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 1 G 2W 1

Received for publication: September 2, 1999 Accepted: 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 sex- specific 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, post-meiotic transcription, sex-chromosome-specific proteins

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 DNA- Fluorescence 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.

Theriogenology 52:13OQ-1321, 1999 Q 1 QQQ by Elsevier Science Inc.

0093-691 X/QQ/$-see front matter

PII 50093-691 X(99)0021 Q-6

1310 Theriogenology

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-sex- chromosome-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 sperm- membrane material.

MATERIALS AND METHODS

Plasma Membrane 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 ’ was used

& xtender) were obtained from Gencor . For Western blots, the nitrogen cavitation procedure (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.

Theriogenology 1311

sperm. Tubes were shaken on ice for 1 h, centrifuged at 107,000 x g for 1 h at 4°C and protein assays performed.

Electronhoresis. Protein fractions were resolved by SDS-PAGE using the standard Mini-Gel proceduree as described in the manufacturer’s instructions. Gels were stained in 0.2% (wt/vol) Coomassie Blue.

Production of Antisera

Antibodies to fetal-membrane moteins. New Zealand White rabbits’, 3.0 to 3.5 kg were immunized by 4 SC injections followed by an im boost. The antisera were assigned Greek letter symbols as follows: female rabbit anti-male bovine tissue - alpha (a); female rabbit anti-female bovine - beta (p); male rabbit anti-female bovine - gamma (y); male rabbit anti-male bovine - delta (6).

Antibodies to snerm membrane nroteins. Male and female rabbits were immunized with pooled, fresh or cryopreserved sperm (4 x SC and 1 im as previously). Sperm cells were washed twice in HEPES-buffered saline Sperm TALP (Tyrode’s, albumin, lactate, pyruvate; 26) with centrifugation at 200 x g for 10 min at room temperature. This relatively non-stringent treatment was used in order to

preserve F 39.9 x 10 llular viability. Sperm counts were done in a hemacytometer. Each sc injection containe pooled sperm + 200 nL Freund’s incomplete adjuvant; the im boost contained 79.8 x 10

pooled sperm + 200 uL Freund’s incomplete adjuvant (modified from 1,8 and 15). The antisperm antisera from female and male rabbits were assigned the Greek letter symbols epsilon (E) and zeta (0, respectively. In terms of the hypothesis stated in the Introduction, these are putative “anti-Y” and “anti-x” antisera, respectively. To test the specificity of these antisera for sperm membrane proteins, immunocytochemistry was performed on skim-milk-blocked, methanol-fixed smears of cryopreserved sperm. Second antibody was Fluorosceinisothiocyanate (FITC)-conjugated goat anti-rabbit IgGg. The slides were viewed under a ZeissTM IM35 UV microscope.

Table 1. Definitions of antisera types. Recipient

Female rabbit Male rabbit

Donor tissue

Bovine male Bovine female Bovine sperm (unsorted

alPha (a) beti $9 epsilon (e)

delta (6) g-a (Y) =ta (0

e BioRad, Mississauga, Ontario, Canada.

f Maple Lane Rabbitry, R.R. #2, Clifford, Ontario, Canada and Charles River Rabbitry, Charles

River Canada Inc. (Div. of Bausch & Lomb), St. Constant, Quebec, Canada.

g Sigma Chemical Co., Oakville, Ontario, Canada.

1312 Theriogenology

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, high- detergent, 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 3- kiloDalton (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 cut- off 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.

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

J Amicon Inc., Oakville, Ontario, Canada.

Theriogenology 1313

loaded on the other column. The unbound fractions of these reciprocal loadings were collected as putative X- and Y-chromosome-specific antigens, respectively. SDS-PAGE gels were silver-stained. Rf (mobility relative to dye-fro!t) values were measured and molecular weights calculated, using CorelTM Quattro Pro@ soflware .

Sperm Separation

Straws containing bull sperm cryopreserved in liquid nitrogen were thawed in tap water at 35 “C for 1 min. The thawed sperm were washed twice in protein-free HEPES-buffered saline Sperm TALP and pelleted by centrifugation at 800 x g for 10 min. Motile sperm were isolated by glasswool filtration (0.065 g glasswool packed in 0.1 ml of 1 ml syringe). After adjusting sperm to 50 x 106/m1 in protein free HEPES-buffered saline HBS Sperm TALP, sperm were mixed with type y (antifemale) antiserum at 10% (v/v) final concentration and incubated for 60 min at 38.5 “C and 5% CO*. Approximately 50% of the sperm agglutinated; these were separated from free-swimming sperm by glasswool filtration (0.065 g glasswool packed in 0.1 ml of 1 ml of syringe). The tree, filtered sperm were then washed once in protein-free HEPES-buffered saline HBS Sperm TALP to remove unbound Ab.

In-vitro Fertilization of Bovine Embryo Preparation and Sex Diagnosis

Bovine embryos were produced in vitro as previously described (26). Blastocysts were retrieved on day 7 of development, and sexed cytogenetically, using C-banding, or by PCR (3).

Cytogenetic Sexing

Blastocyst chromosomes were prepared by the method of Ring (19), modified in respect of the first fixative, which was 2:l glacial acetic acid:methanol. For C-banding of the chromosomes, slides were treated with 0.1 N HCl for 20 min at room temperature, rinsed in dH20 and incubated in prewarmed 3% (wt/vol) Ba(OH)z for 60 min at 60°C. Slides were again rinsed, incubated in prewarmed 2X SSC (300 mM NaCl, 30 mM sodium citrate) at 60°C for 60 min, rinsed, and stained in Giemsa (4,9,30). Using a Leitz Orthoplan microscope, males were identified by presence of a Y and an X chromosome and females by two Xs. About 40% of embryos could not be sexed due to inadequate spread of chromosomes.

Embryo Sexing by PCR

Bovine Y-chromosome-specific primers were derived from a repetitive bovine Y-sequence, BRY.4a (25). The expected length of the PCR product is 469 base pairs. Autosomal primers were derived from the bovine satellite sequence 1.709 (28). The expected length of the PCR product is 246 base pairs. Male embryos were identified as those with Y-chromosomal and autosomal fragments, and female embryos as those with autosomal fragments only.

k Core1 - Quattro Pro, Ottawa, Ontario, Canada.

1314 Theriogenology

RESULTS

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 1 a) and then by ion- exchange chromatography (Figure 1 b). 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 1 c and 1 d show such blots.

Antibodies raised against purified male and female fetal SSPs (SSAbs c1 and 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 c1 and y antisera, respectively (Figure 2).

Antibodies against purified male and female fetal SSPs (antisera types CI and 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 01 did 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) antiserum. The values indicate numbers of embryos.

Treatment d 9 ?

Experimental embryos 25ya 23 5 27 sexed cytogenetically 17Y 47 1 3

Total 70 6 30 % 92 8

Cytogenetic controls No 3 b 8 4 1 8 4 4

Total 16 8 5 % 66 34

PCR controls No Ab 107 115 9 a 28 35 1

Total 135 150 10 % 47 53

E 25~ = Gamma SSAb, serial number 25. a= Alpha SSAb (control).

? = Sex could not be diagnosed because of overlap of chromosomes.

Theriogenology 1315

64 s-m P

PI

(4 STD ?

229 124 67

STD 0 8

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.

1316

STDHa HP Hy H6 Ta TS Ty T6 STD

Theriogenology

21

14

Figure 2. Western blot of sperm membrane antigens reacted to anti-fetal antibodies. Antigen: sperm head (H) and tail and midpiece (T) membrane proteins. Antibodies: female anti-male (a), female anti-female (p), male anti-female (y), male anti-male (6). Arrows indicate bands of molecular weight comparable to fetal SSPs.

Anti-sperm (E and (;) antisera each exhibited a l/50,000 titre. Immuno-cytochemistry revealed minimal binding of preimmune sera and no binding of second antibody alone. Anti- sperm antisera demonstrated high cell-surface and intra-acrosomal reactivity in some but not all sperm cells. SDS-PAGE analysis of fractions from 6 affinity-chromatography experiments revealed a putative X-SCSP band in all 6 (mean molecular weight 32.2 kD f 0.750 SD) (Figure 3).

1 2 3 4

Figure 3. Putative X (lanes 1 and 3) and Y (lanes 2 and 4) chromosome-specific sperm membrane proteins. Arrows show -32KDa X sperm molecule in lanes 1 and 3, absent from lanes 2 and 4.

Theriogenology 1317

The 6 experiments all utilized pooled sperm from the same 3 bulls. Experiments 1 to 5 used the same solubilization sample; in experiment 6 the material was separately solubilized. For experiments 2 to 6, the affinity-column ligands comprised the same 2 pooled E or < sera; in experiment 1, only 1 < serum was used. In experiment 1, the material was also passed through p and 8 columns prior to E and c. Five of these experiments attempted to isolate Y-chromosome- specific antigens (i.e. utilized E sera); 3 of the 5 demonstrated enrichment of proteins ranging from 50 to 80 kD.

The calculated variance of the Rt-s of the 6 -32 kD bands was less than the variances of the two molecular weight standards nearest in size (26 kD and 38 kD), known to be the same protein in each experiment.

DISCUSSION

Affinity chromatography of male and female protein preparations, using identical IgG ligands (p, 6 or both), produced differential enrichment for molecules of different molecular weights in the two sexes. This supported part 2 of the hypothesis and provided a basis for purification of SSPs.

Anti-male and -female SSAbs (CI and y, respectively) demonstrated bands in sperm membrane protein Western blots of - 50 to 60 kD and - or ~35 kD, corresponding to the sizes of male and female fetal membrane proteins seen in SDS-PAGE. This supported the possibility that SCSPs, detectable by SSAbs c1 and y, may be present on the surfaces of X and Y sperm (part 1 of hypothesis).

In live sperm experiments, y SSAbs appeared to cause preferential agglutination of X- sperm, since IVF embryos produced from the unagglutinated sperm were predominantly male. The Experimental Group (Table 1) comprised pooled data from 8 experiments, done on separate days. Of 76 embryos that could be sexed, 70 (92%) were male. This proportion is statistically significant: the data plot well into the “Significant Region” of Figure 2 in Moore and Gledhill (22), and show a lower limit of 82% for probable true frequency (of “desired” sex) at the 95% confidence interval, in Table 1 of Quaas and Foote (24). Although many embryos could not be sexed because of chromosomal overlap, this evidently did not bias the result, since the number of undiagnosable embryos was highest in the one experiment of the 8 in which a high number of females was observed (data not shown).

Affinity columns using E and < antisera as ligands enriched for different sperm proteins. The [ column, putatively capable of binding X-SCSPs, consistently isolated (n=6 of 6 trials) a molecule of -32 kD (mean molecular weight = 32.2). The RF of these 6 bands had a smaller variance than the 26-kD and 38-kD molecular weight standards. This indicates that these 6 -32 kD bands could represent the same molecule, which we interpret to be a putative X-SCSP protein. Our data suggest that it is a cell-surface molecule. The E and [ antisera were produced by injecting intact, live sperm. B cells are capable of responding to cell surface antigens with which they may come in contact (whereas T cells require presentation of phagocytosed material

1318 Theriogenology

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, sex- determining 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-chromosome- specific 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).

Theriogenology 1319

Previous studies (13, 15) reported unsuccessful searches for proteins specific to X or Y sperm. The reasons for the differences between their results and ours are not clear. However, the molecules we believe we have identified are present in very minute quantities. One possibility is that the immunological approach we have used is more sensitive in detecting small amounts of protein than other methods of protein analysis, such as 2-dimensional gel electrophoresis.

We envisage the possibility that the technique described here could, in the future, lead to the development of a commercially viable system for sperm “sexing”. The method would utilize mass produced monoclonal or recombinant antisera, which would be centrally handled in a production facility. The procedure would be minimally invasive. It is also possible that the yield would be high, if high titre antibodies can be produced.

In summary, the results preliminarily reported here are compatible with the proposition that X and Y sperm cells do carry different proteins on their cell surfaces. The data suggest that it may be possible to develop an immunologically based system for sperm separation.

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