Letters to the Editor
Am. J. Hum. Genet. 47:349-351, 1990
Sex Ratio among Sperm Cells To the Editor:
Barry Bean presents the argument that the gametic sex ratio may not be 1:1 as theoretically expected. He uses the term "progenitive sex ratio" to refer to the Y:X chromosomal ratio among mature and functional sperm cells. Bean's argument is based on two sources of evidence: Y-body (or F-body) analysis of human sperm and analysis of the chromosome complements of human sperm after fusion of human sperm with hamster oocytes. He maintains that both of these techniques demonstrate an excess of X-bearing sperm. The majority of laboratories using the Y-body technique have reported significantly less than 50% of sperm cells with a fluorescent Y body. Bean has stated that this is consistent with a progenitive sex ratio of 0.9 or less. With his definition of progenitive sex ratio, there is no reason to assume that the Y-body stains only "functional sperm." The quinacrine stain would surely not distinguish either a motile sperm from an immotile sperm or a sperm capable of capacitation from a sperm missing an acrosome. Thus the Y-body test would provide information on gametic - and not on "progenitive" sex ratio. A far more significant problem is the reliability of the Y-body assay. As Bean mentions, several researchers have questioned the validity of this technique (Wyroi 1990 by The American Society of Human) Genetics., All rights reserved. 0002-9297/90/4702-0024$02.00
bek et al. 1984; Martin 1985; Brandriff et al. 1986; Templado et al. 1988; Beckett et-al. 1989). Thomsen and Niebuhr (1986) reported false-negative results in 11% of interphase nuclei from 457 males. In haploid sperm cells, 30%-50% of cells demonstrate a Y-body (Beckett et al. 1989). It is possible that this lower frequency of sperm cells with a Y-body reflects a true gametic sex ratio that is less than one; however, it is more likely that it represents uneven uptake of the quinacrine dye, fading of the dye before analysis, or variations in Yqh + chromosomal material. Bean remarks on the need for "definitive experiments on the correlation of quinacrine F-bodies and direct molecular tests for the sex chromosomes." I have some unpublished observations on the correlation between the percentage of Y-bearing sperm when' fluorescent Y-body analysis is used and that when molecular analysis with a Y-preferential DNA probe (pS4) is used. In a study on the assessment of the sephadex technique for selection of X-bearing human sperm, my graduate student, Teresa Beckett, used analysis of sperm chromosomes, DNA, and Y-bodies (Beckett et al. 1989). She also performed detailed correlations between the DNA and Y-body analyses on the same sperm samples. Sperm samples were diluted with BWW medium and were passed through a sephadex column. Thirteen 1-ml fractions were collected from the column. An aliquot of the unseparated sperm sample, as well as all of the separated sperm-containing fractions, were used for DNA and Y-body analysis. The sperm samples were lysed for DNA extraction and were digested with restriction endonuclease MboI, and membrane-bound 349
350
DNA was hybridized to a 32P-radiolabeled pS4 probe. The percentage of Y chromosome-bearing sperm was determined by comparing the Y-specific 2.2-kb band with the autosomal 2.0-kb band by using a laser densitometer. A set of standards was established for each sperm specimen by mixing DNA from female peripheral blood and DNA from male peripheral blood. The mixing standards consisted of 100%, 87.5%, 75%, 50%, 37.5%, 25%, and 12.5% male DNA and a 100% female DNA standard. All of these samples were processed concurrently on each gel run with the separated sperm fractions, to provide a ratio ofthe two bands for each experiment. This ratio was plotted against the percentage of male DNA in the mixture by using linear regression analysis to create a standard curve exhibiting a positive linear relationship. All of the standards were pooled according to the percentage of male DNA and then were analyzed again to demonstrate a highly significant positive linear correlation between the pooled ratios and the percentage of male DNA (r = .58, P < .0001). These values were used to determine a linear regression equation which allowed us to convert the ratio of the two bands to a percentage of male DNA. The Y-body test was performed on aliquots from both the unseparated semen sample and all of the spermcontaining fractions, according to a modified version (Beckett et al. 1989) of Barlow and Vosa's (1970) staining technique. To minimize observer bias, the slides from two or three experiments were coded and scored for Y-bodies at one time. Estimates, from both the DNA analysis and the Y-body test, of the percentage of sperm containing a Y chromosome were compared for all of the separated fractions plus the unseparated sample. There was a lack of correlation between the two tests for all samples (Pearson's correlation coefficient [P > .05 for each comparison]). I agree that further tests on the correlation between the indirect Y-body test and direct molecular tests are needed, but our results to date indicate that the Y-body assay is not a reliable indicator for the presence of a Y chromosome and that therefore not much weight can be given to a "progenitive sex ratio" based on Y-body analysis. Bean also makes the observation that a number of studies of haploid human sperm chromosome complements report a slight excess of X-bearing sperm, although these have generally not been statistically significant (Martin et al. 1983; Brandriff et al. 1984, 1986; Kamiguchi and Mikamo 1986; Templado et al. 1988). He reanalyzed the data of Kamiguchi and Mikamo (1986) and found that their Y:X ratio of .88
Letters to the Editor
was, in fact, marginally significant (X2 = 4.4, P = .04). He also determined that a recent study by my graduate student, Judy Chernos, on the effect of cryopreservation on the frequency of chromosomal abnormalities in human sperm showed a significant excess of X-bearing sperm when the data were analyzed in toto (Chernos and Martin 1989). We focused on the comparison of sperm karyotypes from fresh versus cryopreserved sperm and found that the sex ratios did not differ significantly for the two groups; nor did they differ from the theoretical 1 : ratio expected. However, if prefreeze and postfreeze numbers are lumped, the excess of X-bearing sperm is marginally significant (X2 = 4.9, P = .025). These results stimulated me to analyze the most recent data from our laboratory, on 5,750 sperm chromosome complements from 83 normal control donors and on 1,647 complements from 18 donors with constitutional chromosomal abnormalities (such as translocations). In both groups, and also in a combined analysis of 7,397 sperm, there is a statistically significant excess of X-bearing sperm. These data suggest that the gametic sex ratio may be less than one, with a slight excess of X-bearing sperm. Alternatively, in my in vitro culture system there may be a small selective advantage in the ability of X- versus Y-bearing human sperm to penetrate hamster oocytes with the zona pellucida removed (Martin 1983). This could be tested by in situ hybridization of sex chromosome-specific DNA probes to ascertain the sex ratio in sperm, and this research is currently in progress in my laboratory. RENEE H. MARTIN Division of Medical Genetics
University of Calgary
Calgary Acknowledgments I would like to thank Evelyn Ko for analyzing the sex ratio of sperm in our most recent data and Lynne Bell for typing the manuscript. R. H. M. is a Medical Scientist supported by the Alberta Heritage Fund for Medical Research, and her research is supported by the Medical Research Council of Canada and by the Alberta Children's Hospital Research Foundation. References Barlow P, Vosa CG (1970) The Y chromosome in human sperm. Nature 226:961-962 Beckett TA, Martin RM, Hoar DI (1989) Assessment of the
Letters to the Editor sephadex technique for selection of X-bearing human sperm by analysis of sperm chromosomes, deoxyribonucleic acid and Y-bodies. Fertil Steril 52:829-835 Brandriff B, Gordon L, Ashworth L, Watchmaker G, Carrano A, Wyrobek A (1984) Chromosomal abnormalities in human sperm: comparisons among four healthy men. Hum Genet 66:193-201 Brandriff BF, Gordon LA, Haendel S, Singer S, Moore DH, Gledhill BL (1986) Sex chromosome ratios determined by karyotypic analysis in albumin-isolated human sperm. Fertil Steril 46:678-685 Chernos JE, Martin RH (1989) A cytogenetic investigation of the effects of cryopreservation on human sperm. Am J Hum Genet 45:766-777 Kamiguchi Y, Mikamo K (1986) An improved, efficient method for analyzing human sperm chromosomes using zona-free hamster ova. Am J Hum Genet 38:;724-740 Martin RH (1983) A detailed method for obtaining preparations of human sperm chromosomes. Cytogenet Cell Genet 35:253-256 (1985) Recognition of the Y chromosome in human spermatozoa. In: Sandberg AA (ed) The Y chromosome, part A: Basic characteristics of the Y chromosome. Alan R. Liss, New York, pp. 403-417 Martin RH, Balkan W, Burns K, Rademaker AW, Lin CC, Rudd NL (1983) The chromosome constitution of 1000 human spermatozoa. Hum Genet 63:305-309 Templado C, Benet J, Genesca A, Navarro J, Caballin MR, Miro R. Egozcue J (1988) Human sperm chromosomes. Hum Reprod 3:133-138 ThomsenJL, Niebuhr E (1986) The frequency of false-positive and false-negative results in the detection of Y-chromosomes in interphase nuclei. Hum Genet 73:27-30 Wyrobek A, Watchmaker G, Gordon L, Carrano AV, Ashworth L, Brandriff B (1984) Aneuploidy and quinacrinepositive spots in human sperm. Am J Hum Genet 36:118S
Am. J. Hum. Genet. 47:351-353, 1990
Progenitive Sex Ratio among Functioning Sperm Cells To the Editor:
The proportions of mature sperm cells bearing Y versus X chromosomes in normal human ejaculates is often assumed to be 1:1. However, there has been considerable speculation that the sperm sex-chromosome composition may deviate from this proportion because of the influence of differential postmeiotic gene expres-
351
sion, selective loss during sperm development, or differential survival or functioning of the sperm following maturation.
The biological literature includes extensive treatment of human diploid sex ratios, usually expressed as the proportionate representation of male to female individuals at a specified stage of the life cycle. The sex ratio at conception, called the "primary sex ratio;" is usually approximated only by extrapolation. The sex ratio at birth is commonly labeled the "secondary sex ratio" and is calculated from birth records to be about 1.06, or 106 male births/100 female births, for many human groups. The natural and clinical variables that influence secondary sex ratio have recently been discussed by Zarutskie et al. (1989). Depending on the stage of development, the sex-chromosome ratios in the meiotic and postmeiotic germ line might better be called the haplophase, gametic, progenitive, or prezygotic sex ratios of the relevant cell population within individuals of the heterogametic sex. I will use the term "progenitive sex ratio" to refer specifically to the Y:X chromosomal ratio among mature and functional sperm cells. The progenitive sex ratio is a conceptual device, an abstraction useful to focus thinking. For humans, especially, we cannot expect to glean precise direct information about sperm function and early conceptions. We can attempt inferences about progenitive sex ratio indirectly from related experimental observations. Compelling data on gametic sex ratio have been rare in the past, but improved techniques, enabling more sophisticated analyses, are now at hand. Several methodologies are possible and ideally should be used conjointly to permit distinction of the sex-chromosome ratios among mature and functioning spermatozoa. For many years, the common method for approximating ratios of Y- versus X-bearing sperm has involved visualization by fluorescence microscopy of "f-bodies" (also known as "Y-bodies") following staining with quinacrines. While scoring reqhes skill, patience, and experience, quinacrine staining can give values that are reproducible to within a few percent. Essentially all laboratories using the f-body methods detect less than 50% presumptive Y-bearing cells, usually reporting values of 44%-48%. Such values are consistent with progenitive sex ratio calculations of about .9. In my laboratory, an improved dual staining method (i.e., the QMEB method) has been developed (Levine and Bean 1988). This method has now been used to study sperm from donors with known chromosomal variants. It is important that a donor lacking the long arm of the Y chromosome gives an f-body score of only
Am. J. Hum. Genet. 47:349-351, 1990
Sex Ratio among Sperm Cells To the Editor:
Barry Bean presents the argument that the gametic sex ratio may not be 1:1 as theoretically expected. He uses the term "progenitive sex ratio" to refer to the Y:X chromosomal ratio among mature and functional sperm cells. Bean's argument is based on two sources of evidence: Y-body (or F-body) analysis of human sperm and analysis of the chromosome complements of human sperm after fusion of human sperm with hamster oocytes. He maintains that both of these techniques demonstrate an excess of X-bearing sperm. The majority of laboratories using the Y-body technique have reported significantly less than 50% of sperm cells with a fluorescent Y body. Bean has stated that this is consistent with a progenitive sex ratio of 0.9 or less. With his definition of progenitive sex ratio, there is no reason to assume that the Y-body stains only "functional sperm." The quinacrine stain would surely not distinguish either a motile sperm from an immotile sperm or a sperm capable of capacitation from a sperm missing an acrosome. Thus the Y-body test would provide information on gametic - and not on "progenitive" sex ratio. A far more significant problem is the reliability of the Y-body assay. As Bean mentions, several researchers have questioned the validity of this technique (Wyroi 1990 by The American Society of Human) Genetics., All rights reserved. 0002-9297/90/4702-0024$02.00
bek et al. 1984; Martin 1985; Brandriff et al. 1986; Templado et al. 1988; Beckett et-al. 1989). Thomsen and Niebuhr (1986) reported false-negative results in 11% of interphase nuclei from 457 males. In haploid sperm cells, 30%-50% of cells demonstrate a Y-body (Beckett et al. 1989). It is possible that this lower frequency of sperm cells with a Y-body reflects a true gametic sex ratio that is less than one; however, it is more likely that it represents uneven uptake of the quinacrine dye, fading of the dye before analysis, or variations in Yqh + chromosomal material. Bean remarks on the need for "definitive experiments on the correlation of quinacrine F-bodies and direct molecular tests for the sex chromosomes." I have some unpublished observations on the correlation between the percentage of Y-bearing sperm when' fluorescent Y-body analysis is used and that when molecular analysis with a Y-preferential DNA probe (pS4) is used. In a study on the assessment of the sephadex technique for selection of X-bearing human sperm, my graduate student, Teresa Beckett, used analysis of sperm chromosomes, DNA, and Y-bodies (Beckett et al. 1989). She also performed detailed correlations between the DNA and Y-body analyses on the same sperm samples. Sperm samples were diluted with BWW medium and were passed through a sephadex column. Thirteen 1-ml fractions were collected from the column. An aliquot of the unseparated sperm sample, as well as all of the separated sperm-containing fractions, were used for DNA and Y-body analysis. The sperm samples were lysed for DNA extraction and were digested with restriction endonuclease MboI, and membrane-bound 349
350
DNA was hybridized to a 32P-radiolabeled pS4 probe. The percentage of Y chromosome-bearing sperm was determined by comparing the Y-specific 2.2-kb band with the autosomal 2.0-kb band by using a laser densitometer. A set of standards was established for each sperm specimen by mixing DNA from female peripheral blood and DNA from male peripheral blood. The mixing standards consisted of 100%, 87.5%, 75%, 50%, 37.5%, 25%, and 12.5% male DNA and a 100% female DNA standard. All of these samples were processed concurrently on each gel run with the separated sperm fractions, to provide a ratio ofthe two bands for each experiment. This ratio was plotted against the percentage of male DNA in the mixture by using linear regression analysis to create a standard curve exhibiting a positive linear relationship. All of the standards were pooled according to the percentage of male DNA and then were analyzed again to demonstrate a highly significant positive linear correlation between the pooled ratios and the percentage of male DNA (r = .58, P < .0001). These values were used to determine a linear regression equation which allowed us to convert the ratio of the two bands to a percentage of male DNA. The Y-body test was performed on aliquots from both the unseparated semen sample and all of the spermcontaining fractions, according to a modified version (Beckett et al. 1989) of Barlow and Vosa's (1970) staining technique. To minimize observer bias, the slides from two or three experiments were coded and scored for Y-bodies at one time. Estimates, from both the DNA analysis and the Y-body test, of the percentage of sperm containing a Y chromosome were compared for all of the separated fractions plus the unseparated sample. There was a lack of correlation between the two tests for all samples (Pearson's correlation coefficient [P > .05 for each comparison]). I agree that further tests on the correlation between the indirect Y-body test and direct molecular tests are needed, but our results to date indicate that the Y-body assay is not a reliable indicator for the presence of a Y chromosome and that therefore not much weight can be given to a "progenitive sex ratio" based on Y-body analysis. Bean also makes the observation that a number of studies of haploid human sperm chromosome complements report a slight excess of X-bearing sperm, although these have generally not been statistically significant (Martin et al. 1983; Brandriff et al. 1984, 1986; Kamiguchi and Mikamo 1986; Templado et al. 1988). He reanalyzed the data of Kamiguchi and Mikamo (1986) and found that their Y:X ratio of .88
Letters to the Editor
was, in fact, marginally significant (X2 = 4.4, P = .04). He also determined that a recent study by my graduate student, Judy Chernos, on the effect of cryopreservation on the frequency of chromosomal abnormalities in human sperm showed a significant excess of X-bearing sperm when the data were analyzed in toto (Chernos and Martin 1989). We focused on the comparison of sperm karyotypes from fresh versus cryopreserved sperm and found that the sex ratios did not differ significantly for the two groups; nor did they differ from the theoretical 1 : ratio expected. However, if prefreeze and postfreeze numbers are lumped, the excess of X-bearing sperm is marginally significant (X2 = 4.9, P = .025). These results stimulated me to analyze the most recent data from our laboratory, on 5,750 sperm chromosome complements from 83 normal control donors and on 1,647 complements from 18 donors with constitutional chromosomal abnormalities (such as translocations). In both groups, and also in a combined analysis of 7,397 sperm, there is a statistically significant excess of X-bearing sperm. These data suggest that the gametic sex ratio may be less than one, with a slight excess of X-bearing sperm. Alternatively, in my in vitro culture system there may be a small selective advantage in the ability of X- versus Y-bearing human sperm to penetrate hamster oocytes with the zona pellucida removed (Martin 1983). This could be tested by in situ hybridization of sex chromosome-specific DNA probes to ascertain the sex ratio in sperm, and this research is currently in progress in my laboratory. RENEE H. MARTIN Division of Medical Genetics
University of Calgary
Calgary Acknowledgments I would like to thank Evelyn Ko for analyzing the sex ratio of sperm in our most recent data and Lynne Bell for typing the manuscript. R. H. M. is a Medical Scientist supported by the Alberta Heritage Fund for Medical Research, and her research is supported by the Medical Research Council of Canada and by the Alberta Children's Hospital Research Foundation. References Barlow P, Vosa CG (1970) The Y chromosome in human sperm. Nature 226:961-962 Beckett TA, Martin RM, Hoar DI (1989) Assessment of the
Letters to the Editor sephadex technique for selection of X-bearing human sperm by analysis of sperm chromosomes, deoxyribonucleic acid and Y-bodies. Fertil Steril 52:829-835 Brandriff B, Gordon L, Ashworth L, Watchmaker G, Carrano A, Wyrobek A (1984) Chromosomal abnormalities in human sperm: comparisons among four healthy men. Hum Genet 66:193-201 Brandriff BF, Gordon LA, Haendel S, Singer S, Moore DH, Gledhill BL (1986) Sex chromosome ratios determined by karyotypic analysis in albumin-isolated human sperm. Fertil Steril 46:678-685 Chernos JE, Martin RH (1989) A cytogenetic investigation of the effects of cryopreservation on human sperm. Am J Hum Genet 45:766-777 Kamiguchi Y, Mikamo K (1986) An improved, efficient method for analyzing human sperm chromosomes using zona-free hamster ova. Am J Hum Genet 38:;724-740 Martin RH (1983) A detailed method for obtaining preparations of human sperm chromosomes. Cytogenet Cell Genet 35:253-256 (1985) Recognition of the Y chromosome in human spermatozoa. In: Sandberg AA (ed) The Y chromosome, part A: Basic characteristics of the Y chromosome. Alan R. Liss, New York, pp. 403-417 Martin RH, Balkan W, Burns K, Rademaker AW, Lin CC, Rudd NL (1983) The chromosome constitution of 1000 human spermatozoa. Hum Genet 63:305-309 Templado C, Benet J, Genesca A, Navarro J, Caballin MR, Miro R. Egozcue J (1988) Human sperm chromosomes. Hum Reprod 3:133-138 ThomsenJL, Niebuhr E (1986) The frequency of false-positive and false-negative results in the detection of Y-chromosomes in interphase nuclei. Hum Genet 73:27-30 Wyrobek A, Watchmaker G, Gordon L, Carrano AV, Ashworth L, Brandriff B (1984) Aneuploidy and quinacrinepositive spots in human sperm. Am J Hum Genet 36:118S
Am. J. Hum. Genet. 47:351-353, 1990
Progenitive Sex Ratio among Functioning Sperm Cells To the Editor:
The proportions of mature sperm cells bearing Y versus X chromosomes in normal human ejaculates is often assumed to be 1:1. However, there has been considerable speculation that the sperm sex-chromosome composition may deviate from this proportion because of the influence of differential postmeiotic gene expres-
351
sion, selective loss during sperm development, or differential survival or functioning of the sperm following maturation.
The biological literature includes extensive treatment of human diploid sex ratios, usually expressed as the proportionate representation of male to female individuals at a specified stage of the life cycle. The sex ratio at conception, called the "primary sex ratio;" is usually approximated only by extrapolation. The sex ratio at birth is commonly labeled the "secondary sex ratio" and is calculated from birth records to be about 1.06, or 106 male births/100 female births, for many human groups. The natural and clinical variables that influence secondary sex ratio have recently been discussed by Zarutskie et al. (1989). Depending on the stage of development, the sex-chromosome ratios in the meiotic and postmeiotic germ line might better be called the haplophase, gametic, progenitive, or prezygotic sex ratios of the relevant cell population within individuals of the heterogametic sex. I will use the term "progenitive sex ratio" to refer specifically to the Y:X chromosomal ratio among mature and functional sperm cells. The progenitive sex ratio is a conceptual device, an abstraction useful to focus thinking. For humans, especially, we cannot expect to glean precise direct information about sperm function and early conceptions. We can attempt inferences about progenitive sex ratio indirectly from related experimental observations. Compelling data on gametic sex ratio have been rare in the past, but improved techniques, enabling more sophisticated analyses, are now at hand. Several methodologies are possible and ideally should be used conjointly to permit distinction of the sex-chromosome ratios among mature and functioning spermatozoa. For many years, the common method for approximating ratios of Y- versus X-bearing sperm has involved visualization by fluorescence microscopy of "f-bodies" (also known as "Y-bodies") following staining with quinacrines. While scoring reqhes skill, patience, and experience, quinacrine staining can give values that are reproducible to within a few percent. Essentially all laboratories using the f-body methods detect less than 50% presumptive Y-bearing cells, usually reporting values of 44%-48%. Such values are consistent with progenitive sex ratio calculations of about .9. In my laboratory, an improved dual staining method (i.e., the QMEB method) has been developed (Levine and Bean 1988). This method has now been used to study sperm from donors with known chromosomal variants. It is important that a donor lacking the long arm of the Y chromosome gives an f-body score of only
