WO1991017188A1

WO1991017188A1 - Separation of mammalian semen into fractions enriched either for spermatozoa containing an x chromosome or for spermatozoa contai ning a y chromosome

Info

Publication number: WO1991017188A1
Application number: PCT/AU1991/000171
Authority: WO
Inventors: Kenneth Clifford Reed; Mark Philip Bradley
Original assignee: A.B. Technolody Pty. Limited
Priority date: 1990-05-08
Filing date: 1991-04-29
Publication date: 1991-11-14

Abstract

The present invention relates to a method for the fractionation of semen which comprises contacting semem sample with a specific antibody preparation such that said antibody binds to spermatozoa bearing a Y chromosome, and thereafter separating antidody-spermatozoa complexes from unbound spermatozoa.

Description

SEPARATION Of MAMMALIAN SEMEN INTO FRACTIONS ENRICHED

EITHER FOR SPERMATOZOA CONTAINING AN X CHROMOSOME OR FOR

SPERMATOZOA CONTAINING A Y CHROMOSOME

The present invention relates to the enrichment of semen either for spermatozoa containing an X chromosome or for spermatozoa containing a Y chromosome_/ and is particularly concerned with the pre-determination of sex in domestic livestock species by artificial insemination with such fractionated suspensions of spermatozoa to bias the sex ratio of progeny. Previous publications disclose procedures designed to attain a similar goal (1-4) .

References cited herein are collected at the end of the specification.

In the Australian dairy industry, some 55% of pregnancies result from artificial insemination (approximately 500,000 in total). Given the inherent predisposition of the dairy industry to female progeny, it would be most advantageous if the semen used for artificial insemination produced a biased sex ratio in progeny such that a preponderance of female calves were born. Dairy producers could then select replacement progeny for their stock from a higher proportion of females which possessed desirable genetic traits. In the beef cattle industry the use of artificial insemination is not so prevalent (approximately 1% of pregnancies, equivalent to approximately 100,000 progeny), but pre¬ selection or biasing of progeny sex would allow studs and producers to increase the rate of genetic gain and develop more economical breeding programs. Similarly, in the sheep and goat industries the availability of sex-biased semen would enable producers to select the most desirable progeny for their stock. The pig industry would benefit by being able to produce a higher proportion of female progeny for markets averse to^*"boar taint" in pig meat products and of male progeny for markets which are less sensitive to boar taint where the industry could benefit from the higher growth rate of males.

The present invention concerns the preparation of fractions containing viable spermatozoa, i.e. spermatozoa that have been recovered from an ejaculate of semen of animal or human origin and are capable of fertilizing an oocyte provided by a female of the same species to produce normal progeny. The said fractions contain a disproportionate abundance either of spermatozoa containing an X chromosome or of spermatozoa containing a Y chromosome, as distinct from the usual composition of unfractionated semen or spermatozoa which contain approximately equal proportions of spermatozoa bearing an X chromosome and spermatozoa bearing a Y chromosome. The efficacy of said enrichment procedure is monitored and quantified by the application of simple tests described herein, whereby DNA comprising a portion of the Y chromosome of male animals is detected by hybridization with a similar sequence, isolated by

SUBSTITUTE SHEET molecular cloning, under conditions whereby the formation of hybrid DNA molecules can be quantified, or by amplification of a specific sequence by polymerase chain reaction followed by its detection (5) . The validity of utilizing these tests to assess the degree of said semen enrichment. is established by using analysed semen fractions to inseminate female animals that have been super-ovulated. Embryos resulting from this fertilization are recovered and their chromosomal sex constitution determined as described in a previous disclosure (5) .

According to one aspect a the present invention there is provided a method for the fractionation of semen, which comprises contacting a semen sample with a specific antibody preparation ("anti-MEA" antibody) such that said antibody binds to spermatozoa bearing a Y chromosome, and thereafter separating antibody-spermatozoa complexes from unbound spermatozoa. In another aspect, the invention relates to fractionated semen produced according to the above process and embryos when fertilized by said fractionated semen.

In a still further aspect of the invention, there is provided anti-MEA antibody preparations. The term *anti-MEA" antibody refers to polyclonal or monoclonal antibody or combination of one or more such antibodies capable of binding to the "male enhanced antigen" (MEA) , an antigen expressed on spermatozoa and precursors thereof and on other cell types of male mammals. Preferably, but in no way limiting this invention, the anti-MEA antibodies are polyclonal and are raised in non-mammalian species such as the domestic fowl. The aforementioned antibodies may be labeled with one or more cytotoxic agents to inactivate spermatozoa bound thereto and/or may be bound to or associated with a support such as glass or polystyrene beads, chromatography matrices, or the like.

In a preferred 'embodiment of this invention, the procedure used to fractionate semen utilizes polyclonal antibodies generated in domestic fowls against a synthetic peptide whose sequence has been deduced from the known sequence of a cloned DNA segment isolated from adult mouse testis as cDNA, that is expressed preferentially in the testis of adult male mammals and is known as "male enhanced antigen" or MEA. The antibodies have been shown to interact with a high degree of specificity with certain tissues and cell types of male mammals, and specifically with a proportion of round spermatids and spermatozoa of mammals as described above.

The polyclonal antibodies in this preferred aspect of the invention ("primary antibodies") may be isolated from the egg yolk or from the plasma or serum of domestic fowls, and are allowed to interact with the spermatozoa. The antibodies may or may not be subjected to additional fractionation procedures to isolate a particular class or classes of immunoglobulin.

Spermatozoa which bind the antibodies of this invention may be separated from spermatozoa which do not bind the antibodies by exposure of antibody-treated spermatozoa to a matrix to which is affixed a reagent which binds specifically to the fowl antibody preparation. This is most commonly a second antibody preparation ("secondary antibody") prepared by immunizing a second species, commonly a mammal, with the antibody preparation that has been allowed to interact with the spermatozoa.

The matrix to which the secondary antibody or other reagent is affixed may be a sheet of cellulose or

S cellulose derivative, a plastic tray or individual wells within a plastic tray, microspheres of high molecular weight carbohydrate such as dextran or modified carbohydrate such as agarose, magnetic microspheres, glass microspheres, or other substance to which such reagent may be firmly attached.

The procedure used to separate spermatozoa which bind the primary antibody from those which do not utilizes the nature of the matrix to which the secondary antibody or other reagent is bound. This may comprise a simple washing procedure, column filtration, magnetic attraction, or other common procedure, or a combination thereof.

EXAMPLE 1.

Isolation of a cDNA species encoding "male-enhanced antigen" (MEA)

A preparation of pooled "anti-H-Y" sera that had been generated by-repeated intraperitoneal-immunization of female mice with syngeneic male spleen cells (4,6) was used by Lau et al . (7) to screen a cDNA expression library constructed in the bacteriophage vector lambda gtll (8) from the polyadenylated RNA of adult mouse testis. Three recombinant phage that were so isolated were found to contain identical DNA sequences that include an open reading frame. The DNA inserts of these phage were subsequently used to screen human and mouse cDNA libraries, constructed in the bacteriophage vector lambda gtlO (8) from the polyadenylated RNA of adult testis, by DNA/DNA hybridization.

The recovered cDNA inserts were found to hybridize with an RNA species that is expressed in the testis of a variety of adult mammalian species, including humans and cattle. In mice, at least, transcription occurs preferentially in the adult testis (7) and appears to be maximal in haploid round spermatids.

The cloned mouse cDNA sequence was found to hybridize at high stringency with discrete restriction fragments of genomic DNA isolated from a variety of mammals, including humans and cattle (7) .

Theoretical translation of the cloned mouse cDNA sequence and of the similar human cDNA sequence yields deduced polypeptide sequences known as "male enhanced antigen" (MEA) .

F.XAMPI.E 2. cDNA and deduced polypeptide -sequence* of mouse MEA

Theoretical translation starting from each of two potential initiation codons (ATG) in the cDNA sequence of mouse MEA give rise to peptides of 174 and 164 amino acids, with calculated molecular masses of 18.6 and 18.3 kDa, respectively (7) . The longer sequence was analysed for regions of potential antigenicity (Figure 1⁾ . One such region of potentially high antigenicity is that known as "MEA peptide.c", detailed in Figure 2.

ΓVAMPLE 3.

Immunization of domestic fowlβ with MEA peptide.c and preparation of antiaβr*

MEA peptide.c was synthesized commercially. 1 mg of the peptide was dissolved in 1 ml of PBS and emulsified with 2 ml of Freund's complete adjuvant.

Virgin female White Leghorn chickens and mature male Rhode Island Leghorn chickens were each immunized with 1 mg ₍total) of MEA peptide.c at several sites in the breast muscles of male and female chickens.

Immunized chickens were bled from the wing veins at approximately 10 day intervals. The blood was allowed to clot at room temperature and the serum recovered and centrifuged to remove any contaminating cellular debris. The sera were then aliquoted and stored at -80^βC until used for assay. Re-immunizations with a protocol similar to that described above were carried out as dictated by the results of ELISA assays (refer below) . In many cases α-MEA peptide.c serum titres returned to near control levels some six weeks after initial immunization, necessitating re-immunization.

Figure 3 discloses typical immune responses of male and female domestic fowls to MEA peptide.c.

EXAMPLE 4. Assay of chicken anti-MEA peptide.c sera

A 1 mg/ml stock solution of MEA peptide.c was prepared in standard phosphate-buffered saline (PBS) . 100 μl of this solution was added to 4.9 ml of carbonate buffer to give a final concentration of 20 μg/ml; 50μl of this solution was placed in each well of a Nunc Maxisorp™ Immunoplate and the plate incubated at

37^βC for 2 hr.

The plate was rinsed with ELISA wash buffer and

100 μl of a It BSA solution in PBS was pipetted into each well of the plate. The plate was incubated for a further

1 hr at 37°C then rinsed with wash buffer.

Serial dilutions of the test sera were made in ELISA diluting buffer. These commonly began at 1:40 and extended to a 1:20,000 dilution. Fifty microlitres of each dilution was then placed in wells of the ELISA plate extending across one row/sample. A pre-immune serum sample or a non-immune serum sample was treated identically to serve as a control. The plate was then incubated for 1 hr at 37^βC. On completion the plate was washed three times with ELISA wash buffer.

A 1:1000 dilution of rabbit anti-chicken peroxidase- conjugated serum ⁽Nordic) was made in diluting buffer and 50 μl of this was added to each test well of the ELISA plate. The plate was then incubated for 1 h at 37^βC.

On completion the plate was washed three times with wash buffer and 50 μl of peroxidase substrate (ABTS) was added to each well. The colour reaction was allowed to develop for 20 min at room temperature and the reaction was terminated by the addition of 100 μl of 10% SDS (sodium dodecyl sulphate) .

The developed plate was read in a standard ELISA plate reader at 410 nm.

Coating buffer:

1.5 g Na2C03 2.93 g NaHCθ3 Water to 1 litre; stored at 4^βC.

Wash buffer:

1.07 g Na₂HP04.12H₂0 0.39 g Na_.2PO .2H2O 8.5 g NaCl

50 μl Tween-20 Water to 1 litre.

Sample diluting buffer: Wash buffer as above, containing 0.25% BSA.

SUBSTITUTE SHEET ABTS substrate solution:

Acetate buffer:

0.1 M Na.Acetate (0.68 g/100 ml) 0.05 M NaH₂P04 (0.65 g NaH₂PO₄.2H₂O/100 ml) Adjusted to pH 4.0 with acetic acid

ABTS stock solution:

0.219 g ABTS/10 ml water Stored dark at 4^βC.

H₂0_2: 30% stock solution

Diluted 0.05 ml with 1.75 ml water.

Final substrate buffer: . 2 ml acetate buffer 20 μl H2O2 100 μl ABTS

EXAMPLE 5.

Coupling of anti-chicken antibody to Sepharoae 4B

Rabbit anti-chicken IgG was obtained from Sigma (cat.# C-2288) .

This was coupled to CN-activated Sepharose 4B (Pharmacia cat.# 17-0430-01) at a final concentration of 0.5 mg/ml beads, according to the standard protocol provided by the supplier (Pharmacia) . The resin was finally resuspended in sperm buffer (see below) and stored at 4°C until required.

EXAMPLE 6.

Treatment of bovine sperm with chicken anti-MEA peptide.c serum

All manipulations with semen and sperm were carried out as close to 30°C as possible. Bovine semen was collected with an artificial vagina according to standard procedures. The semen was diluted 2:1 with sperm buffer, washed twice by centrifugation at 400 x g for 8 min and finally resuspended in its original volume in the same buffer.

A suspension of sperm (5 x 10⁷ cells/ml) was incubated with a dilution of chicken anti-MEA peptide.c serum for 1 h at 30^βC (optimal dilutions varied for different serum preparations) . On completion, two volumes of sperm buffer were added to the suspension and it was washed twice by centrifugation at 400 x g for 5 min.

The sperm were resuspended in the original incubation volume with sperm buffer.

Sperm Buffer:

30 mM Tris-HCl, pH 7.2 103 mM NaCl 5 mM KC1 3 mM MgCl2

0.4 mM EDTA 0.1% BSA

EXAMPLE 7. Fractionation of antibody-reacted spexa

All manipulations were carried out as close to 30"C as possible.

The immuno-separation columns were prepared while the sperm were incubating with antiserum. Polyprep™ columns (Bio-Rad) were used for this purpose.

One column was used per 5 x 10⁷ sperm cells/ml. The columns were prepared as:

1 g of SemPrep™ beads (9) , overlaid with

0.5 ml of rabbit anti-chicken-Sepharose 4B resin. The columns were pre-flushed with three column volumes of sperm buffer (30^βC) .

Each column was placed in the top of a 15 ml conical centrifuge tube and the assembly placed in a rack in a 30°C water bath.

The washed anti-MEA peptide.c-reacted sperm were loaded onto the immuno-separation columns and allowed to interact with the column matrix.

Sperm were eluted from the column by flushing with three column volumes of sperm buffer. The sperm which eluted from the column were collected in the centrifuge tube below the column.

At the end of the filtration step, the sperm from all tubes were pooled and concentrated by centrifugation at 400 x g for 5 min.

At this stage an additional washing step was sometimes included (to remove serum components when these were found to cause a. problem by reducing sperm viability) . The cell concentration of the final pooled and washed suspension was determined with a haemocytometer prior to preparation for freezing.

A typical column elution profile is illustrated in Figure 4.

EXAMPLE 8.

Freexing, thawing and motility assay of fractionated bovine sperm

The semen was processed for freezing according to established protocols.

After freezing, post-thaw viability was established by thawing a straw of semen rapidly at 37^βC and taking samples for assessment in a Hamilton-Thorn sperm motility analyser and visually under a phase contrast microscope using warmed microscope slides and a heated microscope stage.

EXAMPLE £>. Assay of fractionated bovine sperm population* for relative Y-chromosomal DNA content by DNA/DNA dot blot hybridization

DNA probe sequences: The DNA sequences used to determine the relative content of Y-chromosomal DNA in a given cell population were a previously disclosed genomic isolate from the sheep Y chromosome, OY11.1, that is- conserved and repeated in the Y chromosome of domestic ruminants and is homologous to an RNA species transcribed in adult bull testis (5), and an isolate of a transcribed genomic interspersed repeated element (cARTY) recovered from an adult bull testis cDNA library, the DNA sequence of which is disclosed in Figure 5.

Preparation of sperm DNA dot blots:

Duplicate samples of untreated and processed sperm containing 2 x 10⁶ cells were placed into microcentrifuge tubes and the sperm were pelleted by centrifugation. It is essential that the untreated control sperm be exposed to the same washes and be suspended in the same buffer as are processed sperm.

To each tube was added 300 μl of 0.4 M NaOH containing 10 mM EDTA. The tubes were heated at 95^βC for 20 min then cooled.

The samples were again mixed, then 250 μl was removed from each and transferred into an individual well of a standard 96-well microtitre tray. Each sample was serially diluted two-fold, to a total number of six (five successive dilutions), by transferring 125 μl into 125 μl of denaturation solution in adjoining wells of the icrotitre plate. Twenty five μl of each dilution of each sample was transferred into a second 96-well microtitre plate in which each well contained 200 μl of denaturation solution.

The entire contents of each well was then filtered discretely onto positive charge-modified nylon (PCMN) membrane ⁽e.g. Zeta-Probe™, Bio-Rad; Hybond™-N⁺,

Amersham) using a dot-blot vacuum filtration manifold (Bio-Rad). In this way 87.5% of each dilution of each sample was fixed onto one membrane (labeled "A": OY11.1) and 12.5% was fixed onto a second membrane (labeled "B": cARTY) .

After filtration the dot-blotted membranes were neutralized by rinsing in 5 x SSC. This process was repeated for paired sets_ of membranes until all samples had been treated and filtered (^wdot-blotted^w) . Care was taken to prevent cross-contamination of DNA between uses of the dot-blot manifold. This was achieved by treatment of the apparatus in 0.25 M HC1 for 10 min and rinsing in water, followed by treatment in 0.4 N NaOH for 10 min and thorough washing in water. This procedure has been found to remove all residual DNA that may have been left on the dot-blot^'apparatus.

Each membrane also contained samples of serially diluted genomic DNA from known male and known female cattle. The concentrations of these controls was such that the first (most concentrated) dot contained 1 μg of DNA. To ensure unequivocal identification of each membrane, the control DNAs were filtered onto a different part of the membrane for each set of samples.

SUBSTITUTE SHEET After all samples had been filtered and the membranes neutralized, the membranes were washed in 5 x SSC, 1% (w/v) SDS at 68^βC for 2 h.

20 x SSC:

3 M NaCl

0.3 M Na3.citrate, pH 7.0

Radioactive labeling of DNA probes by nick translation; All reagents and incubation mixes were kept on ice/water.

A fresh working dilution of DNase I (200 ng/ml) was prepared by successive diluti'ons of 1/100 and 1/100 of the 2 mg/ml stock into sterile deionized water. The following incubation mix was prepared:

18.0 μl probe DNA (0.2 μg) + deionized water 3.5 μl 10 x NTB 2.0 μl 0.1 M DTT 3.5 μl 1 mg/ml BSA 1.0 μl 0.1 mM dNTP mix

2.0 μl 10 mCi/ml [α-³²P]dCTP The solution was mixed and centrifuged briefly and to it was added:

5.0 μl 200 ng/ml DNase I The solution was again mixed and centrifuged briefly, then incubated at 14^βC for 15 min. During this pre- incubation, a trace was removed for PEI-cellulose chromatography. To it was then added: 1.0 μl Pol I (2.5-5 units/μl) The solution was mixed, centrifuged briefly and incubated at 14^βC for 15 min. Reaction was stopped by adding:

4.0 μl of 10 x stop solution and a second minute sample removed for PEI-cellulose chromatography. Immediately before using the labeled probe for hybridization, it was fragmented and denatured by adding:

3.0 μl of 4 M HC1 mixed and stood at room temperature for 10 min, then was added: 9.0 μl.of 4 M NaOH

The solution was mixed and allowed to stand at room temperature for (at least) 10 min.

Six μl of 4 M acetic acid was added to the hybridization solution and mixed; the probe solution was then added to the hybridization solution with thorough mixing.

PET-cβllulosβ thin layer chromatography:

During pre-incubation, a sample (approximately 0.1 μl, although neither the volume nor its accuracy is important) was removed and spotted onto an origin mark on a 4 x 8 cm (approximately) sheet of PEI-cellulose (polyethyleneimine-cellulose coated plastic sheets for thin layer chromatography; Merck #5579) . On completion of the labeling reaction, a second small sample was removed and spotted adjacent to the first. The samples were air- dried, chromatographed for 10-15 min in a foil-covered beaker containing approximately 2 mm (depth) of 0.75 M KH₂P<_₄ adjusted to pH 3.5 with orthophosphoric acid, then wrapped in plastic film and autoradiographed for 10-30 min (Figure 6) .

Labeling efficiency could be estimated by visual inspection of the autoradiograph or by scanning the chromatogram with a hand-held radiation monitor. A quantitative measure of the relative levels of radioactivity in dCMP, dCTP, and DNA could be obtained by scintillation or Cerenkov counting of appropriate areas scraped from the chromatogram, using the autoradiograph (Figure 6) as a template.

10 x NTB:

0.5 M Tris-HCl, pH 7.5 0.075 M magnesium acetate Sterilized by Millipore filtration (0.22 μ) and stored in 100 μl aliquots at -20^βC.

0.1 M DTT:

0.1 M dithiothreitol sterilized by Millipore filtration (0.22 μ) and stored in 20 μl aliquots at -20^βC.

1 mg/ml BSA:

20 mg/ml stock of nuclease-free bovine serum albumin (Boehringer) diluted in sterile deionized water under sterile conditions and stored in 50 μl aliquots at -20^βC.

0.1 mM dNTP Mix :

1 μl 20 mM dATP 1 μl 20 mM dGTP

1 μl 20 mM dTTP 188 μl TE (10 mM Tris-HCl, pH 7.0, 1 mM EDTA) Prepared from sterile stock 20 mM solutions under sterile conditions and stored in 20 μl aliquots at -70°C, with one working aliquot at -20°C. [α-32p] CTP:

10 mCi/ml [α-³ P]dCTP Purchased as an aqueous solution (approximately 3,000 Ci/mmol, e.g. Amersham) and stored at -20^βC.

DNase I:

Deoxyribonuclease I from bovine pancreas. Type II from Sigma (D 4527). Five mg was dissolved in 2.5 ml of 0.15 M NaCl, 50% glycerol using sterile solutions under sterile conditions and stored in 50 μl aliquots at -70°C, with one working aliquot at -20^βC.

Pol I:

DNA polymerase I from E. colj. endonuclease-free, from Boehringer (642711), approximately 5,000 units/ml; stored at -20°C.

10 x Stop Solution:

0.25 ml 0.5 M EDTA 0.25 ml 20% SDS

0.5 ml distilled water

Hybridization of Dot Blots: Hybridization Solution: Hybridization solution was prepared as follows (10) and pre-equilibrated at 68°C: 2 x PE

7% (w/v) SDS 1% (w/v) BSA (PE ^« 0.133 M sodium phosphate, pH 6.9, 1 mM EDTA; it was prepared as 10 x stock) . Standard Hybridization Procedure:

A sheet of filter paper (e.g. Schleicher & Schull #589; cut slightly larger than the PCMN membrane) was placed onto a sheet of heat-sealable plastic. The PCMN membrane was pre-wetted thoroughly in hybridization solution (minus probe) and laid squarely on the filter paper. A second sheet of filter paper was placed onto the pre-wetted PCMN membrane and the sandwich covered with a second sheet of plastic. The plastic was heat-sealed on three sides adjacent to the filters, leaving one long unsealed flap. Hybridization solution containing probe (approximately 10- ml for 100 cm² membrane) was added to the bag and a large pipette was used to roll out any bubbles that may have been trapped between the filters. The flap was sealed adjacent to the filters, excess plastic was trimmed off and the bag was incubated, at 68^βC _for 4-16 h,

High Sensitivity Hybridization Procedure:

A piece of thin filter paper (e.g. Schleicher & Schull #589; cut slightly larger than the PCMN membrane) was placed on a sheet of heat-sealable plastic and onto it was pipetted half of the hybridization solution containing probe (this required approximately 1.2 ml/100 cm²; the filter paper did not need to be uniformly wetted) . The PCMN membrane was thoroughly pre-wetted in hybridization solution (minus probe) and laid squarely on the filter paper (two membranes could be hybridized simultaneously by placing them back-to-back, i.e_n DNA surfaces outermost) . A second sheet of thin filter paper was placed onto the PCMN membrane and onto it was pipetted the remaining hybridization solution containing probe. The sandwich was covered with a second sheet of plastic and heat-sealed on three sides adjacent to the edges. Before sealing the last side, care was taken to spread the solution uniformly and remove bubbles trapped between the filters by rolling a large pipette back and forth across the bag (bubbles at the sides of the filters are not important) . Excess plastic was trimmed off and the bag was incubated at 68^βC for- -16 h.

Hybridization with Multiple Membranes:

Hybridization of multiple membranes with the same probe was done most conveniently in a single bag or box by interleaving the pre-wetted PCMN membranes (placed back-to-back in pairs) with sheets of filter paper saturated with probe solution. When a bag was used, membranes and filters were stacked successively as above. Alternatively, hybridization solution containing probe was placed in a suitable box and sheets of dry filter paper and PCMN membrane (pre- wetted with hybridization solution minus probe) were added successively, taking care to exclude bubbles between sheets. A sheet of filter paper was placed on top of the stack, and the container was sealed and incubated at 68^βC for 4-16 h.

Washing and Autoradiography:

The hybridized membrane(s) were removed from the bag(s) and rinsed briefly in 2 x SSC. They were washed in 2 x SSC, 0.1% SDS at room temperature for 15 min with vigorous agitation; the membranes were virtually free of background by this stage. They were then washed in pre-heated 0.5 x SSC, 1% SDS at 68°C for 10 min with vigorous agitation then rinsed briefly in 0.5 x SSC, 0.1% SDS at room temperature, blotted lightly and wrapped in GladWrap. The membranes were exposed to X-ray film (Fuji RX) ; for maximum sensitivity, Kodak XAR-5 film was used with DuPont Quanta III intensifying screen (s) at -70^βC.

Typical autoradiographs of fractionated bovine sperm dot blots hybridized as described with OY11.1 and cARTY are shown in Figure 7.

Quantitation of Dot Blots:

After autoradiography the segment of hybridized membrane containing each dot was cut out (using the autoradiograph as a template) and counted individually by liquid scintillation spectrometry to 5% accuracy, for which each piece of membrane was placed in a plastic scintillation mini-vial with 2 ml of scintillation fluid. This was mandatory, since the procedure for sperm fractionation described above results in enrichment for X-bearing sperm, not complete separation. It was not possible to assess the degree of enrichment simply by visual inspection of an autoradiograph. The autoradiograph itself could be scanned with an ELISA plate reader but this was unsatisfactory, both empirically and theoretically (signal intensity is not linearly proportional to the amount of radiation giving rise to the signal, i.e. X-ray film does not have a linear response) . For each paired set of data, the ratio of radioactivity (corrected for background) resulting from hybridization with OY11.1 to that obtained with cARTY was calculated and the mean of the six dilution replicates for each sample was determined. Comparison of that mean ratio

SUBSTITUTE SHEET for each treated sample with the control (untreated) mean ratio gave the degree of enrichment relative to untreated (assumed to be 50%) . Typical results of dot blot quantitation are illustrated in Figure 8.

An accurate control was essential for each preparation. The amount of radioactivity resulting from hybridization to each dot is subject to a wide range of variables such that no comparison is possible between raw data from different experiments.

Scintillation fluid:

15 g butyl-PBD 1,500 ml toluene 1,000 ml 2-methoxyethanol

EXAMPLE 10.

Validation of DNA/DNA dot blot hybridization procedure*

Using mixtures of male and female bovine lymphocytes: Whole blood was collected from male and female cattle by venipuncture into Vacutainers™ containing Li-EDTA. Red blood cells were lysed by the addition of 5-10 volumes of ACE to whole blood and allowing the mixture to stand at room temperature for 10 minutes. The peripheral blood lymphocytes were then collected by centrifugation and cell concentration determined with a haemocytometer. Each sample of male and female cells was adjusted to a final concentration of 2 x 10⁶ cells/ml.

Male and female cells were mixed in defined ratios such that each sample contained a total of 5 x 10⁵ cells/tube in the following proportions:

The contents of each tube were mixed thoroughly and the cells were pelleted by centrifugation. The supernatants were then removed and the samples were processed for dot blot analysis as described above for the preparation of sperm DNA dot blots (Figure 9) .

ACE:

0.75 mM KH2PO₄, pH 7.0 150 mM NH₄CI

2.5 mM Na3.EDTA

Usinp female bovine lymphocytes mixed with bovine spermatozoa:

Frozen semen was obtained in straws from an artificial breeding company, thawed and washed free of cryoprotectant by dilution in PBS. The cells were collected by centrifugation.and counted with a haemocytometer. The sperm suspension was then adjusted to a final concentration of 4 x 10⁶ cells/ml (i.e. twice the concentration used for lymphocytes since sperm are haploid and hence contain half the amount of DNA per cell as do lymphocytes) .

The sperm and female lymphocytes were mixed in defined ratios such that each sample contained a final cell number of 5 x 10⁵ cells/tube in the following proportions:

SUBSTITUTE SHEET

The contents of each tube were mixed thoroughly and the cells were pelleted by centrifugation. The supernatants were then removed and the samples were processed for dot blot analysis as described above for the preparation of sperm DNA dot blots (Figure 10) .

EXAMPLE 11.

Asaay for sex of bovine einbryoa resulting from artificial insemination with fractionated spera Statistical validation of sperm enrichment by analysis of the sex of fertilized embryos, foetuses or progeny requires the analysis of substantial numbers, e.g. for statistical validation of 70% enrichment at the 5% confidence level approximately 125 fertilized progeny must be sexed; for statistical validation of 80% enrichment at the 5% confidence level approximately 50 fertilized progeny must be sexed.

The most appropriate course is to analyse the sex of fertilized embryos resulting from insemination of super- ovulated cows since results can be obtained rapidly, in quantity and at relatively low cost using procedures for determination of the genetic sex of ruminant embryos disclosed previously (5) . 24

EFERENT

1. Bryant, B. .J. ₍ι₉₈₀₎ Preparation of mono_Spe_Cif_ic male-specific antibody and the use thereof for increasing the percentage of mammalian offspring of eⁱther sex, U.S.A. Patent No. 2,070,641 A.

2. Wachtel, S.S. ⁽1983⁾ Monoclonal antibodies to the H-Y antigen, methods of production, applications, and products of manufacture, Internaf1 Patent Appl_ic No. PCT/US84/0U31; Aust. Patent Applic. No WO 85/00502.

*^• Goldberg, E.H. ⁽1985⁾ ATCC HB8116 and its monoclonal antⁱ-H-Y antibody, Hyclonalan, U.S.A. Patent No 4,511,661.

. unknown Inventor ⁽1986) Antibody to middle piece of Y chromosome bearing- spermatozoa, internaf1 Patent- Applic. No. 74532/87; Aust. Patent Applic. No AU-A-74532/87.

^• Reed, K.C., Lord, E.A., Matthaei, K.I., Mann, D.A., Beaton, S., Herr, CM. and Matthews, M.E. ₍i₉₈8₎ Determination of genetic sex in ruminants using Y-chromosome-specific polynucleotides, Internaf1 Patent Applic. No. PCT/AU89/00029; Aust. Patent Applic. No. WO 89/07154.

Koo, G., Stackpole, c, Boyse, E., Hammerling, u. and ardⁱs, M.P. ⁽1973⁾ Topographical location of H-γ antⁱgen on mouse spermatozoa by immunoelectron- mⁱcroscopy, Proc. Natl. Acad. Sci. USA 2Δ, 1502 7. Lau, Y.-F., Chan, K., Kau, Y.W. and Goldberg, E, (1986) Isolation of a male-specific and conserved gene using an anti H-Y antibody. Am. J. Hum. Genet. 12 (Suppl.), A142.

8. Huynh, T.V., Young, R.A. and Davis, R.W. (1985) Construction and screening cDNA libraries in lambda gtlO and lambda gtll, in "DNA Cloning Techniques:

A Practical Approach", Glover, D.M., ed. (IRL Press, Oxford) .

9. Bradley, M.P. ⁽1989) Sperm enrichment, Aust. Patent Applic. No. PJ6981/89.

10. Church, G.M. and Gilbert, W. (1984) Genomic sequencing, Proc. Natl. Acad. Sci. USA £1, 1991-1995.

11. Jameson, B.A. and Wolf, H. (1988) The antigenicity index: a novel algorithm for predicting antigenic determinants, Comput. Applic. Biosci. ±, 181-186.

In the Figures:

Figure 1. Antigenicity plot of theoretical translation peptide of mouse MEA (11). The shaded region on the horizontal axis is that designated "MEA peptide.c" (amino acids 63-92 inclusive; refer to text) whose sequence is represented below in standard single letter code.

Figure 2. Potential antigenic site MEA peptide.c. Superscripted numbers refer to amino acid residues (represented in standard three letter code) from the first of the two potential ATG start codons in the cDNA sequence of mouse MEA. The nucleotide sequence from which the peptide sequence is deduced is shown in standard notation below the peptide sequence.

Figure 3. Responses of domestic fowls to immunisation with MEA peptide.c. (a) Time course of immune response (arrow indicates reimmunisation). Antisera were assayed at a dilution of 1:160 (refer to (b)).

Figure 4. Typical elution profile of bovine sperm on immuno-separation column. The number of sperm recovered from the column by elution ("eluted") is compared with the number of anti-MEA peptide.c-treated sperm applied to the column ("loaded").

Figure 5. Duplex DNA sequence of cARTY. This sequence was cloned as a blunt-ended double-stranded cDNA (prepared from the polyvinyl (A) RNA of adult bull testis) into the Smal site of the plasmid vector Bluescript™ (Stratagene).

Figure 6. Diagram of an autoradiograph of a PEI- cellulose chromatogram following labelling of probe DNA by nick translation.

910813,ejhspe.02l -BTe__spec^6 Figure 7. Typical dot blot hybridisation analysis of fractionated bovine sperm: autoradiographs. Autoradiographs of dot blot hybridisations with Y- chromosomal (left) and "autosomal" (right) DNA probes. Each column of dots represents a series of eight doubling dilutions of untreated (control) sperm ("US"), eluated from immuno-separation columns with three different sperm samples ("El, E2, E3"), male bovine lymphocytes ("ML") and female bovine lymphocytes ("FL"), the latter two serving as hybridisation controls. Refer to text for details.

Figure 8. Typical dot blot hybridisation analysis of fractionated bovine sperm: quantitation by scintillation spectrometr . The mean ratios of radioactivity resulting from hybridisation of six replicate diluted dot blots with the Y-chromosomal DNA sequence 0Y11.1 and with the interspersed repetitive ("autosomal") DNA sequence cARTY (refer to text) is illustrated for unfractionated sperm ( "pre-loading") and for sperm eluted from the immuno- column ("eluted"). Standard deviations are indicated by the error bars.

Figure 9. Validation of dot blot hybridisation analyses using mixtures of male and female bovine lymphocytes.

(a) Autoradiograph of dot blot hybridisations with Y- chromosomal (left) and "autosomal" (right) DNA probes. Each column of dots represents a series of eight doubling dilutions of a cell sample whose composition is indicated beneath the illustration. Refer to text for details. Validation of dot blot hybridisation analyses using mixtures of male and female bovine lymphocytes.

(b) Quantitation by scintillation spectrometry. the mean ratio of radioactivity resulting from hybridisation of the eight dot blot dilutions (a) with Y-chromosomal and "autosomal" DNA probes is illustrated for each of the six cell mixtures. Standard deviation is indicated by the hatched block above each column.

Figure 10. Validation of dot blot hybridisation analyses using mixtures of female bovine lymphocytes and bovine sperm. (a) Autoradiograph of dot blot hybridisations with Y-chromosomal (left) and "autosomal" (right) DNA probes. Each column of dots represents a series of eight doubling dilutions of a cell sample whose composition is indicated beneath the illustration. Refer to text for details.

Validation of dot blot hybridisation analyses using mixtures of female bovine lymphocytes and bovine sperm, (b) Quantitation by scintillation spectrometry. The mean ratio of radioactivity resulting from hybridisation of the eight dot blot dilutions (a) with Y-chromosomal and "autosomal" DNA probes is illustrated for each of the six cell mixtures. Standard deviation is indicated by the hatched block above each column.

Claims

CLAIMS :

1. A method for the fractionation of semen which comprises contacting a semen sample with a specific antibody preparation such that said antibody binds to spermatozoa bearing a Y chromosome, and thereafter separating antibody-spermatozoa complexes from unbound spermatozoa.

2. The method according to claim 1 wherein the antibody is capable of binding to the male enhanced antigen on spermatozoa.

3. The method according to claim 2 wherein the antibody is a polyclonal antibody.

4. The method according to claim 3 wherein the antibody is raised in a non-mammalian species.

5. The method according to claim 4 wherein the antibody is raised in domestic fowl.

6. The method according to claim 1 wherein the antibody is labelled with one or more spermatozoa- inactivating cytotoxic agents.

7. A method for the fractionation of semen which comprises contacting a semen sample with a domestic fowl anti-male enhanced antigen antibody for a time and under conditions sufficient for a spermatozoa bearing a Y chromosome and an antibody complex to form and thereafter separating said complex from unbound spermatozoa.

8. The method according to claim 7 wherein the antibody is a polyclonal antibody.

9. The method according to claim 7 or 8 wherein the antibody is labelled with a spermatozoa-inactivating cytotoxic agent.

10. The method according to claim 1 or 7 wherein the antibody is bound to a solid support.

11. The method according to claim 1 or 7 wherein the bound spermatozoa are separated from unbound spermatozoa by contacting the treated semen sample with a second antibody specific to the first antibody.

12. The method according to claim 11 wherein said second antibody is a polyclonal antibody.

13. The method according to claim 12 wherein said second antibody is bound to a solid support.

14. The method according to claim 13 wherein the solid support is a sheet of cellulose or a cellulose derivative, a plastic tray or a well therein, microspheres of high molecular weight carbohydrate, magnetic microspheres or glass microspheres.

15. An antibody capable of binding to the male enhanced antigen expressed on spermatozoa.

16. The antibody according to claim 15 which is a polyclonal antibody.

17. The antibody according to claim 16 raised in a non-mammalian species.

18. The antibody according to claim 17 wherein the non-mammalian species is domestic fowl.

19. The antibody according to claim 15 labelled with a spermatozoa-inactivating cytotoxic agent.

20. The antibody according to claim 15 or 19 wherein the antibody is bound to a solid support.

21. Fractionated semen produced in accordance with the method of claim 1 or 7.

22. A embryo fertilized by the fractionated semen according to claim 21.

23. A kit for fractionating semen, which kit comprises in compartmental form a first container adapted to receive a semen sample; a second container adapted to contain an antibody specific to spermatozoa bearing a Y chromosome; and optionally a third container adapted to contain a second antibody specific to said first antibody wherein said second antibody is immobilized to the inside of said third container or to a solid support therein.

24. The kit according to claim 23 wherein the first and second antibodies are polyclonal antibodies.

25. The kit according to claim 24 wherein the first antibody is specific to male enhanced antigen.

26. The kit according to claim 25 wherein the first antibody is labelled with a spermatozoa-inactivating cytotoxic agent.

27. The kit according to claim 25 or 26 wherein the antibody is raised in a non-mammalian species.

28. The kit according to claim 27 wherein the non- mammalian species is domestic fowl.

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