The Effect of Steroid Hormones on Motility and Selective Migration of X- and Y-Bearing Human Spermatozoa*

FERTILITY AND STERILITY Copyright ~ 1976 The American Fertility Society

Vol. 27, No.4, April 1976 Printed in U.SA.

THE EFFECT OF STEROID HORMONES ON MOTILITY AND SELECTIVE MIGRATION OF X- AND Y-BEARING HUMAN SPERMATOZOA* KARL J. BECK, M.D.,t SIEGFRIED HERSCHEL, RAINER HUNGERSHOFER, AND EBERHARD SCHWINGER, M.D. Frauenklinik and Gerichtsmedizinisches Institut der Universitat Bonn, D-5300 Bonn-Venusberg, Federal Republic of Germany

Motility and selective migration of X- and Y-bearing human spermatozoa were studied in the presence of physiologic levels of sex steroid hormones (17{3-estradiol; estriol, 0.5 nglml; testosterone; progesterone; lynestrenol; and norgestrel, 5 nglml) and 200-fold higher concentrations. Estrogens and, to a smaller extent, testosterone accelerated spermatozoal migration, while gestagens had an inhibitory effect. 17{3-Estradiol was most effective in stimulating the motility of human spermatozoa, while norgestrel caused the strongest inhibition offorward movement. Migration of x- and Y_ bearing spermatozoa was most significantly altered after longer distances of migration, shown by an increase in the percentage of Y-bearing spermatozoa from 43.7% to 63.3% at a distance of90 mm. However, prolongation of migration time to 36 hours caused a reduction in the percentage of Y-bearing spermatozoa at the 90-mm distance, from 63.3% to 46%. Specific differential effects of sex steroid hormones on the pattern of selective migration and the distribution of X- and Y-bearing spermatozoa were not observed.

Previous studies from this laboratory have shown that sex steroid hormones are found in the uterine cervical mucus in approximately the same concentrations as in the blood. 1 Furthermore, the hormones are secreted in the cervical mucus after exogenous administration. 2 However, no data are available on the function during the reproductive process of sex steroids present in genital fluids. Therefore, the effects of steroid hormones on the motility of human spermatozoa and on the selective migration of X- and V-bearing spermatozoa were studied.

Received June 24, 1975. *Supported by Grant Be 611/2 from the Deutsche Forschungsgemeinschaft. tReprint requests: Dr. K. J. Beck, UniversitatsFrauenklinik, 53 Bonn-Venusberg, West Germany.

MATERIALS AND METHODS

The following steroids were supplied by Schering AG (Berlin, West Germany) and Organon GmbH (Oss, Netherlands): 17,8-estradiol, estriol, testosterone, progesterone, lynestrenol, and norgestrel. The hormones were dissolved in 20% human albumin (ORHA powder; BehringWerke AG, Marburg, West Germany) and further diluted with Tyrode-Ringer solution. "Low concentrations" used in this study corresponded to the maximal concentrations of 17,8-estradiol and progesterone reached in the blood serum during the menstrual cycle (estrogens, 0.5 ng/ml; gestagens and testosterone, 5 ng/ml); 200-fold higher concentrations d (estrogens, 0.1 JLglml; gestagens an testosterone, 1 JLg/ml), designated "high concentrations," were also included in the study. Tyrode-Ringer solution containing

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tions after the 60- and gO-mm marks had been reached by the spermatozoa. The content of each section was transferred to a microscope slide for staining and evaluation of the Y-chromatin by the following procedure 4 • 5 : The spermatozoa were fixed in an ascending series of alcohol. They were then washed in phosphate buffer From the side (pH 7.0) and stained with aqueous 0.01% quinacrine mustard solution. After a ~F======II!lI!!I=. staining period of approximately 10 minutes, the preparation was rinsed for 10 1'''1 'i"ii"I""j""I,II'i"jiiiil""I"'I"'j""I"'I"iii"j"'I,III"'I'''1""lillIl"'1 minutes in distilled water, embedded in o 10 20 30 40 50 60 70 80 .90 100 110 mm phosphate buffer (pH 7.0), and evaluated FIG. 1. Modified Kremer tube. Left, with a fluorescence microscope (Zeiss Unisperm depository in which the capillary tube is inserted. versal, vertical incidence illumination, HBO-200 filter). At lOOO-fold magnifi0.001 respectively 0.2% albumin served cation, the percentage of Y-bearing speras controls. matozoa was calculated from 300 spermatEjaculates were obtained from fertile, ozoa, counted in each section independenthealthy donors after 5 days of sexual ab- ly by two investigators. stinence. Sperm concentrations varied between 100 and 150 x 106 cells/ml and RESULTS fructose levels between 3 and 4 mg/ml. The maximal distances of spermatozoal The ejaculates were used for determinamigration are presented in Table 1 for tion of the speed of migration within 30 and in Table 2 for lower concentrations minutes after obtaining the specimen. The speed of migration was measured TABLE 1. The Influence of Low Concentrations of in a modified Kremer tube3 (Fig. 1). A Steroid Hormones on Spermatozoal Migration a 50-~1 sample of ejaculate was pipetted into Maximal distance reached after Substance a conical plastic incubation tube (Sarstedt 15 min 30 min 60 min no. 30/8, reduced to a volume of 0.2 ml) mm that served as the sperm depository. Sper- Albumin 27.4 ± 2.1 37.5 ± 2.2 53.5± 2.6 (0.001%) matozoal migration was observed in 13032.0 ± 2.0b 46.5 ± 1.6b 69.9 ± 2.5b mm long capillary tubes with calibrated 17f3-Estradiol (0.5 ng/ml) diameter and volume (100 ~l). The capil- Estriol 31.7 ± 1.3b 45.8 ± 1.5b 65.0 ± 2.5 b (0.5 ng/ml) lary tubes were filled with the solutions Testosterone 31.5 ± l.4b 45.3 ± 2.2b 59.2 ± 2.4b of the steroid hormones tested. The maxi(5 nglml) mal distance of migration was evaluated Progesterone 27.1 ± 1.5 c 35.0 ± l.4 d 50.1 ± 1.5 d (5 nglml) after 15, 30, and 60 minutes. Cell density Lynestrenol 27.0 ± 1.8c 35.8 ± 2.5" 51.2 ± 2.2d was determined by counting the total (5 ng/ml) number of spermatozoa in a visual field Norgestrel 25.2 ± 1.7 d 32.2 ± 2.0b 44.1 ± LIb (5 ng/ml) at 120-fold magnification at the 30-mm migration distance after 30 minutes and aValues are means ± standard deviation of 10 50-mm migration distance after 60 min- experiments. Statistical significance was compared with albumin controls, calculated by Student's t-test. utes. bp < 0.0005. Differential migration of X- and Y-bear"Not significant. ing spermatozoa was determined by cutdp < 0.005. ting the capillary tubes into 10-mm secep < 0.05. From above

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STEROIDS AND SELECTIVE SPERM MIGRATION

higher concentrations of steroid hormones. Values determined after 15 minutes were not significantly different among the different hormones. After a period of 30 minutes a significant difference was observed between hormones stimulating sperm motility (estrogens and testosterone) and those suppressing motility (gestagens). The most distinct difference was found after 60 minutes. Under these conditions, the maximal distance of migration was 84.3 mm in the presence of high concentrations of 17f3estradiol, but only 43.5 mm in the presence of high concentrations of norgestrel. Estrogens and testosterone had a slightly more pronounced stimUlatory effect on the migration of spermatozoa at high concentrations, whereas the suppressing effects of the two concentrations of gestagens were not much different. Figures 2 and 3 show the density of spermatozoa in the presence of high and low hormone concentrations. Identical patterns of effects were observed with both concentrations of hormones. 17f3-Estradiol increased the density of the spermato-

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BECKETAL.

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FIG. 4. Distribution of X- and Y-bearing spermatozoa at different sections of the capillary tubes in the presence of 17{3estradiol (0.1 J.Lg/ml), progesterone (1 J.Lg/ mI), testosterone (1 J.Lg/mI), and albumin (0.2%). Results are expressed as percentage of Y-bearing spermatozoa. Values are means ± standard deviations of 10 experiments.

zoa under all conditions. The effect of estriol was less pronounced, but significant increases were obtained with high concentrations after 30 minutes (30 mm) and 60 minutes (50 mm) and with low concentrations only after 60 minutes (50 mm). Testosterone produced a tendency toward increased sperm density after 60 minutes at 50 mm with both high and low concentrations. Norgestrel inhibited spermatozoal motility, resulting in a significant reduction in cell number at the 30-mm distance and an absence of cells at the 50-mm

distance with both high and low concentrations. Lynestrenol and progesterone tended to reduce the cell density without causing significant differences in comparison with the albumin control. The hormones had no significant effects on the differential migration pattern of X- and Y -bearing spermatozoa when migration was stopped after the cells had reached the 60-mm distance in the capillary tube (Fig. 4). The average percentage of Y-bearing cells was 43.7 ± 0.9% in the ejaculates tested. There was a linear increase in the percentage of Y-bearing spermatozoa with increasing migration distance, ranging from 44.7 to 46.2% in the first 10-mm section, to 56.5 to 58.3% in the last 20-mm section. The increase in the percentage of Y -bearing spermatozoa was even more pronounced with increased migration distances, reaching 63% at 90 mm (Fig. 5). However, ifmigration was allowed to proceed for 36 hours, the percentage of Y-bearing spermatozoa decreased from 63% to 46% at the 90-mm mark, since at that time more X-bearing spermatozoa had reached that point. DISCUSSION

As shown in this study, spermatozoal motility is accelerated by estrogens andless significantly-by testosterone, but is retarded by gestagens. The biochemical

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STEROIDS AND SELECTIVE SPERM MIGRATION

mechanism involved is not yet fully understood. Ericsson et aI.,6 using tritiated sex steroid hormones, found that spermatozoal plasma and nuclei have a greater affinity for estradiol and progesterone than for testosterone. The hormonal effects on spermatozoal motility may be mediated by the adenyl ate cyclase system of cells known to be involved in spermatozoal motility. The inhibitory effect of gestagens on human spermatozoal motility in cervical mucus has also been described by Kesseru et ai. 7 According to Kesseru et aI., progesterone is more effective than norgestrel in suppressing spermatozoal motility. This observation is not in agreement with our findings. The discrepancy may be due to different experimental conditions, since the release ofnorgestrel into the medium, as reported by Kesseru et aI., was only 10% of that of progesterone, resulting in striking differences in the concentration of the medium. Our results confirm that differences exist in the speed offorward movement between X- and Y-bearing spermatozoa. The faster migration of the Y-bearing spermatozoa may be due to the different DNA content, thus causing a difference in size and mass between X- and Y-bearing spermatozoa. s , 9 Therefore, the larger, X-bearing spermatozoa would have to displace a larger volume of fluid during their migration, resulting in a greater demand for energy and slower movement, compared with Y -bearing spermatozoa. It is also possible that unknown differences in cell physiology might explain the different rates of migration. There are no differences in the effects of steroid hormones on type of spermatozoa with regard to stimulation or retardation of movement. The uneven distribution of X- and Y-bearing spermatozoa at the beginning of the experiments (43.7% instead the expected 50%) could be due to the fact that some of the Y-chromatin was not demonstrable by the staining technique and microscopic

411

evaluation. The increase in the percentage of Y-bearing spermatozoa with increasing migration distance supports the thesis of Shettles 10 and of other authors ll , 12 that more males are conceived when intercourse occurs immediately before or after ovulation, since Y-bearing spermatozoa reach the egg sooner than X-bearing spermatozoa. Conversely, more females would result from intercourse taking place a longer time before ovulation, since the eggs more likely would be fertilized by the slower, X-bearing spermatozoa. Our findings demonstrate that optimal conditions for sperm migration not only are induced by biochemical and biophysical changes in the cervical mucus, but also may be improved by the presence of estrogens in high concentrations.

REFERENCES 1. Beck KJ, Leyendecker D, Nocke W: Verglei-

2.

3.

4.

5.

6.

7.

8.

chende Untersuchungen iiber die Konzentrationen vor Oestradiol-1 7f3 und Progesteron im Zervixschleim und Serum. In Third European Congress of Sterility, Athens, October 1972, p 321 Beck KJ, Patt V, Kniille E: Der Ubertritt tritiierten Athinylostradiols und 16-a-Athylthio Retroprogesterons in den Cervixschleim nach oraler Application. In preparation Kremer J: The in vitro spermatozoal penetration test in fertility investigations. Proefschrift, Rijksuniversiteit te Groningen, 1968 Caspersson T, Zech L, Johansson C: Differential binding of alkylating fluorochromes in human chromosomes. Exp Cell Res 60:315, 1970 Pearson PL, Bobrow M: Fluorescent staining of the Y-chromosome in meiotic stages of the human male. J Reprod Fertil 22:177, 1970 Ericsson RJ, Cornette JC, Bathala DA: Binding of sex steroids to rabbit sperm. Acta Endocrinol (Kbh) 56:424, 1967 Kesserii E, Camacho-Ortega P, Laudahn G, Schopflin G: In vitro action of progestagens on sperm migration in human cervical mucus. Fertil Steril 26:57, 1975 Roberts AM: Gravitational separation of X and Y-spermatozoa. Nature 238:223, 1972

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9. Summer AT, Robinson JA, Evans HJ: Distinguishing between X, Y and YY-bearing human spermatozoa by fluorescence and DNA content. Nature [New BioI] 229:231, 1971 10. Shettles LB: Factors influencing sex ratios. Int J Gynecol Obstet 8:643, 1970

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11. Kleegman S: Therapeutic donor insemination. Fertil Steril5:7, 1954 12. Hatzold 0: Die Sexualproportion der Geborenen und ihre Schwankungen als prakonzeptionelles Wahrscheinlichkeitsproblem. Mannheimer Diss., Akademieveroff. Reihe B, Studie 5, Miinchen, 1966