoa # % Migration % + Y chromosome Control 6,000,000 100 47 1/100 3,000,000 50 6.8 1/250 4,000,000 67 24 1/500 5,000,000 100 41 Bovine Experiment Ab dilution Spermatozoa # % Migration % + Y chromosome Control 12,000,000 100 ND

1/100 2,900,000 24 ND
1/250 7,300,000 61 ND
1/500 10,000,000 83 ND
Discussion The sexing of spermatozoa has been an elusive goal for many years. The identification of the H-Y antigens over 50 years ago has not made the task any easier since it has been recognized that the H-Y antigen is a family of gene products that are found on male cells but not female cells. H-Y antigens may be gene products that are encoded on the Y chromosome or are antigens encoded on other chromosome but their expression is controlled or regulated by genes expressed on the Y chromosome.

Simply, H-Y antigens is a family of gene products. Is there a similar family of gene products controlled by the X chromosome? There is no answer to this question as yet.
The similarity of the amino acid sequence of proteins within the male and female within a species and across species has made the identification of H-Y
antigens extremely difficult to do. The method of immunizing a female donor with a male cell extract or a fraction of the whole extract has not resulted in Ab products that can distinguish between X and Y gene products 48. This is probably due to the very few differences in amino acid sequences between gene products originating from X
or Y
chromosome within a species and across a species49,50In addition, due to the near identity of the proteins in the cell extract or fractions, immunological tolerance prevents the formation of a vibrant Ab response.

The sequencing of the human genome and the near complete sequence of the swine and bovine genome provides tools that can be employed to identify genes that are unique to or associated with the Y chromosome of any species. We identified four gene products associated with the Y chromosome and made peptide of the selected hycirophilic regions of these proteins. We focused on the peptide nature of the epitopes and not the carbohydrate epitopes since females and male of any species would share the carbohydrate structure within a species. The sharing of identical sequencing usually causes immunological tolerance and prevents the formation of Ab to the structure(s) in question.

Our data clearly shows that the immunization was successful since all animals produced anti-streptavidin antibodies within the four months. A typical secondary response was produced with higher levels of Abs being detected within seven days after vaccination. The-peptide response as measured by Elisa was not observed but the swim up assay, the microscopic and the flow cytometric assay showed that an anti peptide Ab was produced. All pre-immune serum did not react with spermatozoa from swine or bovine. Swine spermatozoa did not react with immune serum from animals immunized with complexes of peptide three (see Table 4) and streptavidin.
Thus, the Ab activity in immune serum from animals immunized with peptide 1, 4, 5, 6 or 9 and streptavidin was specific for the peptide and not due to Abs to streptavidin or to the adjuvant RIBI. Similarly, bovine spermatozoa only reacted to Ab from animals immunized to peptide one and streptavidin. All other pre-immune and immune serum did not react with bovine spermatozoa. This data suggest highly that we generated Ab specific for spermatozoa and that at least one epitope is common between two species.
We expect that this antigen may also be expressed in other species as well and may represent a universal epitope that can be used for sexing spermatozoa from multiple species.

The anti-motility and the head to head (H-H) and tail to tail (T-T) measurements of the antiserum (Table 4) show that the immunization resulted in products that affected spermatozoa in different fashion. Agglutination was observed in 4 out 5 anti-sera, whereas 1 anti-serum had no observable agglutination activity whereas 2 out of 7 had no detectable anti-spermatozoa activity. The agglutination results of swine spermatozoa mimicked the results obtained by microscopy and flow cytometry.
The swim up motility was modified to use the migration and sedimentation chambers so that we could physically separate sufficient quantities of spermatozoa so that the enumeration of the percentage of spermatozoa with Y chromosome could be determined by FISH. Table 5 shows that at a 1/100 dilution of pooled (anti-serum 1,4,5, 6,9) ammonium sulfate fractionated anti-serum that approximately 50% of the spermatozoa migrated into the inner chamber with only 6.8% of the migrated spermatozoa contained the Y chromosome. An identical experiment with ammonium sulfate fractionated anti-serum 1 and bovine spermatozoa was performed and at a dilution of 1/100 and 1/250 24 and 61 % of the spermatozoa migrated. This result confirmed our observations with the flow cytometer.

In conclusion, as examples, we have prepared 5 unique anti-sera to 5 different epitopes on four Y-chromosomal proteins. The anti-serum can be considered i0 monoclonal with multiple affinities since the anti-sera were raised to defined epitopes and not to whole proteins, cell extracts or fractions of cell extracts.
Antibodies to the streptavidin and the adjuvant had no anti-spermatozoa activity since anti-serum 3 in all assays (flow cytometry, microscopic, anti-motility) did not bind to spermatozoa but did have anti-streptavidin activity (results not shown). This type of antibody will enable the selection of female offspring of e.g. swine and bovine where donor sperm will be treated with the antibodies prior to artificial insemination.

With respect to the composition aspect of the present invention, the composition will normally comprise an effective amount of one or more of the active antibody ingredients which are capable of reacting with the Y-chromosomal sperm of a mammal, together with a pharmaceutically acceptable delivery system. Such compositions can be used for a wide variety of mammalian species, and by way of example, include the treatment of species such as swine, bovine, homo sapiens.

Accordingly, the compositions of the invention can be formulated using adjuvants, emulsifiers, pharmaceutically-acceptable carriers or other ingredients routinely use in this art. Such known or conventional adjuvants or emulsifiers that can be used in the compositions of this invention include Alum aluminium hydroxide, 5 complete Freund's adjuvant, incomplete Freund's adjuvant, Quil A. ISCOm's etc. Such formulations are readily determined by one of ordinary skill in the art and also include formulations for immediate release and/or for sustained release. The present compositions can be administered or used according to conventional techniques well known to those skilled in this art. The compositions of the instant invention contain an 10 effective amount of the active ingredient, the amount of which will be dependent on the type of species to be treated as well as the individual type of the antibody.

It will be understood that various modifications can be made to the above-described embodiments without departing from the spirit and scope of the invention described herein.

DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.

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Claims (17)

1. Epitope polypeptide corresponding to antigenic regions of hydrophilic protein sequences, selected from within mammalian gene Y-chromosome protein sequences.

2. The epitope polypeptide of claim 1, wherein the Y-chromosomal sequences are selected from genes from the group having accession numbers D30811, AB027133, BC074923 and G49470.

3. The epitope polypeptide of claim1, selected to be common to more than one mammalian species.

4. The epitope polypeptide of claim 3, wherein the species are porcine and bovine.

5. The epitope polypeptide of claim 1, having an amino acid sequence selected from the group 1, 4, 5, 6 and 9 in Table 2.

6. The epitope polypeptide of claim 1, having an amino acid sequence comprising one of the underlined sequences in Table 2.

7. Antibody to the epitope polypeptide of claim 1.

8. The antibody of claim 7, which comprises at least one monoclonal antibody.

9. A mixture of monoclonal antibodies of claim 7 or 8, having a single specificity.

10. The antibody of claim 7, 8 or 9, selected to react with Y-chromosomal sperm from more than one species.

11. A composition for artificial insemination comprising sperm and antibody of any one of claims 7 to 10 selected to bind to the surface of only the Y-chromosomal sperm component.

12. A method of selecting epitope polypeptides from Y-chromosome genes comprising:

obtaining an H-Y protein sequence from a male chromosome of a mammalian species of interest;
determining at least one hydrophilic region of the sequence; and preparing epitope polypeptide corresponding to the hydrophilic region.

13. The method of claim 12 followed by immunizing at least one mammalian species with at least one prepared epitope polypeptide to generate corresponding antibodies and recovering the antibodies.

14. The method of claim 13 including the additional step of selecting antibody able to react with sperm of at least two species.

15. A method of treating sperm to increase the incidence of female offspring therefrom, comprising contacting the sperm with at least one antibody recovered according to claim 13.

16. A method of decreasing motility of Y-chromosomal sperm comprising treating the sperm with antibody of claim 7.

17. A pharmaceutical composition comprising as the active ingredient the antibody of any one of claims 7 to 10, together with a carrier therefor.