Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02568512 2006-12-04
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
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THAN ONE VOLUME.
THIS IS VOLUME OF _2
NOTE: For additional volumes please contact the Canadian Patent Office.
CA 02568512 2006-12-04
1
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.
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2
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
CA 02568512 2006-12-04
3
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.
CA 02568512 2006-12-04
4
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
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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
CA 02568512 2006-12-04
6
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;
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7
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:
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8
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
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9
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
CA 02568512 2006-12-04
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
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11
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.
CA 02568512 2006-12-04
12
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
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13
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.
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14
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
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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
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16
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
20
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.
CA 02568512 2006-12-04
17
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 2 1 2 1 2
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
CA 02568512 2006-12-04
18
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:
CA 02568512 2006-12-04
19
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
CA 02568512 2006-12-04
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:
CA 02568512 2006-12-04
21
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.
CA 02568512 2006-12-04
22
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
CA 02568512 2006-12-04
23
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
CA 02568512 2006-12-04
24
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.
CA 02568512 2006-12-04
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.
CA 02568512 2006-12-04
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