ELSEVIER

A NEW APPROACH

TO IMMUNOLOGICAL

SEXING

OF SPERM

S.R. Blechera, R. Howie, S. Li, J. Detmar and L.M. Blahut Department of Molecular Biology and Genetics University of Guelph, Guelph, Ontario, Canada N 1G 2W 1 Received for publication: Accepted: September 2, 1999 October 13, 1999

ABSTRACT A non-invasive, immunological method for sexing mammalian sperm would be of benefit to agricultural industries. This paper presents a new approach, based on the hypothesis that sexspecific proteins (SSPs) are evolutionarily more highly conserved than non-SSPs. Antibodies to non-SSPs were raised and used in an afftnity procedure to remove non-SSPs and enrich for SSPs. Thereafter, using column chromatography, purified SSPs were obtained. Sex-specific antibodies (SSAbs) raised against these SSPs appear to bind to sex-chromosome-specific proteins (SCSPs) on the sperm membrane and make possible a sperm-sexing procedure. Antibodies to SCSPs were raised and used to identify putative SCSPs by affinity chromatography. The preliminary results presented here suggest that a viable immunological sperm sexing procedure can be developed.
Q 1999 by Elsevier Science Inc.

Key words:

sperm sexing, immunology, proteins

post-meiotic

transcription,

sex-chromosome-specific

INTRODUCTION “Sexing” of mammalian sperm, i.e. separation of sperm into X- and Y-chromosome-bearing cells (referred to below as X and Y sperm), is a long-standing goal in agricultural industries, including cattle breeding. Many methods have been devised on the basis of supposed differences in various sperm characteristics (reviewed in 32, 11). Of these methods, only the DNAFluorescence Activated Cell Sorting technique (17, 18) has, to date, proven to be consistently reproducible, and even this method has certain drawbacks, as the procedure is relatively slow, potentially invasive, and requires specialized, expensive and immobile equipment and highly skilled operators. In this report, we describe a novel immunological approach, based on a hypothesis developed by S.R. Blecher and report preliminary results.

Acknowledgments Supported by funding from the National Science and Engineering Research Council of Canada; the Ontario Ministry of Agriculture, Food and Rural Affairs; and Gensel Biotechnologies Inc. We thank Dr. Luis Martin for comments on the manuscript, Ian Smith for help with the illustrations and Sandra Brown for secretarial assistance. a Correspondence and reprint requests.
0093-691 X/QQ/$-see
PII 50093-691

Theriogenology 52:13OQ-1321, Q 1 QQQ by Elsevier Science

1999 Inc.

front

matter

X(99)0021

Q-6

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Ohno (23) showed that a large proportion of genes on the mammalian X chromosome are highly evolutionarily conserved; an X-linked gene in any one species predicts the existence of a homologue in all others. This proposition is known as Ohno’s Law. The hypothesis for the present work is: 1) that the sex chromosomes encode or control the expression of X- and Y-sexchromosome-specific proteins (X- or Y-SCSPs) that are expressed on the surface of somatic and sperm cells, and 2) as an extrapolation of Ohno’s Law, that these proteins are more highly conserved between species than non-sex-specific molecules. As a consequence of the latter, when a same-sex recipient is immunized with tissue from a different species, the recipient’s immune system would mainly perceive SSPs as “self’ and would not raise antibodies to these molecules. On the other hand, antibodies would be raised against non-SSPs. The latter would reflect species differences in the non-SSPs; these antibodies could be used to immunoprecipitate non-SSPs and leave SSPs in a partially purified form. As described below, we have used this approach to enrich for SSPs and, after purification, to raise SSAbs in opposite sex recipients. These SSAbs have been successfully used in preliminary sperm-sexing experiments. We also preliminarily report on use of an extension of the approach to identify putative SCSPs in spermmembrane material.

MATERIALS Plasma Membrane

AND METHODS

Protein Preparation and Analysis

Plasma membrane protein nrenaration from fetal organs. Gonads, kidney, spleen and liver were dissected from bovine fetuses (10 to 20 cm crown-rump length) and either used immediately or stored at -70°C until used. Plasma membranes were derived from organs of each sex separately, using the method of Maeda (21), with minor modifications, including the addition of protease inhibitors (0.016 mM N-ethylmaleimide [NEM] and 1 mM Pep&tin) to the homogenization buffer. Plasma membrane proteins were solubilized usi,ng 0.5% final concentration (wt/vol) Triton X- 100 (14) and protein concentrations were determined Plasma membrane nrotein prenarations from snerm. Fresh sperm and cryopreserved sperm (in egg-yolk xtender) were obtained from Gencor ’ . For Western blots, the nitrogen cavitation procedure & was used (6) on fresh bovine sperm. For sperm membrane solubilization, procedures were modified from Klint (20) Fenner (10) and Hendriksen (12). Ejaculates from 3 bulls were washed 3 times with HEPES-buffered saline (mass/L. in ddH20: 8.76 g NaCl; 2.38 g HEPES, pH 7.2) pooled and centrifuged at 600 x g for 10 min at 25 “C. Triton X-100 was added to a final concentration of 0.5% (v/v). Ten uL of 100X protease inhibitor “cocktail” (mass/mL in ddH20: 30.2 mg EDTA; 357 ug phenyl methane sulphonyl fluoride; 8 1.2 mg NEM; 8 11 ug Pepstatin A) was added per mL of washed

b

MJx BioLynx,

Brockville,

Ontario, Canada.

’ Gencor, Guelph, Ontario, Canada.
d

Buhr MM, personal communication.

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sperm.Tubes wereshaken icefor 1h, centrifuged 107,000 g for 1 h at 4°C andproteinassays on at x performed. Electronhoresis. Proteinfractions wereresolved SDS-PAGEusing standard by the Mini-Gel proceduree described themanufacturer’s as in instructions. Gelswerestained 0.2%(wt/vol) in Coomassie Blue. Production Antisera of Antibodiesto fetal-membrane moteins.NewZealand Whiterabbits’,3.0 to 3.5 kg were immunized 4 SC by injections followedby anim boost. Theantisera wereassigned Greekletter symbols follows: female as rabbitanti-male bovinetissue alpha female (a); rabbitanti-female bovine- beta(p); malerabbitanti-female bovine- gamma malerabbitanti-male (y); bovine- delta (6). Antibodiesto snerm membrane nroteins.Male andfemale rabbits wereimmunized with pooled, freshor cryopreserved sperm x SC 1 im aspreviously). Sperm (4 and cellswerewashed twice in HEPES-buffered saline Sperm TALP (Tyrode’s,albumin, lactate, pyruvate;26)with centrifugation at 200x g for 10min at roomtemperature. relatively non-stringent This treatment used orderto was in llular viability. Sperm counts weredonein a hemacytometer. Eachscinjectioncontaine preserve F 39.9x 10 pooledsperm 200nL Freund’s + incomplete adjuvant; im boostcontained x 10 the 79.8 pooledsperm 200 uL Freund’s + incomplete adjuvant(modified from 1,8 and15). The antisperm antisera from femaleandmalerabbits wereassigned Greeklettersymbols the epsilon andzeta(0, (E) respectively.In termsof the hypothesis stated the Introduction, in these putative“anti-Y” and are “anti-x” antisera, respectively.To testthe specificityof these antisera sperm for membrane proteins, immunocytochemistry performed skim-milk-blocked, was on methanol-fixed smears cryopreserved of sperm.Second antibodywas Fluorosceinisothiocyanate (FITC)-conjugated anti-rabbitIgGg. goat The slides wereviewedundera ZeissTM IM35 UV microscope. Table 1. Definitionsof antisera types. Recipient Female rabbit Donor tissue Bovine male Bovinefemale Bovinesperm (unsorted alPha (a) beti $9 epsilon (e) Male rabbit delta(6) g-a (Y) =ta (0

e BioRad, Mississauga, Ontario, Canada. f Maple LaneRabbitry, R.R. #2, Clifford, Ontario, Canada Charles and River Rabbitry, Charles River Canada (Div. of Bausch& Lomb), St. Constant,Quebec,Canada. Inc. g SigmaChemicalCo., Oakville, Ontario, Canada.

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Immunoblotting (Western blotting). Western blotting was done as described in Sambrook (27). Bound antibodies were detected using protein A-horseradish peroxidase, and Diaminobenzidine as substrate.. All primary antibodies used for immunoblotting were previously analyzed by ELISA (16) to establish titre. Female and male antigens were coated at 1 pg/well (for unpurified solubilized proteins) or 100 r&well (for semi-purified or purified SSP). Fetal-membrane nrotein purification. Preclearance of solul#lized fetalplasma membrane proteins was done using CNBr-activated Sepharose 4B affinity columns with rabbit pretmmune sera as ligand. Unbound and bound $uted fractions were collected. The fractions were analyzed by absorbence (4280) protein assay , SDS-PAGE and immunoblotting for elimination of high- lipid, highdetergent, low-protein, non-SSP fractions. Immunoaffinity enrichment for SSPs was done by HPLCi (Ult.raffinity - EP columns, 0.5 ml or 5.0 ml column capacity). The columns were derivatized, accordpg to,the manufacturer’s instructions, using p and 6 IgG, isolated by Protein A Sepharose , as hgand. Individual p and 6 columns were run in tandem, or l3 and 6 IgG were combined on a single column. Precleared, solubilized protein in 0.1 M potassium phosphate, pH 7.0, was passed through the column. The unbound fraction was collected; the bound proteins were eluted with 1.O potassium phosphate, pH 2.7 containing 0.5 M potassium chloride. Unbound and bound fractions were concentrated with 3kiloDalton (kD) cut-off Centricon-3@ concentratorsl and analyzed by protein concentration determination, SDS-PAGE, Western blots and ELISA. Gel filtration was done using HPLC Column Chromatographyi. Superdex 200 HiLoad 16/60 prep grade or Sephacryl 16160 HR- 1001 were used according to the manufacturer’s instruction with 0.05M sodium phosphate, pH 7.0 containing 0.15 M NaCl. Fractions were concentrated and analyzed as in previous stages. Combined putative SSP fractions were further separated by size exclusion on a Superdex 75 HR IO/30 columnh according to the manufacturer’s instructions (elution buffer and subsequent analyses as above). Anion Exchange Chromatography on a DEAE Sephacryl columnh was done using 0.02M Tris hydroxymethyl aminomethane pH 8.0, with a continuous salt gradient to 1.OM NaCl. Bound and unbound fractions were analyzed as previously. Further separations could be done using manufacturer’s suggested appropriate buffers at pH 7.0 and 5.0 with a salt gradient. Bound and unbound fractions were analyzed for the presence of SSPs. Sperm membrane urotein uurification. “Anti-X” and “anti-Y” immunoaffinity columns, prepared using an A&Gel@ Hz kite and isolated E and B IgG as ligands, were loaded with solubilized sperm membrane protein. The unbound and eluted fractions were collected and concentrated with 3-kD cutoff Centricon-3@ concentratorsl. Bound fractions of each of the “anti-X” and “anti-Y” columns were
h

Amersham

Pharmacia Biotech, Baie de’Urfe, Quebec, Canada. Ontario, Canada.

i

Beckman Instruments Inc., Mississauga, Inc., Oakville, Ontario, Canada.

J Amicon

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loaded the othercolumn. Theunbound on fractions these of reciprocal loadings werecollectedas putativeX- andY-chromosome-specific antigens, respectively.SDS-PAGEgelsweresilver-stained. Rf (mobility relativeto dye-fro!t) valuesweremeasured molecular and weights calculated, using CorelTM QuattroPro@ soflware. SpermSeparation Straws containing sperm bull cryopreserved liquidnitrogenwerethawedin tapwaterat 35“C in for 1 min. The thawedsperm werewashed twice in protein-free HEPES-buffered saline SpermTALP andpelleted centrifugation 800x g for 10min. Motile sperm by at wereisolated glasswool by filtration (0.065g glasswool packed 0.1 ml of 1 ml syringe).After adjusting in sperm 50x 106/m1 to in proteinfreeHEPES-buffered saline HBS Sperm TALP, sperm weremixedwith type y (antifemale) antiserum 10%(v/v) final concentration incubated 60min at 38.5“C and5%CO*. at and for Approximately50%of the sperm agglutinated; these wereseparated free-swimming from sperm by glasswool filtration (0.065g glasswool packed 0.1ml of 1ml of syringe).The tree,filtered sperm in werethenwashed oncein protein-free HEPES-buffered saline HBS Sperm TALP to removeunbound Ab. In-vitro Fertilizationof Bovine EmbryoPreparation SexDiagnosis and Bovine embryos wereproduced vitro aspreviouslydescribed in (26). Blastocysts wereretrieved on day7 of development, sexed and cytogenetically, usingC-banding, by PCR(3). or Cytogenetic Sexing Blastocyst chromosomes prepared themethod Ring (19),modifiedin respect the were by of of first fixative, whichwas2:l glacialaceticacid:methanol. C-banding the chromosomes, For of slides weretreated with 0.1N HCl for 20 min at roomtemperature, rinsed dH20 andincubated in in prewarmed (wt/vol) Ba(OH)zfor 60 min at 60°C. Slides 3% wereagainrinsed, incubated in prewarmed SSC(300mM NaCl, 30 mM sodium 2X citrate)at 60°C for 60 min, rinsed, stained and in Giemsa (4,9,30). Usinga Leitz Orthoplan microscope, males wereidentifiedby presence a Y and of anX chromosome females two Xs. About 40%of embryos and by couldnot be sexed to due inadequate spread chromosomes. of EmbryoSexingby PCR Bovine Y-chromosome-specific primers werederivedfrom a repetitivebovineY-sequence, BRY.4a (25). The expected lengthof thePCRproductis 469base pairs. Autosomal primers were derivedfrom the bovinesatellite sequence 1.709(28). Theexpected lengthof thePCRproductis 246 base pairs. Male embryos wereidentifiedasthose with Y-chromosomal autosomal and fragments, andfemale embryos thosewith autosomal as fragments only.

k

Core1 Quattro Pro, Ottawa, Ontario, Canada. -

1314 RESULTS

Theriogenology

Affinity chromatography of solubilized membrane proteins from the tissues mentioned, using same-sex antibodies (types p and 6) as ligand, produced the predicted enrichment for sex-typical molecules (Figure la). This partially purified material (80% non-SSP removed) was used to immunize opposite-sex rabbits. The resulting serum was used in Western blots to determine whether like-sized molecules, exposed in male and female samples on 1-dimensional electrophoresis, following affinity chromatography, were sex specific or non-specific. Further enrichment for SSPs was done by gel filtration (Figure 1a) and then by ion- exchange chromatography (Figure 1b). Repeatable, characteristic profiles were seen on SDS-PAGE for male and female SSPs, in higher (50 to 60 kD and above) and lower (35 kD and below) molecular weight ranges, respectively. Antibodies raised to male and female purified SSP samples, (i.e. material purified by affinity chromatography, gel filtration and ion exchange) were used in Western blots against other purified SSPs, i.e. samples prepared from different tissue preparations. Figures 1c and 1d show such blots. Antibodies raised against purified male and female fetal SSPs (SSAbs c1and y, respectively) as well as non-SSAbs (p and S) were used in Western blots of sperm membrane proteins. Bands of approximately the same sizes as those of some male and female fetal SSPs were detected in these blots by c1and y antisera, respectively (Figure 2). Antibodies against purified male and female fetal SSPs (antisera types CIand y) were tested in experiments on live sperm. Type y produced agglutination of approximately half of the sperm (as estimated by microscopical observation). Following separation of agglutinated and unagglutinated sperm by glasswool filtration, the free sperm produced IVF embryos that were predominantly male (Table 2). Antiserum type 01did not produce agglutination and this serum, as well as types p and 6, were thereafter used as controls. Table 2. Separation of X and Y sperm by Type y (male anti-female) indicate numbers of embryos. Treatment d 25ya 17Y Total % No3 b Total % PCR controls 23 47 70 92 8 8 16 66 antiserum. 9 5 1 6 8 4 4 8 34 115 35 150 53 The values ? 27 3 30 1 4 5 9 1 10

Experimental embryos sexed cytogenetically

Cytogenetic

controls

107 No Ab 28 a 135 Total % 47 E 25~ = Gamma SSAb, serial number 25. a= Alpha SSAb (control). ? = Sex could not be diagnosed because of overlap of chromosomes.

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64

s-m

P

PI
229 124 67

STD

0

8

(4

STD

?

Figure 1. (a) Stepwise purification of female and male sex-specific proteins (SSPs): 1st pair: unpurified; 2nd pair: void (unbound) material following beta-delta ultraffinity purification; 3rd pair: material purified by gel filtration. (b) SSPs further purified by ion exchange, showing bands unique to each sex. (c) Western blot of purified female gonadal tissue, reacted against gamma (anti-female SSP) antiserum raised against material other than that used as antigen in the blot. The arrow indicates an immunoreactive protein of similar MW to previously observed female SSP. (d) Western blot of purified male gonadal tissue reacted against alpha (anti-male SSP) antiserum raised against material other than that used as antigen in the blot. The arrows indicate immunoreactive proteins of similar MWs to previously observed male SSPS.

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STDHa

HP

Hy H6 Ta

TS Ty T6 STD

21 14 Figure 2. Westernblot of sperm membrane antigens reactedto anti-fetal antibodies.Antigen: spermhead(H) andtail andmidpiece(T) membrane proteins. Antibodies: female anti-male(a), femaleanti-female(p), maleanti-female(y), maleanti-male(6). Arrows indicatebands molecularweight comparable fetal SSPs. of to Anti-sperm (E and(;) antisera eachexhibiteda l/50,000 titre. Immuno-cytochemistry revealedminimal bindingof preimmune andno bindingof second sera antibody alone. Antispermantiserademonstrated cell-surfaceandintra-acrosomal high reactivity in some not all but spermcells. SDS-PAGE analysis fractionsfrom 6 affinity-chromatographyexperiments of revealeda putative X-SCSP bandin all 6 (mean molecularweight 32.2 kD f 0.750 SD) (Figure 3). 1 2 3 4

2 sperm Figure 3. Putative X (lanes1 and3) and Y (lanes and4) chromosome-specific membrane proteins. Arrows show -32KDa X spermmoleculein lanes1 and3, absentfrom lanes2 and4.

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The 6 experiments utilized pooledsperm all from the same bulls. Experiments1 to 5 used 3 the samesolubilizationsample; experiment6 the materialwasseparately in solubilized. For experiments to 6, the affinity-column ligandscomprised same pooledEor < sera;in 2 the 2 experiment1, only 1 < serum wasused. In experiment1, the materialwasalsopassed through p and 8 columns prior to E andc. Five of theseexperiments attempted isolateY-chromosometo specificantigens (i.e. utilized Esera);3 of the 5 demonstrated enrichment proteinsranging of from 50 to 80 kD. The calculated varianceof the Rt-s the 6 -32 kD bands of wasless than the variancesof the two molecularweight standards nearest size(26 kD and38 kD), known to bethe same in protein in eachexperiment. DISCUSSION Affinity chromatography maleand femaleproteinpreparations, of usingidenticalIgG ligands(p, 6 or both), produceddifferential enrichment molecules different molecular for of weightsin the two sexes.This supported 2 of the hypothesis provideda basis part and for purification of SSPs. Anti-male and-femaleSSAbs(CIandy, respectively)demonstrated bandsin sperm membrane protein Westernblots of - 50 to 60 kD and - or ~35 kD, corresponding the sizesof to maleand femalefetal membrane proteinsseen SDS-PAGE. This supported possibility that in the SCSPs,detectable SSAbsc1 y, may be present the surfaces X andY sperm by and on of (part 1 of hypothesis). In live spermexperiments, SSAbsappeared cause y to preferentialagglutinationof Xsperm,sinceIVF embryosproducedfrom the unagglutinated sperm werepredominantlymale. The ExperimentalGroup (Table 1) comprised pooleddatafrom 8 experiments, doneon separate days. Of 76 embryosthat could be sexed,70 (92%) weremale. This proportionis statistically significant:the dataplot well into the “Significant Region” of Figure 2 in Moore andGledhill (22), andshowa lower limit of 82% for probable true frequency(of “desired” sex)at the 95% confidenceinterval, in Table 1 of Quaas Foote (24). Although many embryoscould not be and sexedbecause chromosomal of overlap,this evidently did not biasthe result,sincethe numberof undiagnosable embryoswashighestin the oneexperimentof the 8 in which a high numberof females was observed (datanot shown). Affinity columnsusingE and< antisera ligandsenrichedfor different sperm as proteins. The [ column,putatively capable bindingX-SCSPs,consistentlyisolated(n=6 of 6 trials) a of moleculeof -32 kD (mean molecularweight= 32.2). The RF of these6 bandshada smaller variancethan the 26-kD and38-kD molecularweight standards. This indicates these6 that -32 kD bandscould represent same the molecule, which we interpretto be a putative X-SCSP protein. Our datasuggest it is a cell-surface that molecule. The E and[ antisera wereproduced by injecting intact, live sperm. B cellsarecapable responding cell surfaceantigens of to with which they may comein contact (whereas cellsrequirepresentation phagocytosed T of material

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by antigen presenting cells). Immuno-cytochemically, the resulting antisera did indeed react preferentially with cell-surface proteins (and the acrosome). The E column, a putative ligand for Y-SCSPs, did not demonstrate comparable 32 kD bands, but did enrich for higher molecular weight bands in 3 of 5 trials. We predicted that antibodies could be raised to somatic SSP or SCSPs. We used such antisera in Western blotting of sperm membrane proteins, separation of X and Y sperm by agglutination, and affinity column enrichment of a putative X-SCSP. The preliminary results of these experiments support the proposition that SCSPs are present on the sperm-cell surface. Although the experimental numbers are small, the fact that the data from three different techniques concur adds credence to the conclusion. If such molecules are present, it is likely that they have an evolutionarily conserved tunction. One possibility is that they are involved in establishing the sex of the zygote. In mammals, sexdetermining genes and hormones have interlocking functions in sex differentiation (5). Because fertilization occurs in an ambience of female hormones, it is likely that a strong selective pressure would favor a very early genetic signal to establish sex. A surface molecule would serve this purpose. The presence of different SCSPs on the surfaces of X and Y sperm implies post-meiotic transcription and/or translation of these molecules, and that they do not cross inter-spermatid cytoplasmic bridges. There is now ample evidence that post-meiotic transcription and/or translation occurs (12), and evidently not all post-meiotic transcription and/or translation molecules cross the cytoplasmic bridges (33). If, as suggested above, there is a selective advantage for the existence of these molecules, there also would be a selective pressure for them not to cross the bridges. One mechanism that might ensure this is that molecules destined for the plasma membrane might be placed there immediately after synthesis (7). Alternatively, SCSP transcripts may be stored in spermatids in a “translationally repressed state” (29) and released from this state in the appropriate sperm cell. Finally, the sex chromosomes are mainly heterochromatic throughout meiosis; it is possible that the relevant loci may be inactivated, and that transcription occurs only after the bridges are closed. The ability of both somatic female tissue and X sperm to raise sex- or sex-chromosomespecific immune responses in male animals implies that, at some level of genetic expression, two X chromosomes or alleles differ from one. There are several hypothetical ways to account for this. A locus on the Y chromosome could suppress the expression of a somatic X. It is also possible, by analogy with the Xist locus, that a coding X locus is transcribed from the “inactive” X in female somatic tissue (though in the case of Xist the transcript is not translated). The X chromosome is also inactivated in spermatogenesis. A third possibility is a genomically imprinted locus on the X chromosome. An X-chromosomal locus for an immunogenic epitope could be suppressed on the maternal X (Xm) and active on the paternal (X’). The immune system of male (XmY) animals would perceive as foreign the gene product of the Xp allele, present in both X sperm and female somatic (XpXm) tissue. Genomic imprinting of X loci has been reported.(2,3 1).

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Previousstudies (13, 15) reportedunsuccessful searches proteinsspecificto X or Y for sperm. The reasons the differencesbetween for their resultsandoursarenot clear. However, the molecules believe we have identified arepresent very minutequantities. One possibility is we in that the immunologicalapproach haveusedis moresensitivein detectingsmallamounts we of protein than other methods protein analysis, of suchas2-dimensional electrophoresis. gel We envisage possibilitythat the techniquedescribed the herecould, in the future, leadto the development a commerciallyviable system sperm“sexing”. The methodwould utilize of for mass producedmonoclonalor recombinant antisera, which would be centrally handledin a productionfacility. The procedurewould be minimally invasive. It is alsopossible the yield that would be high, if high titre antibodies be produced. can In summary,the resultspreliminarily reportedherearecompatiblewith the propositionthat X andY spermcellsdo carry different proteinson their cell surfaces.The data suggest it that may be possible developan immunologicallybased to systemfor spermseparation. REFERENCES 1. AmbroseJD, Rajamahendran Yoshiki T, Lee CYG. Anti-bull sperm R, monoclonal antibodies:I. Identification of majorantigenicdomains bull sperm manifestation of and of interspecies cross-reactivity. J Androl 1996;17:567-578. 2. Ariel M, Robinson McCarrey JR, CedarH. Gamete-specific E, methylation correlates with imprinting of the murineXist gene. Nat Genet 1997;9:3 15. 12-3 3. Avery B, Jorgensen MadisonV, Greve T. Morphologicaldevelopment sexof CB, and bovine in vitro-fertilized embryos. Molec ReprodDev 1992;32:265-270. 4. Avery B, MadisonV, GreveT. Sex anddevelopment bovine in-vitro fertilized embryos. in Theriogenology1991;35:953-963. 5. Blecher SR, Wilkinson L. Non-hormone mediated chromosomal sex effectsin development:Another look at the Y chromosome-testicular hormoneparadigm. In: Wachtel SS(ed), Evolutionary Mechanisms SexDetermination. BocaRaton, FL: CRC Press, in Inc., 1987;219-229. 6. Buhr MM, Curtis EF, KakudaNS. Composition behaviorof headmembrane and lipids of freshandcryopreserved boar semen.Cryobiology 1994;31:224-238. 7. CaldwellKA, HandelMA. Protamine transcriptsharing amongpostmeioticspermatids. ProcNat1Acad SCUSA 1991;88:2407-2411. 8. CastlePE, Whaley KJ, HoenTE, Moench TR, ConeRA. Contraceptiveeffect of spermagglutinatingmonoclonal antibodies rabbits. Biol Reprod1997;56: in 153-l 59. 9. Craig-Holmes Moore FB, ShawMW. Polymorphism humanC-banded A, of heterochromatin.I. Frequencyof variants. Am J HumanGenet 1973;25:181-192. 10. FennerGP, Johnson HruschkaWR, Bolt DJ. Two-dimensional LA, electrophoresis and densitometric analysisof solubilizedbovine sperm plasma membrane proteinsdetected by silver stainingandradioiodination. Arch Androl 1992;29:21-32. 11. Flaherty SP,Matthews CD. Application of modemmoleculartechniques evaluatesperm to sex selectionmethods.Molec HumanReprod1996;2:937-942.

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12. Hendriksen PJM, Hoogerbrugge JW, Themmen APN, Koken MHM, Hoeimakers JHJ, Oostra BA, Van Der Lende T, Grootegoed JA. Postmeiotic transcription of X and Y chromosomal genes during spermatogenesis in the mouse. Dev Biol 1995; 170:730-733. 13. Hendriksen PJ, Welch GR, Grootegoed JA, Van der Lende T, Johnson LA. Comparison of detergent-solubilized membrane and soluble proteins from flow cytometrically sorted X- and Y-chromosome bearing porcine spermatozoa by high resolution 2-D electrophoresis. Molec Reprod Dev 1996;45:342-350. 14. Hjelmeland LM. Solubilization of native membrane proteins. In: Deutscher MP (ed), Methods in Enzymology, Volume 182: Guide to protein purification. San Diego: Academic Press, Inc., Harcourt Brace Jovanovich, Publishers, 1990;253-264. 15. Howes EA, Miller NG, Dolby C, Hutchings A, Butcher GW, Jones R. A search for sexspecific antigens on bovine spermatozoa using immunological and biochemical techniques to compare the protein profiles of X and Y chromosome-bearing sperm populations separated by fluorescence-activated cell sorting. J Reprod Fertil 1997; 110: 195-204. 16. Hudson L, Hay FC. Practical Immunology, 2nd Edition. Oxford: Blackwell Scientific Publications, 1980;237-238. 17. Johnson LA. Gender preselection in domestic animals using flow cytometrically sorted sperm. J Anim Sci 1992;7O(Suppl2):8-18. 18. Johnson LA, Flook JP, Hawk HW. Sex preselection in rabbits: Live births from X and Y sperm separated by DNA and cell sorting. Biol Reprod 1989;4 1:199-203. 19. King WA, Linages T, Gustavsson I, Bane A. A method for preparation of chromosomes from bovine zygotes and blastocysts. Vet Sci Comm 1979;3:51-56. 20. Klint M, Sege K, Curan B, Ploen L, Peterson PA. Solubilization and enrichment of boar sperm membrane proteins. Gam Res 1985;11:335-348. 2 1. Maeda T, Balakrishan K, Mehdi SQ. Biochim Biophys Acta 1983;73 1:115-l 20. 22. Moore DH, Gledhill BL. How large should my study be so that I can detect an altered sex ratio? Fertil Steril 1988;50:21-25. 23. Ohno S. Sex Chromosomes and Sex-Linked Genes. Berlin: Springer-Verlag, 1967;46-73. 24. Quaas RL, Foote RH. Testing for altered sex ratios: Statistical considerations. In: Amann RP and Seidel GE (eds), Prospects for Sexing Mammalian Sperm. Boulder, CO: Colorado Associated University Press, 1982;277-283. 25. Reed KC, Matthaei KI, Mann DA, Beaton S, Matthews MA. Determination of genetic sex in ruminants using Y-chromosome specific polynucleotides. Patent Cooperation Treaty No. WO 89/07154 Melbourne, Australia, 1989. 26. Rieger E, Grisart B, Semple E, Van Langendonckt A, Betteridge KJ, Dessy F. Comparison of the effects of oviductal cell co-culture and oviductal cell-conditioned medium on the development and metabolic activity of cattle embryos. J Reprod Fertil 1995;105:91-98. 27. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Edition. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989;3: 18.60-l 8.75. 28. Skowronski J, Plucienniczak A, Bednarek A, Jaworski J. Bovine 1.709 satellite. J Molec Biol 1984;177:399-416. 29. Steger K. Transcriptional and translational regulation of gene expression in haploid spermatids. Anat Embryo1 (Berl) 1999;199:471-487. 30. Summer AT. A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res 1972;75:304-306.

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31. Thornhill AR, Burgoyne PS. A paternallyimprintedX chromosome retardsthe development the early mouse of embryo. Development1993;118:171- 174. 32. Windsor DP, EvansG, White IG. Sexpredetermination separation X andY by of chromosome-bearing sperm:A review. ReprodFertil Dev 1993;5:155-171. 33. ZhengY, DengX, Martin-DeLeonPA. Compartmentalization SPAMl (PH-20) RNA in of spermatids leadingto transmission distortion(TRD). In: GagnonC (ed), Proc 8th Int ratio Symp Spermatol,Montreal, Canada,1998;PI:3 abstr. 1