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Stability of Y Chromosome Fluorescence During Freeze-Thawing and Frozen Storage of Human Spermatozoa*
Vol. 25, No.4, April 1974 Printed in U.S.A.
FERTILITY AND STERILITY
Copyright
©
1974 The American Fertility Society
STABILITY OF Y CHROMOSOME FLUORESCENCE DURING FREEZE.THAWING AND FROZEN STORAGE OF HUMAN SPERMATOZOA* J. K. SHERMAN, PH.D.,
I
r
..
•
AND
FLORENCE CHAR, M.D.
Departments of Anatomy and Pediatrics, University of Arkansas Medical Center Little Rock, Arkansas 72201 '
Human spermatozoa have been successfully preserved by freezing and storing at -196°C (the temperature of liquid nitrogen) after glycerol treatment. This method is now a routine laboratory technique with proven clinical applications. About 600 births have resulted from semen which had been stored at -196°C for periods up to 10 years, with fewer abnormal progeny and spontaneous abortions than that of the general population? There is no evidence that mutations are induced by freezing and frozen-storage of any cell type, including about 200 mammalian cell cultures from 40 species, and over 1,600 strains of microorganisms currently banked in an international repository at the American Type Culture Collection, Rockville, Maryland. A study on frozen-storage of 82 normal and neoplastic mammalian tissue culture cells detected no changes in growth rates, isoantigens, strain specific histocompatibility, chromosomal number, and marker chromosomes structure. 2 Practical commercial cattle breeding has revealed no evidence of increased abnormalities or sex ratio change in the millions of progeny from insemination of frozen-stored bovine semen. In basic experimentation, the unchanged nature of single and double stranded DNA after freezing and thawing3 suggests the genetically innocuous nature of cryopresReceived July 10, 1973 . 'Supported in part by the National Science Foundation (GB-36925).
ervation. Frozen-storage has a potential for pr-eserving the original genetic expression as well as for minimizing certain effects of aging on this system, rather than a propensity to induce mutagenic alterations.! In spite of these findings to the contrary, some emotional concern about genetic abnormalities exists. This is reflected, for example, in recent warnings by the United States Public Health Association against the use of frozen-stored human spermatozoa.! The possibility of genetic damage by various chemicals, commonly used drugs, the "pill," and frozen-stored semen theoretically exists. Most probably, the potential mutagenic effect of the "pill" during meiosis or maturation of eggs is much greater than that of freezing and thawing on mature nondividing sp.ermatozoa, and yet the "pill' enjoys WIdespread encouraged use. Nevertheless, evaluation of this possibility with frozen semen should be pursued with current acceptable clinical application. The recently developed technique for the microscopic identification of the Y chromosome in human spermatozoa by induced or secondary fluorescence4 may be useful in the evaluation of one aspect of genetic ( chromosomal) stability during freeze-thawing of human spermatzoa. The purpose of this report is to present ~a~a about the possible effect that cryoInJury and cryoprotection may have on the integrity of the Y chromosome in the human spermatozoon, in terms of its
311
312
April 1974
SHERMAN AND CHAR
capacity to fluoresce. Within defined and appreciated limits, one aspect of genetic stability in cryopreservation of human spermatozoa was, for the first time, directly observed on the chromosome. MATERIALS AND METHODS
Our project was designed to evaluate the effect of freezing and thawing on the fluorescence of the Y chromosome fluorescent (F) -body in human spermatozoa by two different methods. One method is used routinely to preserve successfully a high percentage of spermatozoa in semen samples for clinical therapeutic inseminations. The other method employs the same rates of freezing and thawing as the first, but insures only token survival because it omits glycerol (cryoprotective) pretreatment. In this way, samples of both surviving and nonsurviving spermatozoa can be compared for cryoinjury and cryoprotection on the basis of the ability of their Y chromosomes to fluoresce characteristically. Ejaculates from five healthy young men, 20 to 25 years of age, were used. Each ejaculate was split into two experimental samples. One sample was pretreated at room temperature (22°C) with 15% glycerol by volume; the other sample was not pretreated with glycerol. Aliquots (0.8 cc) to be frozen and thawed were placed on metal canes, frozen in
liquid nitrogen vapor (-196°C), and thawed in a room temperature (22°C) water bath. 5 For frozen-storage evaluation, aliquots from three ejaculates (nos. 1, 3, and 5) were stored in liquid nitrogen for 10 months prior to thawing. In addition, three other samples which had been stored at -196°C for 10, 11, and 13 years were similarly examined after thawing. Three preparations (for motility and fluorescence evaluations) were made for each sample before freezing and after thawing. Percent motility was measured as the average of microscopic (x400) observations, made in triplicate, for each preparation. Fluorescence observations were made on semen smears on glass slides which were fixed in 95 % ethyl alcohol, stained for 15 minutes in 0.5% aqueous quinacrine mustard, placed in citric acid-phosphate buffer (pH 7.0), and mounted with the buffer as coverslip preparations. A Leitz Ortholux microscope with a vertical illuminator, a HBO 200-watt mercury vapor lamp, and a 1.5 mm BG-12 exciter filter were used. About 300 cells were counted on each preparation as to the presence of the F-body, with the average value from three preparations per ejaculate calculated as the percent of the population with F-hodies. Representative photographs were taken with Ektachrome high-speed film for subsequent comparison of fluorescence patterns in nuclei.
TABLE 1. Fluorescence of Y Chromosome in Frozen and Thawed Human Spermatozoa Percent F·boilies···
Percent motilitya
Prefreeze
Post-thaw
Post-thaw
Prefreeze
Ejaculate no.
No glycerol
Glycerol
No glycerol
Glycerol
No glycerol
Glycerol
No glycerol
Glycerol
1 2 3 4 5
70 65 65 55 70
65 65 60 55 70
<1 <1 <1 <1 <1
40 45 50 40 30
39.6 36.3 41.2 37.6 40.9
38.5 34.7 40.4 38.3 37.2
40.1 35.9 38.6 36.5 43.1
37.0 37.1 40.8 36.9 40.5
Average
65
63
<1
41
39.1
37.8
38.8
38.5
aValues based upon observations on three preparations as described in text. /iF-Body = fluorescent body of Y chromosome.
,-------------
Vol. 25, No.4
-
-
--
Y CHROMOSOME FLUORESCENCE STABILITY WHEN FROZEN
313
RESULTS
Neither pretreatment with glycerol nor freezing and thawing with or without glycerol, with accompanying high or low number of motile cells, alters the capacity of Y chromosomes in human spermatozoa to fluoresce in a manner requisite for their identification (Table 1). Also, no difference was noted, by microscopic and photographic examination, in the intensity and pattern of fluorescence 6 exhibited by the general population of spermatozoal nuclei in the human semen examined after all treatments. The percentage of cells seen with Y chromosome F-bodies was within the range of average values previously reported. 4 The figure is less than the expected 50% indicative of the Y chromosomes present because some of the F-bodies were hidden by the cells in the smear. Aliquots from three ejaculates (nos. 1, 3, and 5), which were kept stored in liquid nitrogen (-196°C) for 10 months before thawing, showed no change either in percent motility or in percent Y chromosome F-bodies. The fluorescence pattern of nuclei also remained the same. Spermatozoa from samples stored in liquid nitrogen for 10, 11, and 13 years showed the same qualitative pattern of fluorescence and the same range of values in percentage of F-bodies as the other samples evaluated (Fig. 1). The percent motility of these samples also showed little or no change from the original (O-hour) postthaw value." DISCUSSION
There is no evidence as yet for ultrastructural differentiation of the Y chromosome F-body based upon electron microscopy,',8 but there is evidence for its molecular differentiation as to induced fluorescence. Quinacrine mustard is an alkylating fluorochrome which binds specifically to the DNA of chromosomes,
FIG. 1. Quinacrine-mustard-stained human spermatozoa showing the general pattern of fluorescence and the more intense F-body (arrow) of the Y chromosome. A = before freezing; B = after thawing from 13 years of frozen-storage at -196°C. (X 900).
probably by reacting with guanine-cytosine (G·C)-rich areas. 9 - 12 There also may be an interaction of the fluorochrome with chromosomal protein." Structural abnormalities in DNA and protein in this area of the Y chromosome, therefore, may be reflected by reduction, complete loss, or relocation of the intense fluorescence of the F-body. Our finding that the distinguishing fluorescence of the Y chromosome in human spermatozoa is not altered either by methods which favor or by those which prevent successful cryopreservation up to 13 years has definite but limited meaning as to genetic stability. There is little
314
April 1974
SHERMAN AND CHAR
doubt that the structural configuration of DNA and protein which permits the binding of the fluorochrome, for expression of the distinctive F-body pattern in Y chromosome fluorescence, is not disturbed by glycerol pretreatment, by freezing (and thawing), or by frozen-storage by the nitrogen-vapor technique, with and without glycerol. Also, the maintenance of the general pattern of fluorescence in the nuclei suggests no insult to the molecular basis of such dye-binding in chromatin of other chromosomes, but this should be verified with quantitative techniques. On the basis of staining human spermatozoa with oxidation-reduction potential indicator dyes, it has been demonstrated that freezing and thawing does not modify those proteins of the spermatozoal nucleus which are stainedY Of course, molecular perturbation of the genetic apparatus within or outside of the nucleus could occur during freezing, frozen-storage, and thawing without interference with molecular relations requisite to the expression of fluorescence. Our data do not negate the remote possibility of mutagenic effects of cryopreservation, to be sure, but the data do reduce the likelihood of their occurrence, and support the efficacy of cryopreservation of human semen as a safe clinical application. 1 SUMMARY
One aspect of genetic stability in the cryopreservation of human spermatozoa was treated for the first time at the chromosome level. Characteristic fluorescence of the Y chromosome (F-body), as well as of the other nuclear chromatin, was the criterion used. Comparisons were made before and after freeze-thawing, with and without glycerol, and after up to 13 years of frozen-storage. No evidence for altered integrity of chromosomal fluorescence was noted either
in cryolllJury or in cryoprotection. Data did not rule out possible cryoinduced mutagenetic effects which remain undetected. However, data did support the efficacy and genetic safety of cryopreservat ion of human spermatozoa in the clinical use of frozen-stored semen. REFERENCES 1.
2.
3.
4.
5.
6.
Sherman JK: Synopsis of the use of frozen human semen since 1964: state of the art of human semen banking. Fertil Steril 24: 397, 1973 Hauschka TS, Mitchell JT, Niederpruem DJ: A reliable frozen tissue bank: viability and stability of 82 neoplastic and normal cell types after prolonged storage at -78°C. Cancer Res 19:643, 1959 Ashwood-Smith MJ, Trevino J, Warby C: Effect of freezing on the molecular weight of bacterial DNA. Cryobiology 9:141, 1972 Barlow P, Vosa CG: The Y chromosome in human spermatozoa. Nature (Lond) 226: 961, 1970 Sherman JK: Improved methods of preservation of human spermatozoa by freezing and freeze-drying. Fertil Steril 14:49, 1963 George KP: Cytochemical differentiation along human chromosomes. Nature (Lond) 226:80, 1970
7.
Bedford JM: Observations on the fine structure of spermatozoa of the bush baby (Galago senegalensis), the African green monkey (Cercopithecus aethiops) and man. Am J Anat 121 :443, 1967 8. Pedersen H: Ultrastructure of the ejaculated human sperm. Z Zellforsch Mikrosk Anat 94: 542, 1969 9. Brookes P: In: International Symposium on Chemotherapy of Cancer. Proceedings. Edited by P A Plattner. Amsterdam, Elsevier, 1964 10. Caspersson T, Farber S, Foley GE, et al: Chemical differentiation along metaphase chromosomes. Exp Cell Res 49:219, 1968 11. Caspersson T, Zech L, Modest EJ, et al: Chemical differentiation with fluorescent alkylating agents in Vicia faba metaphase chromosomes. Exp Cell Res 58: 128, 1969 12. Loveless A: Genetic and Allied Effects of Alkylating Agents. University Park, Pennsylvania State University Press, 1966 13. Sherman JK, Bunge RG: Effect of glycerol and freezing on some staining reactions of human spermatozoa. Proc Soc Exp BioI Med 84: 179, 1953
FERTILITY AND STERILITY
Copyright
©
1974 The American Fertility Society
STABILITY OF Y CHROMOSOME FLUORESCENCE DURING FREEZE.THAWING AND FROZEN STORAGE OF HUMAN SPERMATOZOA* J. K. SHERMAN, PH.D.,
I
r
..
•
AND
FLORENCE CHAR, M.D.
Departments of Anatomy and Pediatrics, University of Arkansas Medical Center Little Rock, Arkansas 72201 '
Human spermatozoa have been successfully preserved by freezing and storing at -196°C (the temperature of liquid nitrogen) after glycerol treatment. This method is now a routine laboratory technique with proven clinical applications. About 600 births have resulted from semen which had been stored at -196°C for periods up to 10 years, with fewer abnormal progeny and spontaneous abortions than that of the general population? There is no evidence that mutations are induced by freezing and frozen-storage of any cell type, including about 200 mammalian cell cultures from 40 species, and over 1,600 strains of microorganisms currently banked in an international repository at the American Type Culture Collection, Rockville, Maryland. A study on frozen-storage of 82 normal and neoplastic mammalian tissue culture cells detected no changes in growth rates, isoantigens, strain specific histocompatibility, chromosomal number, and marker chromosomes structure. 2 Practical commercial cattle breeding has revealed no evidence of increased abnormalities or sex ratio change in the millions of progeny from insemination of frozen-stored bovine semen. In basic experimentation, the unchanged nature of single and double stranded DNA after freezing and thawing3 suggests the genetically innocuous nature of cryopresReceived July 10, 1973 . 'Supported in part by the National Science Foundation (GB-36925).
ervation. Frozen-storage has a potential for pr-eserving the original genetic expression as well as for minimizing certain effects of aging on this system, rather than a propensity to induce mutagenic alterations.! In spite of these findings to the contrary, some emotional concern about genetic abnormalities exists. This is reflected, for example, in recent warnings by the United States Public Health Association against the use of frozen-stored human spermatozoa.! The possibility of genetic damage by various chemicals, commonly used drugs, the "pill," and frozen-stored semen theoretically exists. Most probably, the potential mutagenic effect of the "pill" during meiosis or maturation of eggs is much greater than that of freezing and thawing on mature nondividing sp.ermatozoa, and yet the "pill' enjoys WIdespread encouraged use. Nevertheless, evaluation of this possibility with frozen semen should be pursued with current acceptable clinical application. The recently developed technique for the microscopic identification of the Y chromosome in human spermatozoa by induced or secondary fluorescence4 may be useful in the evaluation of one aspect of genetic ( chromosomal) stability during freeze-thawing of human spermatzoa. The purpose of this report is to present ~a~a about the possible effect that cryoInJury and cryoprotection may have on the integrity of the Y chromosome in the human spermatozoon, in terms of its
311
312
April 1974
SHERMAN AND CHAR
capacity to fluoresce. Within defined and appreciated limits, one aspect of genetic stability in cryopreservation of human spermatozoa was, for the first time, directly observed on the chromosome. MATERIALS AND METHODS
Our project was designed to evaluate the effect of freezing and thawing on the fluorescence of the Y chromosome fluorescent (F) -body in human spermatozoa by two different methods. One method is used routinely to preserve successfully a high percentage of spermatozoa in semen samples for clinical therapeutic inseminations. The other method employs the same rates of freezing and thawing as the first, but insures only token survival because it omits glycerol (cryoprotective) pretreatment. In this way, samples of both surviving and nonsurviving spermatozoa can be compared for cryoinjury and cryoprotection on the basis of the ability of their Y chromosomes to fluoresce characteristically. Ejaculates from five healthy young men, 20 to 25 years of age, were used. Each ejaculate was split into two experimental samples. One sample was pretreated at room temperature (22°C) with 15% glycerol by volume; the other sample was not pretreated with glycerol. Aliquots (0.8 cc) to be frozen and thawed were placed on metal canes, frozen in
liquid nitrogen vapor (-196°C), and thawed in a room temperature (22°C) water bath. 5 For frozen-storage evaluation, aliquots from three ejaculates (nos. 1, 3, and 5) were stored in liquid nitrogen for 10 months prior to thawing. In addition, three other samples which had been stored at -196°C for 10, 11, and 13 years were similarly examined after thawing. Three preparations (for motility and fluorescence evaluations) were made for each sample before freezing and after thawing. Percent motility was measured as the average of microscopic (x400) observations, made in triplicate, for each preparation. Fluorescence observations were made on semen smears on glass slides which were fixed in 95 % ethyl alcohol, stained for 15 minutes in 0.5% aqueous quinacrine mustard, placed in citric acid-phosphate buffer (pH 7.0), and mounted with the buffer as coverslip preparations. A Leitz Ortholux microscope with a vertical illuminator, a HBO 200-watt mercury vapor lamp, and a 1.5 mm BG-12 exciter filter were used. About 300 cells were counted on each preparation as to the presence of the F-body, with the average value from three preparations per ejaculate calculated as the percent of the population with F-hodies. Representative photographs were taken with Ektachrome high-speed film for subsequent comparison of fluorescence patterns in nuclei.
TABLE 1. Fluorescence of Y Chromosome in Frozen and Thawed Human Spermatozoa Percent F·boilies···
Percent motilitya
Prefreeze
Post-thaw
Post-thaw
Prefreeze
Ejaculate no.
No glycerol
Glycerol
No glycerol
Glycerol
No glycerol
Glycerol
No glycerol
Glycerol
1 2 3 4 5
70 65 65 55 70
65 65 60 55 70
<1 <1 <1 <1 <1
40 45 50 40 30
39.6 36.3 41.2 37.6 40.9
38.5 34.7 40.4 38.3 37.2
40.1 35.9 38.6 36.5 43.1
37.0 37.1 40.8 36.9 40.5
Average
65
63
<1
41
39.1
37.8
38.8
38.5
aValues based upon observations on three preparations as described in text. /iF-Body = fluorescent body of Y chromosome.
,-------------
Vol. 25, No.4
-
-
--
Y CHROMOSOME FLUORESCENCE STABILITY WHEN FROZEN
313
RESULTS
Neither pretreatment with glycerol nor freezing and thawing with or without glycerol, with accompanying high or low number of motile cells, alters the capacity of Y chromosomes in human spermatozoa to fluoresce in a manner requisite for their identification (Table 1). Also, no difference was noted, by microscopic and photographic examination, in the intensity and pattern of fluorescence 6 exhibited by the general population of spermatozoal nuclei in the human semen examined after all treatments. The percentage of cells seen with Y chromosome F-bodies was within the range of average values previously reported. 4 The figure is less than the expected 50% indicative of the Y chromosomes present because some of the F-bodies were hidden by the cells in the smear. Aliquots from three ejaculates (nos. 1, 3, and 5), which were kept stored in liquid nitrogen (-196°C) for 10 months before thawing, showed no change either in percent motility or in percent Y chromosome F-bodies. The fluorescence pattern of nuclei also remained the same. Spermatozoa from samples stored in liquid nitrogen for 10, 11, and 13 years showed the same qualitative pattern of fluorescence and the same range of values in percentage of F-bodies as the other samples evaluated (Fig. 1). The percent motility of these samples also showed little or no change from the original (O-hour) postthaw value." DISCUSSION
There is no evidence as yet for ultrastructural differentiation of the Y chromosome F-body based upon electron microscopy,',8 but there is evidence for its molecular differentiation as to induced fluorescence. Quinacrine mustard is an alkylating fluorochrome which binds specifically to the DNA of chromosomes,
FIG. 1. Quinacrine-mustard-stained human spermatozoa showing the general pattern of fluorescence and the more intense F-body (arrow) of the Y chromosome. A = before freezing; B = after thawing from 13 years of frozen-storage at -196°C. (X 900).
probably by reacting with guanine-cytosine (G·C)-rich areas. 9 - 12 There also may be an interaction of the fluorochrome with chromosomal protein." Structural abnormalities in DNA and protein in this area of the Y chromosome, therefore, may be reflected by reduction, complete loss, or relocation of the intense fluorescence of the F-body. Our finding that the distinguishing fluorescence of the Y chromosome in human spermatozoa is not altered either by methods which favor or by those which prevent successful cryopreservation up to 13 years has definite but limited meaning as to genetic stability. There is little
314
April 1974
SHERMAN AND CHAR
doubt that the structural configuration of DNA and protein which permits the binding of the fluorochrome, for expression of the distinctive F-body pattern in Y chromosome fluorescence, is not disturbed by glycerol pretreatment, by freezing (and thawing), or by frozen-storage by the nitrogen-vapor technique, with and without glycerol. Also, the maintenance of the general pattern of fluorescence in the nuclei suggests no insult to the molecular basis of such dye-binding in chromatin of other chromosomes, but this should be verified with quantitative techniques. On the basis of staining human spermatozoa with oxidation-reduction potential indicator dyes, it has been demonstrated that freezing and thawing does not modify those proteins of the spermatozoal nucleus which are stainedY Of course, molecular perturbation of the genetic apparatus within or outside of the nucleus could occur during freezing, frozen-storage, and thawing without interference with molecular relations requisite to the expression of fluorescence. Our data do not negate the remote possibility of mutagenic effects of cryopreservation, to be sure, but the data do reduce the likelihood of their occurrence, and support the efficacy of cryopreservation of human semen as a safe clinical application. 1 SUMMARY
One aspect of genetic stability in the cryopreservation of human spermatozoa was treated for the first time at the chromosome level. Characteristic fluorescence of the Y chromosome (F-body), as well as of the other nuclear chromatin, was the criterion used. Comparisons were made before and after freeze-thawing, with and without glycerol, and after up to 13 years of frozen-storage. No evidence for altered integrity of chromosomal fluorescence was noted either
in cryolllJury or in cryoprotection. Data did not rule out possible cryoinduced mutagenetic effects which remain undetected. However, data did support the efficacy and genetic safety of cryopreservat ion of human spermatozoa in the clinical use of frozen-stored semen. REFERENCES 1.
2.
3.
4.
5.
6.
Sherman JK: Synopsis of the use of frozen human semen since 1964: state of the art of human semen banking. Fertil Steril 24: 397, 1973 Hauschka TS, Mitchell JT, Niederpruem DJ: A reliable frozen tissue bank: viability and stability of 82 neoplastic and normal cell types after prolonged storage at -78°C. Cancer Res 19:643, 1959 Ashwood-Smith MJ, Trevino J, Warby C: Effect of freezing on the molecular weight of bacterial DNA. Cryobiology 9:141, 1972 Barlow P, Vosa CG: The Y chromosome in human spermatozoa. Nature (Lond) 226: 961, 1970 Sherman JK: Improved methods of preservation of human spermatozoa by freezing and freeze-drying. Fertil Steril 14:49, 1963 George KP: Cytochemical differentiation along human chromosomes. Nature (Lond) 226:80, 1970
7.
Bedford JM: Observations on the fine structure of spermatozoa of the bush baby (Galago senegalensis), the African green monkey (Cercopithecus aethiops) and man. Am J Anat 121 :443, 1967 8. Pedersen H: Ultrastructure of the ejaculated human sperm. Z Zellforsch Mikrosk Anat 94: 542, 1969 9. Brookes P: In: International Symposium on Chemotherapy of Cancer. Proceedings. Edited by P A Plattner. Amsterdam, Elsevier, 1964 10. Caspersson T, Farber S, Foley GE, et al: Chemical differentiation along metaphase chromosomes. Exp Cell Res 49:219, 1968 11. Caspersson T, Zech L, Modest EJ, et al: Chemical differentiation with fluorescent alkylating agents in Vicia faba metaphase chromosomes. Exp Cell Res 58: 128, 1969 12. Loveless A: Genetic and Allied Effects of Alkylating Agents. University Park, Pennsylvania State University Press, 1966 13. Sherman JK, Bunge RG: Effect of glycerol and freezing on some staining reactions of human spermatozoa. Proc Soc Exp BioI Med 84: 179, 1953