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The Effect of Ion-Exchange Column Chromatography on Separation of X and Y Chromosome-Bearing Human Spermatozoa*
Vol.
FERTILITY AND STERILITY The American Fertility Society Copyright ®
1976
27, No. 10, October 1976 Printed in U.S.A.
THE EFFECT OF ION-EXCHANGE COLUMN CHROMATOGRAPHY ON SEPARATION OF X ANDY CHROMOSOME-BEARING HUMAN SPERMATOZOA* DOUGLAS C. DOWNING, M.S., DONALD L. BLACK, PH.D., WALTER H. CAREY, PH.D., AND DONNA L. DELAHANTY, M.S.
Laboratory for Reproductive Physiology, University of Massachusetts, Amherst, Massachusetts 01002
Separation of X and Y chromosome-bearing spermatozoa has been attempted using ion-exchange column chromatography, with cation- and anion-exchange resins oflow, intermediate, and high ionic strength. Examination ofF-bodies on the Y chromosome of treated human sperm and progeny resulting from insemination of treated rabbit spermatozoa indicates that in none of the cases investigated did the treatment cause a separation of X and Y chromosome-bearing spermatozoa. The treatment does appear to filter out dead rabbit (and bull) spermatozoa, but the possible beneficial effects of this phenomenon are as yet uninvestigated.
The only unquestioned difference between X andY chromosome-bearing spermatozoa is that of chromosome type, and efforts to exploit this difference to permit sex preselection have been extensive. Many of these efforts have involved electrophoresis of various types of spermatozoa1-7; none has been both successful and repeatable. The recent finding that Y chromosome-bearing spermatozoa of human 8 and some other species9 possess an F -body now affords us the means for detecting alterations in the ratio of X and Y chromosome-bearing spermatozoa in a sample. Separation of X- and Y-bearing spermatozoa by Sephadex gel filtration has been reported. 10 In light of recent reports of some degree of success in separation of X- and Y-bearing human sperm, the question of whether there is an exploitable charge differential between the two types of spermatozoa remains unanswered. Accepted May 19, 1976. *Supported in part by a grant from SPD Technology.
The weight difference between the two types of human spermatozoa has been estimated at roughly 4%, 11 and a charge difference related either to mass or chromosome type is therefore conceivable. It was hypothesized that spermatozoa with differing charge characteristics would react differently when passed through a bed of ion-exchange resin. If all spermatozoa carried like charges that differed in intensity, both types would be influenced by a resin, but separation would be impossible since the magnitude of influence would differ. MATERIALS AND METHODS
To test this hypothesis, cation- and anion-exchange resins of high, intermediate, and low ionic strength were used (see Table 1). The resins were equilibrated to pH 6.0, 7 .2, or 8.0 in 0.5 M phosphate-citrate buffer, and packed to a height of 11.5 em in glass or plastic chromatography columns. Semen samples, donated by members of the laboratory, were pooled and washed
1187
1188
TABLE 1. Ion-Exchange Resins Used in Attempts To Separate X- and ¥-Bearing Spermatozoa Resina
October 1976
DOWNING ET AL.
Exchange group
AG50w-X8
Na+
Biorex 63
Na+
Biorex 70
Na+
AGl-X8
Cl-
Biorex 5
Cl-
AG3-X4A
Cl-
TABLE 3. The Effect of Ion-Exchange Treatment on the Recovery and Survival of Rabbit Spermatozoa
Mesh size
Ionic strength
Resin
Strongly acidic Intermediate acidic Weakly acidic Strongly basic Intermediate basic Weakly basic
ph
%Recovery
Survival: % live before treatment) %live after treatment
6.0
41.0 ± 4.2
106.6 ± 1.0
6.0
29.4
112.6
20-50 20-50 20-50
Anionexchange Cationexchange
20-50 20-50 20-50
a Resins were purchased from Biorad Laboratories, Rockville Center, N.Y.
before and after chromatography and airdried for later use in determining the Fbody ratio. Spermatozoal survival was determined by staining samples of the suspension obtained before and after chromatography with an eosin-bluish aniline, live-dead stain. 12 The ratio of X and Y chromosome-bearing human spermatozoa was determined by staining with quinacrine hydrochloride 1 and examination of the cells for F -bodies. Rabbit spermatozoa were treated in the same manner as described for the human. Treated spermatozoa were inseminated into does ovulated with luteinizing hormone or into does superovulated with pregnant mare serum and luteinizing hormone. Blastocysts were recovered surgically 51h to 6 days later and sexed by staining for sex chromatin with buffered thionine. 13 In some cases, the pups were examined and sexed 2 to 3 days postpartum. These animals were normal in all aspects.
once or twice on 0.5 M phosphate-citrate buffer at pH 7.2 and resuspended in the same buffer at the pH used in equilibration of the ion-exchange resin. The concentration of cells was approximately 20 to 50 x 106 cells/mi. Rabbit semen samples were collected with an artificial vagina and prepared in the same manner. One milliliter of the spermatozoal suspension was layered on top of the resin bed in the column and passed into the resin by allowing the column to flow. After the entire sample had entered the resin, additional buffer was added, and the flow through the column was maintained at 3 to 4 ml/minute. Eight milliliters of effluent were collected and the spermatozoa were concentrated by centrifuging for 15 minutes at 1200 x g. The supernatant was discarded and the sperRESULTS matozoa were resuspended in phosphatecitrate buffer to the desired concentration. Since no differences were found between Smears of the suspensions were made resins of differing ionic strength, they are TABLE 2. The Effect of Ion-Exchange Treatment on the Recovery and Survival ofHuman Spermatozoa Resin
Anionexchange Cationexchange
pH
6.0 7.2 8.0 6.0 7.2 8.0
%Recovery
48.1 52.6 43.5 22.5 45.4 36.3
Survival: % live before treatment!% live after treatment
41.2 43.6 62.6 34.2 48.0 85.7
TABLE 4. The Effect of Ion-Exchange Treatment on the Separation of X andY Chromosome-Bearing Human Spermatozoa % Cells with F ·body
Resin
Anionexchange Cationexchange Control
pH6.0
pH 7.2
pH 8.0
44.5
44.0
46.5
45.4
45.8
46.3
43.7 ± 4.8
45.1 ± 5.2
47.7 ± 5.7
Vol. 27, No. 10
1189
SEPARATION OF X AND Y CHROMOSOME-BEARING SPERMATOZOA
TABLE 5. The Effect of Ion-Exchange Treatment of Rabbit Semen on the Sex of the Early Embryo Resin
Cation-exchange Anion-exchange a Includes
Total no. of blastocysts or young examineda
No. of males
N a. of females
115 431
56 221
210
I
48.7 51.3
24 2- to 3-day young: 16 from cation-exchange and 8 from anion-exchange treatment.
referred to hereafter collectively as anion or cation exchangers. Recovery of spermatozoa following chromatography was generally between 20 and 50% with both the human and rabbit spermatozoa (Tables 2 and 3).
f
59
%Males
to 6-day blastocysts has recently been reported to be 48.6%. 15 DISCUSSION
Ion-exchange column chromatography, under the conditions which were used The effect of chromatography on human in these experiments, appears to have no and rabbit spermatozoal survival differed. the separation of human and effect on The treatment tended to kill a large porrabbit X and Y chromosome-bearing spertion of the human spermatozoa; survival matozoa. This finding is consistent with (% live before treatment/% live after treatmuch of the data suggesting that the ment) ranged from 20 to 90%, with more charge difference between the two types being killed at the lower pH. With rabbit spermatozoa, many of the dead sperm of spermatozoa cannot be utilized to effect were retained on the column, so that the separation. Disagreement with other repercentage oflive spermatozoa after treat- ports claiming success may be attribument was greater (106 to 112%) than in table to differences in accuracy of experithe original sample. This phenomenon is mental methods or possibly to speciespattern not unique to rabbit spermatozoa; in the specific differences in amount and 16 of spermatozoal surface charge. bull the effect is even more pronounced There is considerable doubt as to (103 to 187% ). 14 whether ion-exchange phenomena could Chromatography did not separate X- occur in these experiments. Although the and Y-bearing human spermatozoa (Table smallest resin particles are 3 to 4 times the 4). The percentage of cells showing F- length of the relatively large bull sperbodies ranged from 43.7 ± 4.8 SD to matozoon, a spermatozoon is enormous 47.7 ± 5.7 SD in control samples, and in comparison with the sodium or chloride from 42.3 ± 8.2 SD to 50 ± 4.3 SD exchange groups. Alternatively, there after chromatography. No significant may have been an electrostatic reaction differences were found between groups. between the spermatoon and the exchange Chromatography of rabbit sperm also groups covering the resin surface. If had no apparent effect on the separation this were the case, the reaction between of X- and Y-bearing sperm (Table 5). spermatozoa and resin would be opposite Of 115 blastocysts or young resulting from that expected for ion exchange. Regardinsemination of cation-exchange resin- less of the method of action, the fact treated spermatozoa, 56 were male and 59 remains that there was no significant female, a sex ratio (percentage male) of separation of the X- and Y-bearing sper48.7%. Of 431 blastocysts or young matozoa. A difference in the magnitude resulting from insemination of anion- of the charge, if one actually exists, canexchange resin-treated spermatozoa, 221 not be demonstrated with ion-exchange were male and 210 female, a sex ratio column chromatography under the conof 51.3%. The normal sex ratio for 5lh- ditions which were used in these experi-
DOWNING ET AL.
1190
ments. Only dead rabbit spermatozoa appeared to be influenced, and they were retained on both resins (Table 3). REFERENCES 1. Schroder VN: Physico-chemical analysis of the physiology of spermatozoa. V. Artificial control of sex in mammals. Anim Br Abstr 3:166, 1934 2. Gordon MJ: The control of sex: if sperm cells from a rabbit are placed in an electric field, they separate into two groups. One group, when it is used to inseminate female rabbits, tends to produce male offspring; the other, female. Sci Am 199:87, Nov 1958 3. Bangham AD: Electrophoretic characteristics of ram and rabbit spermatozoa. Proc R Soc Lond [Biol) 155:292, 1961 4. Pilz A: Das Verhalten der Saugetierspermien im Elektrischen Feld. Z Tierzucht Zuchbiol 60:315, 1952 5. Vesselinovitch SD: Microelectrophoresis of bovine spermatozoa. Cornell Vet 49:359, 1959 6. Sevinc A: Experiments on sex control by electrophoretic separation of spermatozoa in the rabbit. J Reprod Fertil16:7, 1968 7. Hafs HD, Boyd LJ: Galvanic separation of Xand Y-chromosome-bearing sperm. In Sex Ratio at Birth-Prospects for Control, Edited by CA Kiddy, HD Hafs. Albany NY, Boyd Printing, 1970, p 85
October 1976
8. Barlow P, Vosa CG: The Y chromosome in human spermatozoa Nature 226:961, 1970 9. Pearson PL, Babrow M, Vosa CG, Barlow PW: Quinacrine fluorescence in mammalian chromosomes. Nature 231:326, 1971 10. Steeno 0, Adimoelja A, Steeno J: Separation of X- andY-bearing human spermatozoa with the Sephadex gel-filtration method. Andrologia 7:95, 1975 11. Evans HJ: Properties of X and Y sperm. In Edinburgh Symposium on the Genetics of the Spermatozoon, Edited by RA Beatty, S Gluecksohn-Waelsch. Copenhagen, Bogtrykkeriet Forum, 1972, p 144 12. Schaffer HE, Almquist JA: Vital staining of bovine spermatozoa with an eosin-aniline blue staining mixture. J Dairy Sci 31:677, 1948 13. Klinger HP, Ludwig KS: A universal stain for the sex chromatin body. Stain Techno! 32:235, 1957 14. Downing DC, Black DL, Carey WH, Delahanty DL: Unpublished data 15. Fechheimer NS, Beatty RA: Chromosomal abnormalities and sex ratio in rabbit blastocysts. J Reprod Fertil 37:331, 1974 16. Yanagimachi R, Noda YD, Fujimoto M, Nicolson GL: The distribution of negative surface charges on mammalian spermatozoa. Am J Anat 135:497, 1973
FERTILITY AND STERILITY The American Fertility Society Copyright ®
1976
27, No. 10, October 1976 Printed in U.S.A.
THE EFFECT OF ION-EXCHANGE COLUMN CHROMATOGRAPHY ON SEPARATION OF X ANDY CHROMOSOME-BEARING HUMAN SPERMATOZOA* DOUGLAS C. DOWNING, M.S., DONALD L. BLACK, PH.D., WALTER H. CAREY, PH.D., AND DONNA L. DELAHANTY, M.S.
Laboratory for Reproductive Physiology, University of Massachusetts, Amherst, Massachusetts 01002
Separation of X and Y chromosome-bearing spermatozoa has been attempted using ion-exchange column chromatography, with cation- and anion-exchange resins oflow, intermediate, and high ionic strength. Examination ofF-bodies on the Y chromosome of treated human sperm and progeny resulting from insemination of treated rabbit spermatozoa indicates that in none of the cases investigated did the treatment cause a separation of X and Y chromosome-bearing spermatozoa. The treatment does appear to filter out dead rabbit (and bull) spermatozoa, but the possible beneficial effects of this phenomenon are as yet uninvestigated.
The only unquestioned difference between X andY chromosome-bearing spermatozoa is that of chromosome type, and efforts to exploit this difference to permit sex preselection have been extensive. Many of these efforts have involved electrophoresis of various types of spermatozoa1-7; none has been both successful and repeatable. The recent finding that Y chromosome-bearing spermatozoa of human 8 and some other species9 possess an F -body now affords us the means for detecting alterations in the ratio of X and Y chromosome-bearing spermatozoa in a sample. Separation of X- and Y-bearing spermatozoa by Sephadex gel filtration has been reported. 10 In light of recent reports of some degree of success in separation of X- and Y-bearing human sperm, the question of whether there is an exploitable charge differential between the two types of spermatozoa remains unanswered. Accepted May 19, 1976. *Supported in part by a grant from SPD Technology.
The weight difference between the two types of human spermatozoa has been estimated at roughly 4%, 11 and a charge difference related either to mass or chromosome type is therefore conceivable. It was hypothesized that spermatozoa with differing charge characteristics would react differently when passed through a bed of ion-exchange resin. If all spermatozoa carried like charges that differed in intensity, both types would be influenced by a resin, but separation would be impossible since the magnitude of influence would differ. MATERIALS AND METHODS
To test this hypothesis, cation- and anion-exchange resins of high, intermediate, and low ionic strength were used (see Table 1). The resins were equilibrated to pH 6.0, 7 .2, or 8.0 in 0.5 M phosphate-citrate buffer, and packed to a height of 11.5 em in glass or plastic chromatography columns. Semen samples, donated by members of the laboratory, were pooled and washed
1187
1188
TABLE 1. Ion-Exchange Resins Used in Attempts To Separate X- and ¥-Bearing Spermatozoa Resina
October 1976
DOWNING ET AL.
Exchange group
AG50w-X8
Na+
Biorex 63
Na+
Biorex 70
Na+
AGl-X8
Cl-
Biorex 5
Cl-
AG3-X4A
Cl-
TABLE 3. The Effect of Ion-Exchange Treatment on the Recovery and Survival of Rabbit Spermatozoa
Mesh size
Ionic strength
Resin
Strongly acidic Intermediate acidic Weakly acidic Strongly basic Intermediate basic Weakly basic
ph
%Recovery
Survival: % live before treatment) %live after treatment
6.0
41.0 ± 4.2
106.6 ± 1.0
6.0
29.4
112.6
20-50 20-50 20-50
Anionexchange Cationexchange
20-50 20-50 20-50
a Resins were purchased from Biorad Laboratories, Rockville Center, N.Y.
before and after chromatography and airdried for later use in determining the Fbody ratio. Spermatozoal survival was determined by staining samples of the suspension obtained before and after chromatography with an eosin-bluish aniline, live-dead stain. 12 The ratio of X and Y chromosome-bearing human spermatozoa was determined by staining with quinacrine hydrochloride 1 and examination of the cells for F -bodies. Rabbit spermatozoa were treated in the same manner as described for the human. Treated spermatozoa were inseminated into does ovulated with luteinizing hormone or into does superovulated with pregnant mare serum and luteinizing hormone. Blastocysts were recovered surgically 51h to 6 days later and sexed by staining for sex chromatin with buffered thionine. 13 In some cases, the pups were examined and sexed 2 to 3 days postpartum. These animals were normal in all aspects.
once or twice on 0.5 M phosphate-citrate buffer at pH 7.2 and resuspended in the same buffer at the pH used in equilibration of the ion-exchange resin. The concentration of cells was approximately 20 to 50 x 106 cells/mi. Rabbit semen samples were collected with an artificial vagina and prepared in the same manner. One milliliter of the spermatozoal suspension was layered on top of the resin bed in the column and passed into the resin by allowing the column to flow. After the entire sample had entered the resin, additional buffer was added, and the flow through the column was maintained at 3 to 4 ml/minute. Eight milliliters of effluent were collected and the spermatozoa were concentrated by centrifuging for 15 minutes at 1200 x g. The supernatant was discarded and the sperRESULTS matozoa were resuspended in phosphatecitrate buffer to the desired concentration. Since no differences were found between Smears of the suspensions were made resins of differing ionic strength, they are TABLE 2. The Effect of Ion-Exchange Treatment on the Recovery and Survival ofHuman Spermatozoa Resin
Anionexchange Cationexchange
pH
6.0 7.2 8.0 6.0 7.2 8.0
%Recovery
48.1 52.6 43.5 22.5 45.4 36.3
Survival: % live before treatment!% live after treatment
41.2 43.6 62.6 34.2 48.0 85.7
TABLE 4. The Effect of Ion-Exchange Treatment on the Separation of X andY Chromosome-Bearing Human Spermatozoa % Cells with F ·body
Resin
Anionexchange Cationexchange Control
pH6.0
pH 7.2
pH 8.0
44.5
44.0
46.5
45.4
45.8
46.3
43.7 ± 4.8
45.1 ± 5.2
47.7 ± 5.7
Vol. 27, No. 10
1189
SEPARATION OF X AND Y CHROMOSOME-BEARING SPERMATOZOA
TABLE 5. The Effect of Ion-Exchange Treatment of Rabbit Semen on the Sex of the Early Embryo Resin
Cation-exchange Anion-exchange a Includes
Total no. of blastocysts or young examineda
No. of males
N a. of females
115 431
56 221
210
I
48.7 51.3
24 2- to 3-day young: 16 from cation-exchange and 8 from anion-exchange treatment.
referred to hereafter collectively as anion or cation exchangers. Recovery of spermatozoa following chromatography was generally between 20 and 50% with both the human and rabbit spermatozoa (Tables 2 and 3).
f
59
%Males
to 6-day blastocysts has recently been reported to be 48.6%. 15 DISCUSSION
Ion-exchange column chromatography, under the conditions which were used The effect of chromatography on human in these experiments, appears to have no and rabbit spermatozoal survival differed. the separation of human and effect on The treatment tended to kill a large porrabbit X and Y chromosome-bearing spertion of the human spermatozoa; survival matozoa. This finding is consistent with (% live before treatment/% live after treatmuch of the data suggesting that the ment) ranged from 20 to 90%, with more charge difference between the two types being killed at the lower pH. With rabbit spermatozoa, many of the dead sperm of spermatozoa cannot be utilized to effect were retained on the column, so that the separation. Disagreement with other repercentage oflive spermatozoa after treat- ports claiming success may be attribument was greater (106 to 112%) than in table to differences in accuracy of experithe original sample. This phenomenon is mental methods or possibly to speciespattern not unique to rabbit spermatozoa; in the specific differences in amount and 16 of spermatozoal surface charge. bull the effect is even more pronounced There is considerable doubt as to (103 to 187% ). 14 whether ion-exchange phenomena could Chromatography did not separate X- occur in these experiments. Although the and Y-bearing human spermatozoa (Table smallest resin particles are 3 to 4 times the 4). The percentage of cells showing F- length of the relatively large bull sperbodies ranged from 43.7 ± 4.8 SD to matozoon, a spermatozoon is enormous 47.7 ± 5.7 SD in control samples, and in comparison with the sodium or chloride from 42.3 ± 8.2 SD to 50 ± 4.3 SD exchange groups. Alternatively, there after chromatography. No significant may have been an electrostatic reaction differences were found between groups. between the spermatoon and the exchange Chromatography of rabbit sperm also groups covering the resin surface. If had no apparent effect on the separation this were the case, the reaction between of X- and Y-bearing sperm (Table 5). spermatozoa and resin would be opposite Of 115 blastocysts or young resulting from that expected for ion exchange. Regardinsemination of cation-exchange resin- less of the method of action, the fact treated spermatozoa, 56 were male and 59 remains that there was no significant female, a sex ratio (percentage male) of separation of the X- and Y-bearing sper48.7%. Of 431 blastocysts or young matozoa. A difference in the magnitude resulting from insemination of anion- of the charge, if one actually exists, canexchange resin-treated spermatozoa, 221 not be demonstrated with ion-exchange were male and 210 female, a sex ratio column chromatography under the conof 51.3%. The normal sex ratio for 5lh- ditions which were used in these experi-
DOWNING ET AL.
1190
ments. Only dead rabbit spermatozoa appeared to be influenced, and they were retained on both resins (Table 3). REFERENCES 1. Schroder VN: Physico-chemical analysis of the physiology of spermatozoa. V. Artificial control of sex in mammals. Anim Br Abstr 3:166, 1934 2. Gordon MJ: The control of sex: if sperm cells from a rabbit are placed in an electric field, they separate into two groups. One group, when it is used to inseminate female rabbits, tends to produce male offspring; the other, female. Sci Am 199:87, Nov 1958 3. Bangham AD: Electrophoretic characteristics of ram and rabbit spermatozoa. Proc R Soc Lond [Biol) 155:292, 1961 4. Pilz A: Das Verhalten der Saugetierspermien im Elektrischen Feld. Z Tierzucht Zuchbiol 60:315, 1952 5. Vesselinovitch SD: Microelectrophoresis of bovine spermatozoa. Cornell Vet 49:359, 1959 6. Sevinc A: Experiments on sex control by electrophoretic separation of spermatozoa in the rabbit. J Reprod Fertil16:7, 1968 7. Hafs HD, Boyd LJ: Galvanic separation of Xand Y-chromosome-bearing sperm. In Sex Ratio at Birth-Prospects for Control, Edited by CA Kiddy, HD Hafs. Albany NY, Boyd Printing, 1970, p 85
October 1976
8. Barlow P, Vosa CG: The Y chromosome in human spermatozoa Nature 226:961, 1970 9. Pearson PL, Babrow M, Vosa CG, Barlow PW: Quinacrine fluorescence in mammalian chromosomes. Nature 231:326, 1971 10. Steeno 0, Adimoelja A, Steeno J: Separation of X- andY-bearing human spermatozoa with the Sephadex gel-filtration method. Andrologia 7:95, 1975 11. Evans HJ: Properties of X and Y sperm. In Edinburgh Symposium on the Genetics of the Spermatozoon, Edited by RA Beatty, S Gluecksohn-Waelsch. Copenhagen, Bogtrykkeriet Forum, 1972, p 144 12. Schaffer HE, Almquist JA: Vital staining of bovine spermatozoa with an eosin-aniline blue staining mixture. J Dairy Sci 31:677, 1948 13. Klinger HP, Ludwig KS: A universal stain for the sex chromatin body. Stain Techno! 32:235, 1957 14. Downing DC, Black DL, Carey WH, Delahanty DL: Unpublished data 15. Fechheimer NS, Beatty RA: Chromosomal abnormalities and sex ratio in rabbit blastocysts. J Reprod Fertil 37:331, 1974 16. Yanagimachi R, Noda YD, Fujimoto M, Nicolson GL: The distribution of negative surface charges on mammalian spermatozoa. Am J Anat 135:497, 1973