Equality in Survival of X and Y Chromosome-Bearing Human Spermatozoa*

Vol. 27, No. 10, October 1976 Printed in U.SA.

FERTILITY AND STERILITY Copyright © 1976 The American Fertility Society

EQUALITY IN SURVIVAL OF X ANDY CHROMOSOME-BEARING HUMAN SPERMATOZOA* DOUGLAS C. DOWNING, M.S.,

AND

DONALD L. BLACK, PH.D.

Department of Veterinary and Animal Sciences, Laboratory for Reproductive Physiology, University of Massachusetts, Amherst, Massachusetts 01002

Human X andY chromosome-bearing spermatozoa survived equally well during washing and resuspension in buffers ofpH 52 and 8.0 and during incubation in these buffers for 11 hours at 37' C. This suggests that alteration ofthe ratio ofliving X- andY-bearing spermatozoa by direct treatment is not an effective method of sex ratio alteration.

A frequently made assumption in studies of spermatozoa and sex predetermination is that X and Y chromosomebearing spermatozoa survive equally well under various conditions. It has not been shown, however, that X- and Y-bearing spermatozoa do in fact survive in equal numbers during normal washing procedures. Furthermore, there is conflicting evidence on the equal survival ofX- andYbearing spermatozoa during incubation in various media and under certain experimental conditions. Sex predetermination has been attempted through pH-related in vivo treatment of the male or female and by in vitro treatment of spermatozoa. Since Unterberger 1 reported successful alteration of the human sex ratio, the effect of pH on the sex ratio has been questioned. Precoital vaginal douches of varying concentrations of acids or bases has produced significant alterations of the sex ratio in some cases 2 • 4 and not in others. 4 Direct treatment of rabbit 5 • 8 and swine spermatozoa9 with substances Accepted May 6, 1976. *Supported in part by Grant 13-056300 from the SPD Technology Fund and Grants 2T01-HD00204 and 2T01-HD00204-07 from the National Institutes of Health.

of varying pH failed to pt
Semen used in these investigations was donated by healthy volunteers. The semen of each man had been regularly examined over a period of 2 years and judged morphologically normaL Incubations were carried out in a phosphate-citrate buffer1 used extensively in this laboratory, containing both benzylpenicillin and streptomycin. Experiment 1: Parameters of Washing and Incubation. Twelve individual semen samples were maintained at 37' C until liquefaction had occurred. The samples were then divided in half, diluted 1:1 with phosphate-citrate buffer at pH 7 .2, and centrifuged at 1200 x g for 15 minutes. After removal of the supernatants, the cells were resuspended in buffer

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DOWNING AND BLACK

October 1976

of pH 5.2 or 8.0. One-milliliter samples were incubated at 37° C for 11 hours in W arburg incubation flasks attached to Warburg respiration manometers. The atmosphere in each flask was air and C02 absorbed by 10% KOH in the center well. The pH of each sample was determined before and after incubation. Experiment II: Survival of X and Y Chromosome-Bearing Spermatozoa. Each of three semen samples was washed, and the cells were resuspended in buffer of pH 5.2 and incubated as before. Livedead stained slides were prepared from the spermatozoal suspension both after washing and after incubation. 11 Only spermatozoa from flasks showing oxygen consumption during incubation were utilized for survival determinations. The partial pressure of oxygen in these flasks never fell below 100 mm Hg-well above the critical range of 4 to 5 mm Hg. 12 This experiment was later repeated, using semen from the same donors and buffer ofpH 8.0. Survival of X and Y chromosome-bearing spermatozoa was determined by a sequential staining technique. 2 Eight live-dead stained slides per sample were used for this purpose.

the buffer pH. Spermatozoal suspensions in buffer of pH 5.2 averaged pH 5.43, and those in buffer of pH 8.0 averaged pH 7 .66. Incubation caused a small, insignificant, decrease in pH: postincubation pH values were 5.38 and 7.51, respectively. The percentage of live spermatozoa in the low-pH sample fell from 51.0% postwash to 26.1% postincubation (Table 1). In the higher-pH sample these figures were 58.9% postwash and 34.9% postincubation, indicating comparable decreases in percentage live of 29.6% and 24.0% in the two groups (Table 1). Of the six samples used in experiment II, the percentage live fell 14 to 40% during incubation, and five of the six changes were significant at the 5% level (Table 2). There were no significant differences postwash or postincubation between percentage of live cells with visible F-bodies at either pH. Classification of roughly 6000 live cells showed the percentage of Y-bearing cells in the live population to be 49.6% in the buffer of pH 5.2 and 49.0% in the buffer of pH 8.0 After incubation, the percentage of live cells with an F-body was 49.6% in both cases (Table

RESULTS

DISCUSSION

2).

Resuspension of washed spermatozoa This work confirms the widely held asin buffer of pH 5.2 and 8.0 slightly altered sumption that X and Y chromosomeTABLE 1. The Effect of Washing and Incubation in Phosphate-Citrate Buffer of pH 52 and 8.0 on Survival of Human Spermatozoa Buffer pH

Experiment and sample no. a

% Live spermatozoa 0 Sperm concentration

Postwash

Postincubation

Change

10 6 cellslml

5.2 5.2 5.2 5.2

I (12 samples) 11-1 11-2 11-3

92.5 ± 47.5 56.6 74.0 85.8

51.0 57.8 56.4 73.6

± ± ± ±

6.3 3.8 1.9 2.8

26.1 20.8 42.4 34.5

± ± ± ±

11.7 4.4 3.3 5.0

-29.6 -37.0 -14.0 -39.1

8.0 8.0 8.0 8.0

I (12 samples) 11-1 11-2 11-3

87.7 ± 43.1 54.7 55.7 204.0

58.9 58.4 65.9 81.4

± ± ± ±

10.3 6.3 2.6 3.5

34.9 29.8 25.4 51.4

± ± ± ±

11.1 2.1 3.3 2.2

-24.0 -28.6 -40.5 -30.0

"Significant difference between means of 11-1, 11-3, 11-2, and 11-3 (}' < 0.05). bValues are means ± standard deviation.

Vol. 27, No. 10

SURVIVAL OF X AND Y CHROMOSOME-BEARING SPERMATOZOA

TABLE 2. The Effect of Washing and Incubation in Buffer of pH 52 and 8.0 on Survival of X and Y Chromosome-Bearing Spermatozoa %F-bodies in live cellsa

Buffer pH

Sample no. Postwash

5.2

1 2 3

Mean 8.0

Mean

1 2 3

Postincubation

49.5 ± 2.1 48.8 ± 3.8 50.4 ± 2.7

48.3 ± 1.9 50.5 ± 1.1 50.1 ± 3.7

49.6 ± 0.8b

49.6 ± 1.2b

50.0 ± 4.1 48.8 ± 2.0 48.2 ± 2.6

49.6 ± 3.5 49.9 ± 3.3 49.2 ± 3.9

49.0 ± 0.9b

49.6 ± 0.4b

avalues are means ± standard deviation. bNo significant differences between means.

bearing spermatozoa survive in equal numbers during normal washing procedures, and that the ratio of live spermatozoa at ejaculation is equality. The exposure of spermatozoa to buffers of differing pH did not result in differential survival of the two types of spermatozoa. This finding suggests that alteration of pH is not an effective method of producing populations rich in X- and Ybearing spermatozoa and cannot be used as an effective method of sex selection. To the extent that these in vitro conditions compare with in vivo conditions, this work further suggests that spermatozoal survival in the female reproductive tract is probably equal, thereby resulting in a 5~ primary sex ratio. This conclusion depends upon two conditions: first, that equal numbers of X and Y spermatozoa at the site of fertilization result in equal fertilization by the two types; second, that motility and speed of arrival at the site of fertilization is not an important factor in determining which spermatozoon participates in fertilization. There is no evidence that X and Y spermatozoa are different in their ability to fertilize on ovum. The ability of the Y spermatozoon to reach the ovum earlier than its heavier counterpart has been proposed. 13 • 14 Differential spermatozoal survival could be of primary importance

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in situations in which insemination precedes ovulation, or in species having a long capacitation time. In vivo aging of spermatozoa, however, has no effect on the sex ratio. 15-17 REFERENCES 1. Unterberger F: Das Problem der wilkiirlichen Beeinflussung des Geschlechts beim Menschen. Dtsch Med Wochenschr 56:304, 1930 2. Schii.pfer W: Beitrag zur wilkiirlichen Beeinflussung des Geschlechts. Z Zuchtungskunde 12:232, 1937 3. Roberts E: The effect of lactic acid and sodium bicarbonate on the sex ratio. J Hered 31:499, 1940 4. Quisenberry JH, Chandiramani SV: Experimental attempt to modify the sex ratio: in rats and rabbits. J Hered 31:503, 1940 5. Cole LJ, Waletzky E, Shakelford M: A test of sex control by modification of the acid-alkaline balance. J Hered 31:501, 1940 6. Casida LE, Murphree RE: Fertility and sex ratios in the rabbit: from semen treated in vitro with lactic acid and sodium bicarbonate. J Hered 33:434, 1942 7. Quisenberry JH: Additional data on sex ratio in rabbits. J Hered 36:160, 1945 8. Emmens CW: Insemination pH and the sex ratio in rabbits. J Hered 51:156, 1960 9. McPhee HC, Eaton ON: Experimental attempts to modify the sex ratio in rabbits and pigs. J Hered 33:429, 1942 10. Shettles LB: Factors influencing sex ratios. Int J Gynecol Obstet 8:643, 1970 11. Wakim PE: Determining the sex of baby rabbits by ascertaining the pH of the vagina of the mother before mating. Am J Osteopath Assoc 72:173, 1972 12. Downing DC, Black DL: Unpublished data 13. Nevo AC: Dependence of sperm motility and respiration on oxygen concentration. J Reprod Fertil 9:103, 1965 14. Ericsson RJ, Langevin CN, Nishino M: Isolation of fractions rich in human Y sperm. Nature 246:421, 1973 15. Tesh JM: Effects of the aging of rabbit spermatozoa in utero on fertilization and prenatal development. J Reprod Fert 20:299, 1969 16. Vickers AD: Delayed fertilization and the prenatal sex-ratio of the mouse. J Reprod Fertil 20:63, 1969 17. Koefoed..Johnsen HH, Pavlok A, Fulka J: The influence of the aging of rabbit spermatozoa in vitro on fertilizing capacity and embryonic mortality. J Reprod Fertil 26:351, 1971