 |
US
Patent & Trademark Office |
| Patent
Number |
4,999,283 |
| Title |
Method
for x and y spermatozoa separation |
| Inventor(s) |
Panayiotis
M. Zavos;
Karl A.Dawson |
| Date
Granted |
March
12, 1991 |
| United States Patent |
4,999,283 |
| Zavos ,
et al. |
March 12, 1991 |
Method for x and y spermatozoa separation
Abstract
A method for separating male and female determining spermatozoa
includes the initial step of exposing freshly ejaculated spermatozoa
in a substantially protein free diluent to an excess concentration
of a monoclonal antibody directed against H-Y antigen that
binds substantially exclusively with male determining spermatozoa.
The method continues with the suspending of the exposed spermatozoa
together with a conjugate of (1) an immunoglobulin G antibody
that binds substantially exclusively to the monoclonal antibody
and (2) an immunoabsorbant substrate in a substantially protein
free diluent. This forms a conjugate/spermatozoa preparation.
The method concludes with the recovering of the separated
male and female determining spermatozoa.
| Inventors: |
Zavos; Panayiotis M.
(Lexington, KY); Dawson; Karl A. (Lexington,
KY) |
| Assignee: |
University of Kentucky Research
Foundation (Lexington, KY) |
| Appl. No.: |
396738 |
| Filed: |
August 18, 1989 |
| Current U.S.
Class: |
435/2; 435/7.21;
436/501; 436/503; 436/514; 436/547; 436/548; 436/811;
436/813; 436/824; 436/906; 530/413 |
| Intern'l Class:
|
A01N 001/02; C12Q
001/00; C12N 005/00 |
| Field of Search:
|
435/7,2,240.1,240.2
436/503,501,510,519,517,518,811,813,824,906 530/387,806,809,413 |
References Cited [Referenced
By]
U.S. Patent Documents
| 3894529 |
Jul., 1975 |
Shrimpton |
435/2. |
| 4083957 |
Apr., 1978 |
Lang |
435/2. |
| 4276139 |
Jul., 1981 |
Lawson |
435/2. |
| 4448767 |
May., 1984 |
Bryant |
435/2. |
| 4511661 |
Apr., 1985 |
Goldberg |
436/503. |
| 4680258 |
Jul., 1987 |
Hammerling et al. |
435/7. |
Other References
"H-Y Antigen: Genetics and Serology",
1977 Immunological Review, Wachtel.
Bennett & Boyse, "Sex Ratio in Progeny of Mice Inseminated
with Sperm Treated with H-Y Antiserum". |
Primary Examiner: Teskin; Robin
Attorney,
Agent or Firm: King & Schickli
Parent Case Text
This is a continuation of application Ser. No. 818,338, filed
Jan. 10, 1986 now abandoned.
Claims
We claim:
1. The method for separating male and female determining spermatozoa
for the purpose of controlling the sex of offspring prior to
conception, comprising the steps of:
exposing the spermatozoa in a protein free diluent to an excess
concentration of a first antibody that binds male determining
spermatozoa;
suspending said exposed spermatozoa together with a conjugate
of a second antibody that binds exclusively to said first antibody
and an immunoabsorbent substrate in a protein free diluent to
form a conjugate/spermatozoa preparation; and
recovering the female determining spermatozoa from the conjugate/spermatozoa
preparation while said male determining spermatozoa are bound
to said immunoabsorbent substrate.
2. The method for separating male and female determining spermatozoa
recited in claim 1, including the additional step of recovering
said male determining spermatozoa from said immunoabsorbent
substrate.
3. The method for separating male and female determining spermatozoa
recited in claim 1, wherein said exposing step includes the
additional step of diluting said spermatozoa to a concentration
of between 10.times.10.sup.6 and 100.times.10.sup.6 active spermatozoa
per milliliter of protein free diluent.
4. The method for separating male and female determining spermatozoa
recited in claim 1, wherein said first antibody is monoclonal
antibody directed against H-Y antigen of said male determining
spermatozoa.
5. The method for separating male and female determining spermatozoa
recited in claim 1, including the steps of recovering said exposed
spermatozoa from said excess concentration of said first antibody
and washing said exposed spermatozoa to remove unbound first
antibody.
6. The method for separating male and female determining spermatozoa
recited in claim 5, wherein said recovering is by centrifugation.
7. The method for separating male and female determining spermatozoa
recited in claim 5, including the step of resuspending said
washed, exposed spermatozoa in a protein free diluent.
8. The method for separating male and female determining spermatozoa
recited in claim 1, including the additional step of incubating
said spermatozoa at approximately 37.degree. C. during said
exposing and suspending steps.
9. The method for separating male and female determining spermatozoa
recited in claim 1, wherein said immunoabsorbent substrate is
agarose beads.
10. The method for separating male and female determining spermatozoa
recited in claim 1, wherein said second antibody is anti-immunoglobulin
antibody capable of binding said first antibody.
11. The method for separating male and female determining spermatozoa
recited in claim 1, wherein said suspending step includes the
additional step of agitating said conjugate/spermatozoa preparation
to prevent the immunoabsorbent substrate from settling.
12. The method for separating male and female determining spermatozoa
recited in claim 1, wherein said female determining spermatozoa
recovering step includes the steps of draining said protein
free diluent and female determining spermatozoa from said immunoabsorbent
substrate and then centrifuging said protein free diluent and
female determining spermatozoa to separate said female determining
spermatozoa from said diluent.
13. The method for separating male and female determining spermatozoa
recited in claim 1, including the additional step of filtering
the separated female determining spermatozoa so as to substantially
remove dead spermatozoa and increase overall efficacy.
14. The method for separating male and female determining spermatozoa
recited in claim 13, wherein said filtering step includes the
step of passing the separated female determining spermatozoa
through an unbound sephadex bead column.
15. The method for separating male and female determining spermatozoa
recited in claim 2, including the additional step of filtering
the separated male determining spermatozoa so as to substantially
remove dead spermatozoa and increase overall efficacy.
16. The method for separating male and female spermatozoa recited
in claim 2, including the additional steps of separately filtering
the separated male and female determining spermatozoa so as
to remove dead spermatozoa and increase overall efficacy and
separately collecting said separated male and female determining
spermatozoa in a protein containing media following filtering,
said protein containing media serving to support the separated
spermatozoa until subsequent use in artificial insemination.
17. The method for separating male and female determining spermatozoa
recited in claim 1, wherein said protein free diluent is a sodium
citrate buffer.
Description
TECHNICAL FIELD
This invention relates to a method for separating male and female
determining spermatozoa for the purpose of selectively increasing
the probability of producing offspring of either sex.
BACKGROUND OF THE INVENTION
The ability to select the sex of offspring produced in fertilization
has long been recognized as having tremendous commercial potential
and application. An excellent example of this may be found in
the field of animal husbandry.
Dairy farm operations require the female of the species, cows,
for milk production. Males of the species, bulls, are only important
for breeding purposes. Thus, it is clear that any method that
safely and effectively increases the production of female offspring
versus male offspring would greatly benefit dairy cattle breeders
and farmers and, therefore, be very valuable. Such a method
would allow more effective and efficient farm operation, save
money and increase farm milk production.
Of course, it should be recognized that humans may also benefit
from such a method of selecting the sex of offspring. Parents
could very simply satisfy their desires to have a son or daughter.
This allows improved family planning, may reduce family tension
and also limit family size for the benefit of all involved.
Numerous attempts have been made in the past to separate x-(male
determining) and y-(female determining) bearing spermatozoa
in order to determine the sex of the offspring before conception.
The methods used include centrifugation, filtration, electrophoresis,
immunological techniques and exposure of spermatozoa to alkaline
or acidic environments.
Mechanical methods of separating male and female determining
spermatozoa, based on the difference in density between the
two types, are disadvantageously characterized by reduced spermatozoa
survivability and by reduced viability of the surviving spermatozoa.
Immunological methods of spermatozoa separation do not suffer
as dramatically from these disadvantages.
U.S. pat. no. 4,448,767 to Bryant discloses two methods of immunological
spermatozoa separation. The first method is a single antibody
separation system wherein male specific antibody is bound to
sephadex beads placed in a column. Native or unseparated spermatozoa
is then added to the column. The male determining spermatozoa
become bound to the male specific antibody/Sephadex bead conjugates
as the female spermatozoa are eluted from the column. The male
determining spermatozoa are then recovered separately.
The second method is a dual antibody separation system wherein
native sperm is treated with male specific antibody. A second
antibody, capable of specifically binding the male specific
antibody is coupled to Sephadex beads placed in a column. The
male specific antibody treated spermatozoa is then added to
the column. The male determining spermatozoa become bound to
the second antibody/Sephadex bead conjugates through the male
specific antibody as the female spermatozoa are eluted from
the column. The male determining spermatozoa are then recovered
separately as in the first method.
While the two Bryant methods do serve to increase the percentage
of mammalian offspring of either sex as desired, the methods
are not without their disadvantages and, therefore, may be improved.
Specifically, Bryant discloses a complicated technique of animal
immunization with histocompatability (H-Y) antigen and multiple
repeated absorptions through female spleen cells to separate
and purify the male specific antibody used in the Bryant method.
Despite this difficult and tedious technique, it should be appreciated
that the resulting antibody is still not completely binding
specific to the H-Y antigen found only in male determining and
not female determining spermatozoa. Thus, it should be appreciated
that a portion of the resulting male specific antibody used
in Bryant could disadvantageously bind through antigens other
than the y-antigen to some female determining spermatozoa. As
a result, some female determining spermatozoa are bound to the
Sephadex beads in the Bryant columns and eluted with the male
determining spermatozoa fraction. Thus, it is clear that the
highest levels of effective separation are not attainable with
the Bryant method.
Bryant also disadvantageously requires the packing of a column
with antibody/Sephadex bead conjugate. The column must be packed
properly so that the rate of flow or spermatozoa elution is
within a range allowing complete immunoreaction between the
spermatozoa and the antibody of the antibody/bead conjugates.
Too fast a flow rate means incomplete immunoreaction and separation
and, therefore, the presence of male determining spermatozoa
in the eluted female determining spermatozoa fraction. The proper
packing of the column requires a great deal of preparation time
and large quantities of expensive eluant or filtration solution
are required to complete the separation and recover the majority
of the spermatozoa from the column. This last consideration
is particularly important when you consider that different eluants
are typically required for each species of spermatozoa being
separated. Thus, large quantities of a number of different and
sometimes expensive eluants must be stored at the separation
lab.
An additional problem in Bryant relates to the utilization of
protein containing media for spermatozoa dilution prior to exposure
of the spermatozoa to the antibody. Disadvantageously, the protein
in the media retards the cross reactivity of the spermatozoa
and the antibody. It is believed that the existing protein fraction
in the spermatozoa diluent coats the spermatozoa covering the
histocompatability H-Y antigen sites on the male determining
spermatozoa and preventing antibody/antigen binding. This causes
incomplete separation as, with either method in Bryant, coated
male determining spermatozoa are eluted with the female determining
spermatozoa.
From the above, it should be appreciated that a need exists
for an improved method of immunologic spermatozoa separation.
SUMMARY OF THE INVENTION
It is, accordingly, one object of the present invention to provide
a method of spermatozoa separation, based on sex determining
properties, overcoming the above-described limitations and disadvantages
of the prior art.
A further object of the present invention is to provide an immunological
method of spermatozoa separation that is less complicated and
more effective than methods of the prior art.
Still another object of the present invention is to effectively
separate female and male determining spermatozoa for subsequent
fertilization to produce offspring of a selected sex with an
increased probability of success.
A still further object of the present invention is to provide
a method of separating male and female determining spermatozoa
wherein the separated spermatozoa have improved efficacy and
viability.
Other objects and advantages of the present invention will become
apparent as the description thereof proceeds.
In satisfaction of the foregoing objects and advantages, there
is provided by this invention a method for separating male and
female determining spermatozoa for the purpose of selectively
increasing the probability of producing offspring of either
sex during subsequent fertilization.
The method includes the step of exposing unseparated, native
spermatozoa to an excess concentration of a first antibody that
binds substantially exclusively with male determining spermatozoa
through the H-Y surface antigen. A second antibody covalently
bound to an immunoabsorbent substrate, such as agarose beads,
binds substantially exclusively to the first antibody. The second
antibody/immunoabsorbent conjugates are suspended together with
the spermatozoa previously exposed to the first antibody in
a protein-free diluent appropriate for the spermatozoa being
separated. For example, bovine spermatozoa may be suspended
in a solution of sodium citrate having a pH of approximately
7.2 and an osmotic pressure of approximately 325 mOsm. Following
immunoreaction in suspension, the female determining spermatozoa
are recovered while the male determining spermatozoa are bound
to the immunoabsorbent substrate through the first and second
antibodies. Lastly, if desired, the male determining spermatozoa
are recovered from the substrate.
More specifically, the native spermatozoa are diluted in a protein-free
diluent to a concentration of between 10.times.10.sup.6 to 100.times.10.sup.6
active spermatozoa per milliliter depending on the species being
separated. Since the diluent is protein free, there is no coating
of the surface of the spermatozoa and, more particularly, the
H-Y antigen sites on the male determining spermatozoa. Thus,
more effective and complete immunoreaction is assured between
the first antibody and the male determining spermatozoa.
Preferably, the first antibody is a male specific monoclonal
antibody directed against H-Y antigen on the male determining
spermatozoa. These monoclonal antibodies, available from the
National Institute of Health, are prepared by outgrowth of primary
hybridomas and recovery of the cultured medium. By using monoclonal
antibodies of this type, no other antibodies are present in
the antisera to adversely effect or block the cross reactivity
of the antibodies with the H-Y antigen on the male determining
spermatozoa or to bind any other antigen such as may be found
on the female determining spermatozoa. Optimum selective binding
is therefore assured.
Following the exposing step, the exposed spermatozoa are separated
from the excess concentration of the first antibody. This separation
may be performed by gentle centrifugation that does not harm
the spermatozoa. The spermatozoa are then washed to remove unreacted
first antibody and resuspended in fresh protein free diluent.
Preferably, the second antibody coupled to the agarose beads
is an anti-immunoglobulin antibody specific for binding with
the male-specific, first antibody bound to the exposed male
spermatozoa. The beads are added to the exposed spermatozoa
in the protein free diluent and the bead/spermatozoa preparation
is agitated to prevent the beads from settling and thereby assure
complete immunoreaction between the two antibodies. Again, the
protein free diluent prevents antigen "coating" that can adversely
effect the cross reactivity of the second antibody with the
first antibody and thereby reduce separation efficiency.
Following the suspending step, the female determining spermatozoa
may be recovered by draining the bead/spermatozoa preparation
solution from the agarose beads now bound to the male determining
spermatozoa. The drained solution is then gently centrifuged
and the undamaged female determining spermatozoa are recovered
in a pellet. The female determining spermatozoa may then be
resuspended in fresh protein free diluent and filtered so as
to substantially remove all dead and weak spermatozoa and increase
the efficacy of the collected fraction. This can done by adding
the spermatozoa to a column of sephadex beads. The viable spermatozoa
are then eluted from the column and collected in a protein containing
medium for freezing and/or subsequent use in artificial insemination.
The male determining spermatozoa are then recovered from the
agarose beads. There are many methods known in the art for such
recovery. The methods include competitive inhibition using a
solution of male specific antibody, enzymatic digestion of the
beads and alteration of the pH or salt concentration of the
diluent.
The male determining spermatozoa may then be filtered and collected
as described above so as to substantially remove all dead spermatozoa
and increase the overall efficacy of the fraction. Specifically,
viable spermatozoa pass through the column at a substantially
faster rate than dead spermatozoa and, therefore, excellent
separation results are attainable.
Still other objects of the present invention will become readily
apparent to those skilled in this art from the following description
wherein there is shown and described a preferred embodiment
of this invention, simply by way of illustration of one of the
modes best suited to carry out the invention. As it will be
realized, the invention is capable of other different embodiments
and, its several details are capable of modifications in various,
obvious aspects all without departing from the invention. Accordingly,
the drawings and descriptions will be regarded as illustrative
in nature and not as restrictive.
BRIEF DESCRIPTION OF THE DRAWING
The accompanying drawing incorporated in and forming a part
of the specification, illustrates several aspects of the present
invention, and together with the description serves to explain
the principles of the invention. In the drawing:
FIG. 1 is a flow sheet illustrating the method of the present
invention;
FIG. 2 is a schematical representation showing the monoclonal
antibody directed against H-Y antigen binding with the male
determining spermatozoa; and
FIG. 3 is a schematical representation showing the binding of
the second antibody/bead conjugates to the exposed and monoclonal
H-Y antibody bound male determining spermatozoa.
DETAILED DESCRIPTION OF THE INVENTION
As shown in FIG. 1, the first step 12 of the method 10 of the
present invention involves exposing live, native spermatozoa
to an excess concentration of an antibody that binds substantially
exclusively to male determining spermatozoa. Specifically, freshly
ejaculated spermatozoa are diluted to a concentration of between
10.times.10.sup.6 and 100.times.10.sup.6 active spermatozoa
per milliliter. The diluent used varies depending on the species
of the spermatozoa being separated. Specific examples of diluents
include sodium citrate solution for bovine spermatozoa, creme
gel buffer for equine spermatozoa and test buffer for human
spermatozoa. The diluent used, however, should be free of protein
as any protein in the diluent tends to coat the spermatozoa
and block the H-Y antigen binding sites on the surface of the
male determining spermatozoa. Thus, by substantially eliminating
this blocking the method of the present invention provides conditions
for optimal cross reactivity between the antibody and antigen
for the best possible immunoseparation results.
An excess concentration of the male specific antibody is contained
in or added to the diluent and spermatozoa. The male specific
antibody is a monoclonal anti-H-Y antibody (immunoglobulin G)
prepared by outgrowth of primary hybridomas and recovered from
culture medium by stepwise precipitation and dialysis. Such
a monoclonal antibody is produced by the mouse lymphocyte hybrid
cell line B6YD1O available from the National Institute of Health.
As shown in FIG. 2, only male determining spermatozoa 14 express
the H-Y antigen 16 on their surface. The female determining
spermatozoa 18 do not express the H-Y antigen believed associated
with the Y or male determining chromosome. Since the monoclonal
antibody 20 is absolutely specific for the H-Y antigen, it binds
only to the male determining spermatozoa 14 and not any of the
female determining spermatozoa 18.
Following a short incubation period for immunoreaction of, for
example, 45 minutes, the spermatozoa/monoclonal H-Y antibody
dilution is centrifuged in step 22 to remove (note FIG. 1) and
collect the exposed spermatozoa from the diluent. The exposed
spermatozoa are then washed (also step 22) using fresh, pure
protein free diluent in order to remove unbound monoclonal antibody.
After washing, the exposed spermatozoa are resuspended in an
equal volume of protein free diluent (as used during the exposing
step) substantially without the presence of unbound H-Y antibody.
Immediately thereafter, antibody/immunoabsorbent conjugates
are added to the dilution so as to form a conjugate/spermatozoa
suspension preparation in step 24. As shown in FIG. 3, the antibody
26 coupled to the immunoabsorbent material or agarose beads
28 binds immunoglobulin G and, therefore, binds the monoclonal
anti-H-Y antibody 20 already bound to the male determining spermatozoa
14. Again, just as with the first antibody 20, the female determining
spermatozoa 18 do not immunoreact with the immunoglobulin G
antibody 26 bound to the beads 28.
In order for the second, immunoglobulin G antibody 26, to properly
react with the monoclonal H-Y antibody 20, the immunoglobulin
G antibody must be species specific for the monoclonal antibody.
Thus, if spermatozoa are exposed to monoclonal H-Y antibody
produced by mice lymphocyte cells as described above, the second
antibody bound to the conjugate must be anti-mouse immunoglobulin
G. Such a conjugate of anti-mouse immunoglobulin G and agarose
beads is available from Sigma Corporation.
The anti-immunoglobulin G/agarose bead conjugate is added to
the diluted spermatozoa at an approximate concentration of 0.4
milliliters of conjugate beads per milliliter of diluted spermatozoa
(assuming a minimum of 0.4 milligrams of mouse immunoglobulin
G may be bound and eluted from each milliliter of antibody-bound
beads). The conjugate/spermatozoa preparation is incubated at
approximately 37.degree. C. and agitated to prevent the beads
from settling and thereby maintain the beads and spermatozoa
in physical contact for complete immunoreaction. During this
time, the anti-immunoglobulin G antibody of the conjugate binds
the monoclonal H-Y antibody bound to the male determining spermatozoa
as shown in FIG. 3. Thus, the antibody/bead conjugate agglutinates
the monoclonal antibody/male determining spermatozoa complexes.
Following immunoreaction and agglutination, the female determining
spermatozoa are recovered at step 30. This step may be completed
by draining the conjugate/spermatozoa preparation solution from
the antibody/agarose bead conjugates now bound to the male determining
spermatozoa. Since the female determining spermatozoa do not
bind to the beads (note FIG. 3), the female determining spermatozoa
are contained in this drained solution. The solution is then
gently centrifuged to collect the female determining spermatozoa
in a pellet at the bottom of the centrifuge tube without significantly
adversely affecting spermatozoa viability.
A Sephadex filtering column is then prepared as is known in
the art. The pelleted female determining spermatozoa are then
resuspended in a diluent and added in step 32 to the filtering
column. Viable spermatozoa pass through or are eluted from the
column at a substantially faster rate than dead or weak spermatozoa
and, therefore, excellent filtering results are obtainable.
The spermatozoa eluted from the column may be collected in a
protein containing medium for maintaining the spermatozoa for
freezing and/or subsequent use in artificial insemination. An
example of such a protein containing medium is the protein free
diluent used above during the exposing and suspending steps
mixed with egg yolk, 20% by volume.
The male determining spermatozoa are also recovered at step
34 from the agarose beads. There are many methods known in the
art for such recovery. The methods include competitive inhibition
using a solution of male specific antibody, enzymatic digestion
of the beads, and alteration of the pH or salt concentration
of the diluent. Following recovery, the male determining spermatozoa
are filtered in step 36 through a sephadex column in the same
manner as the female determining spermatozoa so as to increase
the efficacy of the male determining spermatozoa fraction or
sample.
The following examples are presented to further illustrate the
invention, but it is not to be considered as limited thereto.
EXAMPLE 1
HY antibody is obtained by outgrowth of a monoclonal hybridoma
cell culture (American Type Culture Collection, Number, B6YD10).
The cell culture is grown in RPMI 1640 medium with L-Glutamine
(M.A. Bioproducts) containing 200 Units of Gentomycin per ml
and supplemented with 20% fetal bovine serum. The growth environment
is an incubator gassed via 5% CO.sub.2 in air and maintained
at 37.degree. C. Supernatant is harvested at 48-hour intervals
and frozen at -20.degree. C. environment until processing. The
antibody (globulin fraction) is collected from the supernatant
by precipitation with 50% saturated ammonium sulfate (3.times.).
The ammonium sulfate is then removed via dialysis against sodium
citrate diluent (SCD). During this process, globulin fractions
are concentrated into a volume which is one tenth of the original
culture fluid collected. The recovered globulin fractions are
aliquoted into 1 ml fractions, frozen, and maintained at -70.degree.
C. environment until use.
EXAMPLE 2
Bovine spermatozoa are collected by any appropriate method known
in the art. The semen is collected directly into a prewarmed
vessel at 37.degree. C. and maintained at this temperature until
dilution 10 to 15 minutes later. The freshly ejaculated bovine
spermatozoa are diluted to 40.times.10.sup.6 active spermatozoa
per ml by adding an appropriate quantity of physiologically
balanced sodium citrate solution at 325 m Osm/liter, pH 7.2,
and free of protein. An excess concentration of 1:200 monoclonal
antibody directed against HY antigen as prepared in Example
1 is then added to the diluted spermatozoa. The spermatozoa
preparation is then incubated for a period of 45 minutes at
37.degree. C. The preparation is then centrifuged for 10 minutes
at 1,000 g and the spermatozoa collected in a pellet. The spermatozoa
pellet is then resuspended in an equal volume of sodium citrate
solution without the HY antibody. Immediately thereafter, 0.4
ml of agarose beads covalently bound with anti-mouse IgG (available
from Sigma Chemical Company, St. Louis, Mo.) are added per ml
of diluted spermatozoa. This spermatozoa-agarose bead suspension
is then incubated for 30 minutes at 37.degree. C. Since the
beads settle to the bottom of the suspension if allowed to set
for five minutes or longer, the suspension is agitated or mixed
every 10 minutes to keep the sperm and beads in physical contact.
At the end of the 30 minute incubation period, the sperm fraction
is collected by decanting or draining the supernatant. Following
collection, the sperm fraction is transferred and layered on
top of a Sephadex sperm filtration column and filtered.
Actually, the column consists of three disposable plastic syringes
each 1 cm in diameter and 6.5 cm high. A pad of Johns-Manville
Micro-Fiber, code 112, No. 475 glass, is prepared by taking
thin sheets of 3 to 4 mg fiber and folding the long fibers under
to make a circle of approximately 1 cm in diameter. A pad is
then gently pushed to the bottom of each syringe using a Pasteur
pipet until 1 to 2 mm protrudes through the hole in the syringe.
The fibers are inspected so that no holes should be evident
in the syringe. Twenty g of Sephadex G 15 (40-120 particle size)
are added to 100 ml of diluent, 34 g/liter, 320 mOsm osmotic
pressure. The sephadex is then allowed to swell at 5.degree.
C. for 4 hours. Each syringe is filled with sodium citrate solution
to the 2.5 ml level. A volume of 0.8 ml of rapidly mixing Sephadex
slurry is then added to the diluent in each syringe. The Sephadex
is allowed to settle for 10 minutes and then the filter is ready
for use. The Sephadex column should be set at 8 mm height. More
Sephadex may be added, if necessary, until the level reaches
the 8 mm mark. The filters are firmly held in a vertical position
and a beaker is used to catch the filtrate.
Flow rates are adjusted to approximately 0.5-0.7 ml per minute
measured by the 2.5-1.5 ml markings on the syringe-filters.
Fast flow rates indicate insufficient fiberglass or holes in
the fiberglass pads which would allow Sephadex to pass into
the collection vials. Slow flow rates, however, are caused by
too much fiberglass in the pad and may result in insufficient
filtering. The filters are allowed to drain to the 1 ml mark
and then the filter is refilled with the diluent from a wash
bottle, taking care not to disturb the Sephadex surface. The
filters are allowed to drain to the 2.5 ml level and the beaker
is replaced with a 20 ml volumetric flask and a 1 ml semen sample
containing 40.times.10.sup.6 active spermatozoa is added or
layered to the top of the column. All samples are mixed immediately
before sampling to assure accuracy. Adequate time is allowed
for the filter to drain to 1.2 ml mark and then to be refilled
to the top with additional sodium citrate solution.
Male determining spermatozoa are recovered as follows. Agarose
beads recovered following draining of the female determining
spermatozoa are mixed with monoclonal H-Y antibody twice the
original concentration used during the female determining spermatozoa
separation procedure. As it may be noted, the agarose beads
at the end of the separation procedure formed complexes made
out of the antibody/bead conjugate which agglutinated the monoclonal
antibody/male determining spermatozoa. The added H-Y antibody
in excess (2.times. the original concentration) is mixed gently
via agitation to break the bondage of the male spermatozoa and
the agarose bead. As the bondage is broken, the H-Y antibody
then cross reacts with the anti-immunoglobulin G site on the
agarose bead rendering it inactive (inhibited). The method via
which the process of separation of the male determining spermatozoa
form the bead conjugates is otherwise known as competitive inhibition.
The agitation for purposes of separation of the male spermatozoa
may continue for 30 minutes (at 37.degree. C.) or until no visible
physical bondage between the beads and the spermatozoa is evident.
Following this, the agitated solution is allowed to sit for
an additional 30 minutes to allow the beads to settle to the
bottom of the container and then the supernatant is removed
which would contain the freed male spermatozoa (37.degree. C.).
Following recovery the male determining spermatozoa are filtered
in step 36 through a Sephadex column in the same manner as the
female determining spermatozoa and processed either for artificial
insemination or for cryopreservation purposes.
* * * * *
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