U.S. patent application number 10/295345 was filed with the patent office on 2003-04-10 for method for sex determination of mammalian offspring.
This patent application is currently assigned to Vicam, L.P.. Invention is credited to Basker, Christopher J., Benjamin, Thomas L., George, Susan, Kohn, Barbara, Livingston, David.
Application Number | 20030068654 10/295345 |
Document ID | / |
Family ID | 25388608 |
Filed Date | 2003-04-10 |
United States Patent
Application |
20030068654 |
Kind Code |
A1 |
Benjamin, Thomas L. ; et
al. |
April 10, 2003 |
Method for sex determination of mammalian offspring
Abstract
A method for increasing the percentage of mammalian offspring of
either sex which comprises contacting a semen sample with an
antibody specific for the spermatozoa determinative of one sex and
separating said spermatozoa from spermatozoa determinative of the
other sex, said antibody being bound to a non-porous magnetic bead
support having a diameter of 0.1 to 2 microns.
Inventors: |
Benjamin, Thomas L.;
(Cambridge, MA) ; Kohn, Barbara; (Watertown,
MA) ; Basker, Christopher J.; (Mansfield, MA)
; George, Susan; (Chelmsford, MA) ; Livingston,
David; (Brookline, MA) |
Correspondence
Address: |
John R. Van Amsterdam, Ph.D.
Wolf, Greenfield & Sacks, P.C.
600 Atlantic Avenue
Boston
MA
02210
US
|
Assignee: |
Vicam, L.P.
313 Pleasant Street
Watertown
MA
02472
|
Family ID: |
25388608 |
Appl. No.: |
10/295345 |
Filed: |
November 15, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10295345 |
Nov 15, 2002 |
|
|
|
09643839 |
Aug 21, 2000 |
|
|
|
6489092 |
|
|
|
|
09643839 |
Aug 21, 2000 |
|
|
|
09299181 |
Apr 23, 1999 |
|
|
|
6153373 |
|
|
|
|
09299181 |
Apr 23, 1999 |
|
|
|
08886203 |
Jul 1, 1997 |
|
|
|
Current U.S.
Class: |
435/7.2 ;
119/300; 800/21 |
Current CPC
Class: |
C12N 5/0612 20130101;
G01N 33/56966 20130101; G01N 33/54326 20130101; Y10S 530/852
20130101 |
Class at
Publication: |
435/7.2 ; 800/21;
119/300 |
International
Class: |
G01N 033/53; G01N
033/567; A01K 031/19 |
Claims
What is claimed is:
1. A method for separating spermatozoa determinative of one sex
from spermatozoa determinative of the other sex comprising
contacting a semen sample with antibody specific for a spermatozoa
determinative of one sex, and plastic beads, and separating
spermatozoa bound to the antibody and linked to the beads from the
sample.
2. The method of claim 1, wherein the antibody is linked to the
plastic beads through a linking compound.
3. The method of claim 2, wherein the linking compound is an
antibody.
4. The method of claim 3, wherein the linking antibody is IgG.
5. The method of claim 2, wherein the linking compound is protein
A.
6. The method of claim 1, wherein the antibody specific for the
spermatozoa determinative of one sex is IgG.
7. The method of claim 1, wherein the antibody specific for the
spermatozoa determinative of one sex is specific for Y-bearing
sperm.
8. The method of claim 7, wherein the antibody specific for
Y-bearing sperm is a monoclonal antibody.
9. The method of claim 7, wherein the antibody specific for
Y-bearing sperm is a polyclonal antibody.
10. The method of claim 7, wherein the antibody specific for
Y-bearing sperm is specific for an H-Y antigen.
11. The method of claim 1, wherein the antibody specific for the
spermatozoa determinative of one sex is specific for X-bearing
sperm.
12. The method of claim 11, wherein the antibody specific for
X-bearing sperm is a monoclonal antibody.
13. The method of claim 11, wherein the antibody specific for
X-bearing sperm is a polyclonal antibody.
14. The method of claim 7, further comprising allowing the beads to
settle, and removing the remaining supernatant rich in X-bearing
sperm.
15. The method of claim 14, wherein the supernatant rich in
X-bearing sperm is subjected to the method at least one additional
time.
16. The method of claim 14, further comprising recovering the beads
to collect Y-bearing sperm.
17. The method of claim 1 1, further comprising allowing the beads
to settle, and removing the remaining supernatant rich in Y-bearing
sperm.
18. The method of claim 17, wherein the supernatant rich in
Y-bearing sperm is subjected to the method at least one additional
time.
19. The method of claim 17, further comprising recovering the beads
to collect X-bearing sperm.
20. A method of artificial insemination comprising, contacting a
semen sample with antibody specific for a spermatozoa determinative
of one sex, and beads, and separating spermatozoa bound to the
antibody and linked to the beads from the sample and artificially
inseminating a mammal with the spermatozoa determinative of one
sex.
21. The method of claim 20, wherein the antibody is linked to the
beads through a linking compound.
22. The method of claim 21, wherein the linking compound is an
antibody.
23. The method of claim 22, wherein the linking antibody is
IgG.
24. The method of claim 21, wherein the linking compound is protein
A.
25. The method of claim 20, wherein the antibody specific for the
spermatozoa determinative of one sex is IgG.
26. The method of claim 20, wherein the antibody specific for the
spermatozoa determinative of one sex is specific for Y-bearing
sperm.
27. The method of claim 26, wherein the antibody specific for
Y-bearing sperm is a monoclonal antibody.
28. The method of claim 26, wherein the antibody specific for
Y-bearing sperm is a polyclonal antibody.
29. The method of claim 26, wherein the antibody specific for
Y-bearing sperm is specific for an H-Y antigen.
30. The method of claim 20, wherein the antibody specific for the
spermatozoa determinative of one sex is specific for X-bearing
sperm.
31. The method of claim 30, wherein the antibody specific for
X-bearing sperm is a monoclonal antibody.
32. The method of claim 30, wherein the antibody specific for
X-bearing sperm is a polyclonal antibody.
33. The method of clam 20, wherein the beads are selected from the
group consisting of plastic beads and magnetic beads.
34. The method of claim 33, wherein the magnetic beads have a
diameter of 0.1 to 2.0 microns.
35. The method of claim 34, wherein the magnetic beads have a
diameter of 0.1 to 0.5 microns.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/643,839, entitled "Method for Sex Determination of Mammalian
Offspring," filed on Aug. 21, 2000, now pending, which is a
divisional of U.S. application Ser. No. 09/299,181, filed on Apr.
23, 1999, now issued as U.S. Pat. No. 6,153,373, which is a
continuation of U.S. application Ser. No. 08/886,203, filed on Jul.
1, 1997, now abandoned. The disclosures of each of these
applications are incorporated by reference herein in their
entirety.
BACKGROUND OF THE INVENTION
[0002] The present invention is directed to a method for increasing
the percentage of mammalian offspring of either sex by contacting a
sperm sample with an antibody specific for one sex, the antibody
being bound to a magnetic bead of a diameter which permits
separation of spermatozoa having sufficient motility to permit
successful insemination and fertility.
[0003] Farmers and other animal husbandry persons have long
recognized the desirability of enhancing the probability of
offspring of a selected sex. Methods have been proposed in the past
for increasing the percentage of X-sperm cells or Y-sperm cells to
thereby achieve a greater chance of achieving male or female
offspring, respectively. Examples of prior research are reviewed,
for example, in Garner, D. L. et al., "An Overview of Separation of
X- and Y-Spermatozoa," Proceedings of the Tenth Technical
Conference on Artificial Insemination and Reproduction (National
Association of Animal Breeders), pp. 87-92 (1984) and Pinkel, D. et
al., "Flow Cytometric Determination of the Proportions of X- and
Y-Chromosome Bearing Sperm In Samples of Purportedly Separated Bull
Sperm," J. Animal Scien., 60, pp. 1303-1307 (1985).
[0004] Previous methods have included, for example, methods based
upon density sedimentation (see, for example, Brandriff, B. F. et
al. "Sex Chromosome Patios Determined by Karyotypic Analysis in
Albumin-Isolated Human Sperm," Fertil. Steril., 46, pp. 678-685
(1986)).
[0005] U.S. Pat. No. 3,687,806 to Van Den Bovenkamp discloses an
immunological method for controlling the sex of mammalian offspring
by use of antibodies which react with either the X- or
Y-chromosomes and utilizing an agglutination step to separate bound
antibodies from unaffected antibodies.
[0006] U.S. Pat. No. 4,191,749 to Bryant discloses a method for
increasing the percentage of mammalian offspring of either sex by
use of a male-specific antibody coupled to a solid-phase
immunoabsorbant material to selectively bind male-determining
spermatozoa, while the female-determining spermatozoa remain
unbound in a supernatant.
[0007] U.S. Pat. No. 5,021,244 to Spaulding discloses a method for
sorting living cells based upon DNA content, particularly sperm
populations to produce subpopulations enriched in X- or Y-sperm by
means of sex-associated membrane proteins and antibodies specific
for such proteins.
[0008] However, these methods often result in insufficient
separation of X- and Y-sperm and often damage the sperm, thereby
reducing its motility and fertility success rate.
SUMMARY OF THE INVENTION
[0009] It is, therefore, an object of the present invention to
provide a method for increasing the probability that mammalian
offspring will be of a specific desired sex.
[0010] It is a further object of the invention to provide a method
for the separation of X- and Y-determinative spermatozoa without
compromising the motility or fertilization rate of the separated
spermatozoa.
[0011] It is another object of the present invention to provide a
method for artificial insemination which permits insemination with
a sperm sample enriched in X- or Y-determinative sperm.
[0012] It is still a further object of the invention to provide a
method for separating X- and Y-determinative sperm by means of
antibodies specific for the selected sperm, bound to magnetic
beads, to permit highly specific separation, so as to provide a
sperm sample enriched in one selected spermatozoa type and
substantially free of the other spermatozoa type.
[0013] These and further objects of the present invention are
achieved by a method which contacts a sperm sample with antibodies
specific to a selected spermatozoa type, the antibodies being bound
to beads, preferably magnetic beads having a diameter of from 0.1
to 2 microns, and subsequently removing the beads whereby the
remaining supernatant may be collected and contains spermatozoa of
only X- or Y-determinative type.
DETAILED DESCRIPTION OF THE INVENTION
[0014] This invention is not limited in its application to the
details of construction and the arrangement of components set forth
in the following description or illustrated in the drawings. The
invention is capable of other embodiments and of being practiced or
of being carried out in various ways. Also, the phraseology and
terminology used herein is for the purpose of description and
should not be regarded as limiting. The use of "including,"
"comprising," or "having," "containing", "involving", and
variations thereof herein, is meant to encompass the items listed
thereafter and equivalents thereof as well as additional items.
[0015] The present invention provides for separation of X- and
Y-bearing sperm which are competent to fertilize using standard AI
techniques. As noted above, prior methods of separation often
compromise the motility and fertilization ability of the sperm, so
that fertilization utilizing such separated sperm requires
complicated techniques such as IVF. The method of the invention can
be utilized for separating X- and Y-sperm from a variety of
mammalian species, including various livestock, such as cattle and
sheep, as well as dogs, cats, horses, swine, and other species. The
process is also applicable to humans.
[0016] By means of the present invention, a sperm sample containing
both X- and Y-sperm can be separated to produce an X- or Y-enriched
sperm subpopulation which is substantially pure with respect to the
desired spermatozoa and substantially free of the other
spermatozoa-type. By "substantially free," we mean that use of a
sample enriched, for example, with X-sperm, when utilized for
artificial insemination, has only a small chance of producing male
offspring, because the sperm sample has less than 20%, preferably
less than 10%, of Y-sperm. Separation of the X- or Y-spermatozoa is
accomplished by use of antibodies which bind to X- or Y-specific
proteins from sperm cells. These antibodies can be of any type of
antibody (including IgG and IgM) and can be either polyclonal
antibodies or monoclonal antibodies. If polyclonal antibodies are
to be used, then such antibodies can be prepared according to per
se known procedures. For example, procedures such as those
described in Hurn, B. A. et al., (1980), Meth. in Enzymology, Ed.
Van Vanakis, H. and Langone, J., pp. 10414 142, can be used.
[0017] If desired, monoclonal antibodies can be utilized and
prepared according to methods which are per se known in the art,
such as those originally authored by Milstein and Kohler, published
in Nature (1975), 256, pp. 495-497. This basic procedure involves
injecting an animal, usually a mouse, with an immunogenic
substance. After suitable time for antibody production to the
immunogen, the mouse is sacrificed. Cells are removed from the
spleen and fused with myeloma cells. Hybridoma cells resulting from
this fusion are able to reproduce in vitro, and each express a
genetic information for one specific antibody. The antibodies
produced from one hybridoma fusion thus will only recognize a
single antigenic determinative of the immunogen.
[0018] Cells cultured from individual hybridoma cells can then be
screened for production of antibodies to the target antigenic
determinant. Those hybridomas positive for the target antigen can
be further screened to identify those having the desired level of
affinity.
[0019] Monoclonal antibodies displaying all of these
characteristics can then be screened using actual assay conditions
to determine if the assay condition alters the antibody binding
characteristics or affinity, and to screen out those with
cross-reactivity to possible contaminating antigens.
[0020] Preferred antibodies are those which are specific for and
bind to Y-sperm, such as antibodies which bind to the H-Y antigen.
Such antibodies can be prepared, for example, by the procedure
described in U.S. Pat. No. 4,680,258 to Hammerling et al.
[0021] As noted above, the antibodies specific for either X- or
Y-spermatozoa are immobilized on beads. These beads can be plastic
beads or magnetic beads. Useful plastic beads are Sepharose 6 MB,
or other beads which are large enough to settle out in a batch
purification process. When plastic beads are utilized, the beads
having antibody bound thereto are mixed in a sperm sample and
allowed to settle to the bottom of the container. This step can be
repeated, if desired, to increase the completeness of separation of
sperm according to sex chromosome.
[0022] If magnetic beads are used, the beads are microspheres of
magnetic particles representing an immobilizing matrix. It has been
found, according to the present invention, that magnetic beads
having a diameter of from 0.1 to 2 microns in diameter are
specifically useful for separating the desired species of
spermatozoa without compromising the motility and fertilization
ability of the spermatozoa. Particularly useful magnetic beads are
described, for example, in U.S. Pat. No. 5,071,076; U.S. Pat. No.
5,108,933; U.S. Pat. No. 4,795,698; and PCT Pat. No. Publication
No. WO91/09678. According to the procedures described in these
patents, beads can be prepared having especially a diameter of 0.1
to 0.5 microns.
[0023] The antibodies are bound to the beads by means of procedures
which are per se known in the art. In general, a linking compound
is attached to the magnetic beads during manufacture of the beads.
On to the beads, an antibody (such as an IgG antibody which is
directed against mouse IgM) is bound by mixing beads at about 1 mg
iron/ml with purified antibody at 1 mg/ml protein. After the
antibody is bound to the beads, the beads are washed so only
attached antibody remains. Additional procedures known to those
skilled in the art are described, for example, in U.S. Pat. No.
4,018,886; U.S. Pat. No. 3,970,518; U.S. Pat. No. 4,855,045; and
U.S. Pat. No. 4,230,685. Protein A is a preferred linking compound
which greatly increases the effectiveness of capture by the
attached antibodies. (Forsgren et al., (1977) J. Immunol. 99:19).
Protein A attaches to the Fc portion of IgG subclass antibodies,
thus extending and presenting the Fab portion of these antibodies.
The resulting correct orientation of the antibodies and extension
away from the particles leads to a very effective interaction
between the bound antibodies and their target.
[0024] The method of attachment of Protein A to magnetic particles
may proceed by any of several processes available through known
scientific literature. In one such procedure, magnetic iron oxide
particles of approximately one micrometer diameter are chemically
derivatized by a reaction, first with 3-aminopropyltriethoxysilane,
then with glutaraldehyde. The derivatized magnetic particles are
then mixed with Protein A resulting in a magnetic particle to which
Protein A is covalently attached. The antibodies are then added to
the Protein A magnetic particles and after a short incubation, the
Protein A-antibody complexes form. (Weetall, H. H. (1976) Meth. In
Enzymol. 44:134-48).
[0025] The following is a specific example of the method according
to the invention.
EXAMPLE 1
[0026] About 10.sup.8 to 10.sup.10 spermatozoa in amount 1-10 ml of
volume are washed with buffered culture medium (Dulbecco's Modified
Eagles Medium) by centrifuging at 1,500 rpm for 10 minutes. The
sperm are then resuspended in 4 ml buffered culture medium after
gently decanting the supernatant. The sperm suspension is then
divided into two vials, and into each vial is added 500 .mu.L of
magnetic beads having bound thereto antibodies for Y-bearing sperm.
The vials are capped and then rotated at 3 rpm, end over end, on a
rotator at room temperature for 30 minutes.
[0027] After rotation, the caps are loosened and placed in a
magnetic capture rack, such as that described in U.S. Pat. No.
5,571,481. After 15 minutes of exposure to the magnetic field, the
supernatant is removed which is rich in X-bearing sperm and
substantially free of Y-bearing sperm. The removed supernatant is
then counted for sperm, and the concentration is adjusted as
necessary for insemination. That sample is then loaded into a straw
or straws for the insemination procedure.
[0028] If desired, more complete separation can be obtained by
repeating, in a serial manner, the magnetic capture procedure on
the supernatant.
[0029] Each of the publications/patents referred to above is hereby
incorporated by reference.
[0030] The invention being thus described, it will be clear that
the same may be varied in many ways. Such variations are not to be
regarded as a departure from the spirit and scope of the invention,
and all such modifications as would be obvious to one skilled in
the art are intended to be included within the scope of the
following claims.
[0031] Having thus described several aspects of at least one
embodiment of this invention, it is to be appreciated various
alterations, modifications, and improvements will readily occur to
those skilled in the art. Such alterations, modifications, and
improvements are intended to be part of this disclosure, and are
intended to be within the spirit and scope of the invention.
Accordingly, the foregoing description and drawings are by way of
example only.
* * * * *