FERTILITY AND STERILITY
Vol. 58, No.2, August 1992
Copyright 1992 The American Fertility Society
Printed on ocid·free paper in U.S.A.
Determination of sex ratio of spermatozoa with a deoxyribonucleic acid-probe and quinacrine staining: a comparison
Roelof J. van Kooij, Ph.D.* Bernard A. van Oost, Ph.D.t University Hospital Utrecht, Utrecht, and University Hospital Nijmegen, Nijmegen, The Netherlands
Objective: To evaluate sex selection of spermatozoa. Design: A deoxyribonucleic acid (DNA) probe (pDP34) detecting distinguishable loci on both X and Y chromosome was used to validate the quinacrine-staining method that is often used for determination of the percentage of Y -bearing sperm. Sperm samples were centrifuged over Percoll to obtain samples with a high X:Y ratio according to the quinacrine-staining method. Controls (sperms before processing over Percoll) and processed sperms were subjected to DNA extraction and analysis with the DNA probe. Results: The DNA analysis revealed a 1.0 X:Y ratio ofthe spermatozoa before and after Percoll separation. Conclusion: We conclude that the quinacrine method is not suitable for evaluation of methods that claim to separate X and Y -bearing sperm. Fertil Steril 1992;58:384-6 Key Words: Deoxyribonucleic acid probe pDP34, quinacrine, sex ratio of spermatozoa, Percoll separation
In many of the recent methods describing attempts to separate human X and Y spermatozoa, the success of enrichment is judged solely by quinacrine staining of the Y sperm. This method, originally proposed by Barlow and Vosa (1), is based on determination of the percentage of spermatozoa with a fluorescent spot after quinacrine staining. Because quinacrine staining of human chromosomes causes intense fluorescence in the Y chromosome, it is assumed that the fluorescent spots (F bodies), which can be seen in roughly 40% of the spermatozoa, represent Y bearing gametes. Clinical programs for separation of X and Ybearing sperm cells are based on the F -body test (2). There is, however, considerable doubt that the iden-
Received December 18, 1991; revised and accepted April 15, 1992. * Reprint requests: Roelof J. van Kooij, Ph.D., Department of Reproductive Medicine, Division of Obstetrics and Gynecology, University Hospital Utrecht, Heidelberglaan 100, 3584 ex Utrecht, The Netherlands. t DNA-Diagnostic Laboratory, Department of Human Genetics, University Hospital Nijmegen. 384
van Kooij and van Oost
Sex selection of spermatozoa
tification by quinacrine staining is correct. Not all Y sperm seem to exhibit an F body; on the other hand, the incidence of spermatozoa with two F bodies is high (3). The latter finding is not confirmed by chromosome analysis (4). Beckett et al. (5) did not find a correlation between the F -body test and the content of Y chromosomes estimated with the deoxyribonucleic acid (DNA) probe (pS4). A few years ago it was reported (6) that the Fbody count can be drastically influenced by centrifuging spermatozoa over discontinuous Percoll gradients, whereby the most efficient separation (up to 90% sperm without F body) was obtained after centrifugation of the sperms over as much as 12 layers of Percoll of increasing density. It was reported (6, 7) that the sex ratio of a small series of babies born after insemination with Percoll-separated sperm was changed in favor of the female sex. In this work, we report on the ratio between X and Y spermatozoa, as determined with a single DNA probe in sperm DNA samples, detecting distinguishable loci in both the X and Y chromosome on the one hand and the F -body count on the other hand. We used samples that were enriched for Fertility and Sterility
F -body negative spermatozoa according to the previously mentioned Percoll method (6) and as a control the original semen before Percoll separation. MATERIALS AND METHODS Sperm Selection
Discontinuous Percoll gradients were prepared according to Iizuka et al. (6). Sperm samples of three healthy donors were divided into two parts. One part was used directly for the analysis with the DNA probe. Of the second part, a smear was made for Fbody count, and the rest was diluted with N-[2-hydroxyethyl]piperazine-N 1 _ [2-ethanesulfonic acid] (Hepes)-buffered Ham's F-10 medium (GIBCO, Breda, The Netherlands) and concentrated over 25% Percoll (Pharmacia, Uppsala, Sweden). The pellet was resuspended in 0.7 mL of 25% Percoll and then placed on top of the 12-layer discontinuous gradient. The gradient was centrifuged at 250 X g in a conventional desk top laboratory centrifuge for 30 minutes in a swing-out rotor. The fractions were separated by carefully pipetting the several layers into tubes, and all layers were subjected to a microscopic analysis of concentration and motility. Several fractions were subjected to DNA extraction and analysis (see DNA Isolation). Before starting DNA extraction, a small drop was taken for a smear, fixed, and prepared for F-body count with fluorescence microscopy. F -body counts were scored on coded slides by two observers. DN A Isolation
Twenty to 50 X 106 sperm cells were taken up in 10 mL of 6 M guanidinium HCL, 25 mM sodium citrate, 0.5% Sarkosyl (Sigma Chemical Co., St. Louis, MO), and 0.1 M beta mercaptoethanol (8). Proteinase K (0.25 mg/mL, final concentration; Boehringer, Mannheim, Germany) digestion was carried out overnight at 50°C. DNA was precipitated by addition of 0.7 volumes of isopropanol at room temperature and was subsequently dissolved in 0.5 mL 0.01 M TRIS (pH 7.5) in 1 mM ethylene diamine tetraacetic acid (EDTA). DN A Analysis
Five to 10 11m DNA was digested with 20 to 40 units of TaqI restriction enzyme overnight at 60°C according to the instructions of the manufacturer (GIBCO-BRL, Bethesda). The restricted DNA sample was electrophoresed (1 to 2 V/cm) in 0.9% agarose in 40 mM TRIS pH 7.5/1 mM EDTA. The Vol. 58, No.2, August 1992
separated DNA fragments were denatured and blotted on a nylon membrane (9) in 0.5 N NaOH/1.5 M NaCl. The probe pDP34 (DXYS1) (10, 11) was labeled with 32p by the random priming method (12) to a specific activity of 50 IlCij Ilg, and 5 IlCi of this labeled probe was incubated with the nylon membrane in 0.5 M sodium phosphate pH 7.5/7% sodium dodecyl sulfate/1 mM EDTA overnight at 65°C in roller bottle. Excess and loosely bound radioactivity was removed by three washes with 40 mM sodium phosphate buffer pH 7.5/0.1 % sodium dodecyl sulfate for 10 minutes at 65°C. For 1 to 3 days x ray film was exposed to the blot at -70°C with intensifying screens. The probe pDP34 recognizes distinguishable loci on both X and Y chromosome. RESULTS
In Table 1 results of one of three Percoll separations are presented. Motile spermatozoa concentrated in the fractions with the highest Percoll density. The F-body positive spermatozoa remained in the low-density fractions; in the high-density fraction 86% of the spermatozoa was without F-body. Two similar experiments were done, resulting in high-density fractions with 85% sperm without F body and 90% sperm without F body. DNA isolated from the fractionated and unfractionated spermatozoa was analyzed with the probe pDP34 (DXYS1), with which loci on both the X and the Y chromosome are detected (11). These results on the autoradiogram for TaqI digests of male DNA in bands of 14.6 kbp are specific for the Y chromosome and either a band of 11.8 or 10.6 kbp for the
Table 1 Results of a Separation of Sperm Over a Multilayer Discontinuous Percoll Gradient
Percoll density
Sperm concentration *
%
30 35 40 45 50 55 60 65 70 75 80
0.7 1.4 1.3 6.0 10.0 16.0 21.0 23.0 8.0 14.0 60.0
Motility
Percent without Fbody
%
%
0 10 sp:j: 10 10 20 30 30 40 50 80
NDt ND 16 ND ND 42 ND 56 ND ND 86
* The concentration is given in number of spermatozoa/mL. t ND, not determined. :j: sp, sporadically motile cells. van Kooij and van Oost
Sex selection of spermatozoa
385
Figure 1 Southern blot analysis of sperm DNA. Genomic DNA from total and fractioned sperm from a single donor was isolated and analyzed as described in Materials and Methods. For each fraction, 5-l'g aliquots (lanes 2, 4 and 6) and lO-l'g aliquots (lanes 1,3 and 5) were analyzed. See Results for further details.
X chromosome. In Figure 1, the hybridization signals are shown for un fractionated sperm (51 % without F body, lanes 5 and 6), the midportion ofthe gradient (58% without F body, lanes 3 and 4), and the bottom fraction of the gradient (85% without F body, lanes 1 and 2). No difference in intensity between the Yand X-specific band is discernible, which argues against any selection for the X chromosome-bearing spermatozoa. The experiment was completely repeated for two other semen donors with essentially the same results. DISCUSSION
Our results strongly suggest that F -body count can indeed be influenced by Percoll separation. However, the most important finding is that the Fbody negative spermatozoa selected in this way do not seem to be X-bearing sperm, as shown conclusively by the DNA analysis. Use ofthe probe pDP34 has the advantage that the signals of X and Y chromosome can be compared on the same gel, thus making standards of different mixtures of male and female DNA (as had to be done by Beckett et al. [5]) superfluous. The pattern of separation of the sperm samples on Percoll is entirely in accordance with the finding of Aitken and West (13) that the most motile spermatozoa concentrate in the layers of highest density, whereas damaged or dead cells remain at the top layers. It is rather unlikely that in this fraction of less competent cells the majority of the Y-bearing sperm is present. It might be that damaged spermatozoa can be more easily stained with quinacrine. In accordance with the findings of Iizuka et al. (7), we found that even more simple Percoll gradients (2 layers instead of 12) already influenced the percentage of F -body positive cells (results not shown). In our in vitro fertilization program, sperm are washed on a routine basis with Percoll gradients. However, unlike Iizuka et al. (7) have found after
386
van Kooij and van Oost
Sex selection of spermatozoa
insemination with Percoll-separated sperm cells, we found no change in the sex ratio. In conclusion, it is our opinion that techniques for sex selection cannot be validated by F-body count. Furthermore, in our hands Percoll density centrifugation does not influence the ratio of X-bearing sperm cells. Acknowledgments. The authors thank Ms. Elisabeth Franck, Department of Reproductive Medicine, Division of Obstetrics and Gynecology, University Hospital Utrecht and Mr. P. M. van Zandvoort, DNA-Diagnostic Laboratory, Department of Human Genetics, University Hospital Nijmegen, The Netherlands, for their excellent technical assistance and David C. Page, M.D., Whitehead Institute for Biomedical Research, Cambridge, Massachussets for making the pDP34 probe available to us.
REFERENCES 1. Barlow P, Vosa CG. The Y chromosome in human spermatozoa. Nature 1970;226:961-2. 2. Beernink FJ, Ericsson RJ. Male sex preselection through sperm isolation. Fertil Steril 1982;38:493-5. 3. Beatty RA. F-bodies as chromosome markers in mature human sperm heads. Cytogenet Cell Genet 1977;18:33-49. 4. BrandriffB, Gordon L, Ashworth L, Watchmaker G, Moore D, Wyrobeck AJ, et at. Chromosomes of human sperm: variability among normal individuals. Hum Genet 1985;70:1824. 5. Beckett TA, Martin RH, Hoar DI. Assessment ofthe sephadex technique for selection of X-bearing human sperm by analysis of sperm chromosomes, deoxyribonucleic acid and V-bodies. Fertil Steril1989;52:829-35. 6. lizuka R, Kaneko S, Aoki R, Kobayashi T. Sexing of human sperm by discontinuous Percoll density gradient and its clinical application. Hum Reprod 1989;2:573-5. 7. lizuka R, Kaneko S, Kobanawa K, Kobayashi T. Washing and concentration of human semen by Percoll density gradients and its application to AIH. Arch AndroI1988;20:11724. 8. Bahnak BR, Wu QY, Coulombel L, Drouet L, KerbiriouNabias D, Meyer D. A simple and efficient method for isolating high molecular weight DNA from mammalian sperm. Nucleic Acids Res 1988;16:1208. 9. Southern E. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Bioi 1975;98:503-17. 10. Page D, de Martinville B, Barker D, Wyman A, White R, Francke U, et at. Single-copy sequence hybridizes to polymorphic and homologous loci on human X and Y chromosomes. Proc Natl Acad Sci USA 1982;79:5352-6. 11. Moore GE, Ivens A, Chambers J, Farrall M, Williamson R, Page DC, et at. Linkage of an X-chromosome cleft palate gene. Nature 1987;326:91-2. 12. Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983;132:6-13. 13. Aitken RJ, West KM. Analysis of the relationship between reactive oxygen species production and leucocyte infiltration in fractions of human semen separated on Percoll gradients. Int J Androl 1990;13:433-51.
Fertility and Sterility
Vol. 58, No.2, August 1992
Copyright 1992 The American Fertility Society
Printed on ocid·free paper in U.S.A.
Determination of sex ratio of spermatozoa with a deoxyribonucleic acid-probe and quinacrine staining: a comparison
Roelof J. van Kooij, Ph.D.* Bernard A. van Oost, Ph.D.t University Hospital Utrecht, Utrecht, and University Hospital Nijmegen, Nijmegen, The Netherlands
Objective: To evaluate sex selection of spermatozoa. Design: A deoxyribonucleic acid (DNA) probe (pDP34) detecting distinguishable loci on both X and Y chromosome was used to validate the quinacrine-staining method that is often used for determination of the percentage of Y -bearing sperm. Sperm samples were centrifuged over Percoll to obtain samples with a high X:Y ratio according to the quinacrine-staining method. Controls (sperms before processing over Percoll) and processed sperms were subjected to DNA extraction and analysis with the DNA probe. Results: The DNA analysis revealed a 1.0 X:Y ratio ofthe spermatozoa before and after Percoll separation. Conclusion: We conclude that the quinacrine method is not suitable for evaluation of methods that claim to separate X and Y -bearing sperm. Fertil Steril 1992;58:384-6 Key Words: Deoxyribonucleic acid probe pDP34, quinacrine, sex ratio of spermatozoa, Percoll separation
In many of the recent methods describing attempts to separate human X and Y spermatozoa, the success of enrichment is judged solely by quinacrine staining of the Y sperm. This method, originally proposed by Barlow and Vosa (1), is based on determination of the percentage of spermatozoa with a fluorescent spot after quinacrine staining. Because quinacrine staining of human chromosomes causes intense fluorescence in the Y chromosome, it is assumed that the fluorescent spots (F bodies), which can be seen in roughly 40% of the spermatozoa, represent Y bearing gametes. Clinical programs for separation of X and Ybearing sperm cells are based on the F -body test (2). There is, however, considerable doubt that the iden-
Received December 18, 1991; revised and accepted April 15, 1992. * Reprint requests: Roelof J. van Kooij, Ph.D., Department of Reproductive Medicine, Division of Obstetrics and Gynecology, University Hospital Utrecht, Heidelberglaan 100, 3584 ex Utrecht, The Netherlands. t DNA-Diagnostic Laboratory, Department of Human Genetics, University Hospital Nijmegen. 384
van Kooij and van Oost
Sex selection of spermatozoa
tification by quinacrine staining is correct. Not all Y sperm seem to exhibit an F body; on the other hand, the incidence of spermatozoa with two F bodies is high (3). The latter finding is not confirmed by chromosome analysis (4). Beckett et al. (5) did not find a correlation between the F -body test and the content of Y chromosomes estimated with the deoxyribonucleic acid (DNA) probe (pS4). A few years ago it was reported (6) that the Fbody count can be drastically influenced by centrifuging spermatozoa over discontinuous Percoll gradients, whereby the most efficient separation (up to 90% sperm without F body) was obtained after centrifugation of the sperms over as much as 12 layers of Percoll of increasing density. It was reported (6, 7) that the sex ratio of a small series of babies born after insemination with Percoll-separated sperm was changed in favor of the female sex. In this work, we report on the ratio between X and Y spermatozoa, as determined with a single DNA probe in sperm DNA samples, detecting distinguishable loci in both the X and Y chromosome on the one hand and the F -body count on the other hand. We used samples that were enriched for Fertility and Sterility
F -body negative spermatozoa according to the previously mentioned Percoll method (6) and as a control the original semen before Percoll separation. MATERIALS AND METHODS Sperm Selection
Discontinuous Percoll gradients were prepared according to Iizuka et al. (6). Sperm samples of three healthy donors were divided into two parts. One part was used directly for the analysis with the DNA probe. Of the second part, a smear was made for Fbody count, and the rest was diluted with N-[2-hydroxyethyl]piperazine-N 1 _ [2-ethanesulfonic acid] (Hepes)-buffered Ham's F-10 medium (GIBCO, Breda, The Netherlands) and concentrated over 25% Percoll (Pharmacia, Uppsala, Sweden). The pellet was resuspended in 0.7 mL of 25% Percoll and then placed on top of the 12-layer discontinuous gradient. The gradient was centrifuged at 250 X g in a conventional desk top laboratory centrifuge for 30 minutes in a swing-out rotor. The fractions were separated by carefully pipetting the several layers into tubes, and all layers were subjected to a microscopic analysis of concentration and motility. Several fractions were subjected to DNA extraction and analysis (see DNA Isolation). Before starting DNA extraction, a small drop was taken for a smear, fixed, and prepared for F-body count with fluorescence microscopy. F -body counts were scored on coded slides by two observers. DN A Isolation
Twenty to 50 X 106 sperm cells were taken up in 10 mL of 6 M guanidinium HCL, 25 mM sodium citrate, 0.5% Sarkosyl (Sigma Chemical Co., St. Louis, MO), and 0.1 M beta mercaptoethanol (8). Proteinase K (0.25 mg/mL, final concentration; Boehringer, Mannheim, Germany) digestion was carried out overnight at 50°C. DNA was precipitated by addition of 0.7 volumes of isopropanol at room temperature and was subsequently dissolved in 0.5 mL 0.01 M TRIS (pH 7.5) in 1 mM ethylene diamine tetraacetic acid (EDTA). DN A Analysis
Five to 10 11m DNA was digested with 20 to 40 units of TaqI restriction enzyme overnight at 60°C according to the instructions of the manufacturer (GIBCO-BRL, Bethesda). The restricted DNA sample was electrophoresed (1 to 2 V/cm) in 0.9% agarose in 40 mM TRIS pH 7.5/1 mM EDTA. The Vol. 58, No.2, August 1992
separated DNA fragments were denatured and blotted on a nylon membrane (9) in 0.5 N NaOH/1.5 M NaCl. The probe pDP34 (DXYS1) (10, 11) was labeled with 32p by the random priming method (12) to a specific activity of 50 IlCij Ilg, and 5 IlCi of this labeled probe was incubated with the nylon membrane in 0.5 M sodium phosphate pH 7.5/7% sodium dodecyl sulfate/1 mM EDTA overnight at 65°C in roller bottle. Excess and loosely bound radioactivity was removed by three washes with 40 mM sodium phosphate buffer pH 7.5/0.1 % sodium dodecyl sulfate for 10 minutes at 65°C. For 1 to 3 days x ray film was exposed to the blot at -70°C with intensifying screens. The probe pDP34 recognizes distinguishable loci on both X and Y chromosome. RESULTS
In Table 1 results of one of three Percoll separations are presented. Motile spermatozoa concentrated in the fractions with the highest Percoll density. The F-body positive spermatozoa remained in the low-density fractions; in the high-density fraction 86% of the spermatozoa was without F-body. Two similar experiments were done, resulting in high-density fractions with 85% sperm without F body and 90% sperm without F body. DNA isolated from the fractionated and unfractionated spermatozoa was analyzed with the probe pDP34 (DXYS1), with which loci on both the X and the Y chromosome are detected (11). These results on the autoradiogram for TaqI digests of male DNA in bands of 14.6 kbp are specific for the Y chromosome and either a band of 11.8 or 10.6 kbp for the
Table 1 Results of a Separation of Sperm Over a Multilayer Discontinuous Percoll Gradient
Percoll density
Sperm concentration *
%
30 35 40 45 50 55 60 65 70 75 80
0.7 1.4 1.3 6.0 10.0 16.0 21.0 23.0 8.0 14.0 60.0
Motility
Percent without Fbody
%
%
0 10 sp:j: 10 10 20 30 30 40 50 80
NDt ND 16 ND ND 42 ND 56 ND ND 86
* The concentration is given in number of spermatozoa/mL. t ND, not determined. :j: sp, sporadically motile cells. van Kooij and van Oost
Sex selection of spermatozoa
385
Figure 1 Southern blot analysis of sperm DNA. Genomic DNA from total and fractioned sperm from a single donor was isolated and analyzed as described in Materials and Methods. For each fraction, 5-l'g aliquots (lanes 2, 4 and 6) and lO-l'g aliquots (lanes 1,3 and 5) were analyzed. See Results for further details.
X chromosome. In Figure 1, the hybridization signals are shown for un fractionated sperm (51 % without F body, lanes 5 and 6), the midportion ofthe gradient (58% without F body, lanes 3 and 4), and the bottom fraction of the gradient (85% without F body, lanes 1 and 2). No difference in intensity between the Yand X-specific band is discernible, which argues against any selection for the X chromosome-bearing spermatozoa. The experiment was completely repeated for two other semen donors with essentially the same results. DISCUSSION
Our results strongly suggest that F -body count can indeed be influenced by Percoll separation. However, the most important finding is that the Fbody negative spermatozoa selected in this way do not seem to be X-bearing sperm, as shown conclusively by the DNA analysis. Use ofthe probe pDP34 has the advantage that the signals of X and Y chromosome can be compared on the same gel, thus making standards of different mixtures of male and female DNA (as had to be done by Beckett et al. [5]) superfluous. The pattern of separation of the sperm samples on Percoll is entirely in accordance with the finding of Aitken and West (13) that the most motile spermatozoa concentrate in the layers of highest density, whereas damaged or dead cells remain at the top layers. It is rather unlikely that in this fraction of less competent cells the majority of the Y-bearing sperm is present. It might be that damaged spermatozoa can be more easily stained with quinacrine. In accordance with the findings of Iizuka et al. (7), we found that even more simple Percoll gradients (2 layers instead of 12) already influenced the percentage of F -body positive cells (results not shown). In our in vitro fertilization program, sperm are washed on a routine basis with Percoll gradients. However, unlike Iizuka et al. (7) have found after
386
van Kooij and van Oost
Sex selection of spermatozoa
insemination with Percoll-separated sperm cells, we found no change in the sex ratio. In conclusion, it is our opinion that techniques for sex selection cannot be validated by F-body count. Furthermore, in our hands Percoll density centrifugation does not influence the ratio of X-bearing sperm cells. Acknowledgments. The authors thank Ms. Elisabeth Franck, Department of Reproductive Medicine, Division of Obstetrics and Gynecology, University Hospital Utrecht and Mr. P. M. van Zandvoort, DNA-Diagnostic Laboratory, Department of Human Genetics, University Hospital Nijmegen, The Netherlands, for their excellent technical assistance and David C. Page, M.D., Whitehead Institute for Biomedical Research, Cambridge, Massachussets for making the pDP34 probe available to us.
REFERENCES 1. Barlow P, Vosa CG. The Y chromosome in human spermatozoa. Nature 1970;226:961-2. 2. Beernink FJ, Ericsson RJ. Male sex preselection through sperm isolation. Fertil Steril 1982;38:493-5. 3. Beatty RA. F-bodies as chromosome markers in mature human sperm heads. Cytogenet Cell Genet 1977;18:33-49. 4. BrandriffB, Gordon L, Ashworth L, Watchmaker G, Moore D, Wyrobeck AJ, et at. Chromosomes of human sperm: variability among normal individuals. Hum Genet 1985;70:1824. 5. Beckett TA, Martin RH, Hoar DI. Assessment ofthe sephadex technique for selection of X-bearing human sperm by analysis of sperm chromosomes, deoxyribonucleic acid and V-bodies. Fertil Steril1989;52:829-35. 6. lizuka R, Kaneko S, Aoki R, Kobayashi T. Sexing of human sperm by discontinuous Percoll density gradient and its clinical application. Hum Reprod 1989;2:573-5. 7. lizuka R, Kaneko S, Kobanawa K, Kobayashi T. Washing and concentration of human semen by Percoll density gradients and its application to AIH. Arch AndroI1988;20:11724. 8. Bahnak BR, Wu QY, Coulombel L, Drouet L, KerbiriouNabias D, Meyer D. A simple and efficient method for isolating high molecular weight DNA from mammalian sperm. Nucleic Acids Res 1988;16:1208. 9. Southern E. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Bioi 1975;98:503-17. 10. Page D, de Martinville B, Barker D, Wyman A, White R, Francke U, et at. Single-copy sequence hybridizes to polymorphic and homologous loci on human X and Y chromosomes. Proc Natl Acad Sci USA 1982;79:5352-6. 11. Moore GE, Ivens A, Chambers J, Farrall M, Williamson R, Page DC, et at. Linkage of an X-chromosome cleft palate gene. Nature 1987;326:91-2. 12. Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983;132:6-13. 13. Aitken RJ, West KM. Analysis of the relationship between reactive oxygen species production and leucocyte infiltration in fractions of human semen separated on Percoll gradients. Int J Androl 1990;13:433-51.
Fertility and Sterility
