Serum hormone levels affect sperm function*

Transcription

1 FERTILITY AND STERILITY Copyright 1990 The American Fertility Society Vol. 54. No.1. July 1990 Printed on acid-free paper in U.S.A. Serum hormone levels affect sperm function* Mike T. Mbizvo, D.Phil.t Sharon Thomas, B.S. David L. Fulgham, B.S. Nancy J. Alexander, Ph.D.t The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia If hormone levels affect sperm motility during capacitation, then the serum added to samples prepared for artificial insemination could affect sperm fertilizability. We investigated various sperm functional movement characteristics (percent motile, progressive velocity, linearity, beat cross frequency, lateral head displacement, longevity, and hyperactivation) in specimens incubated with women's sera, as well as exogenous hormone preparations of estradiol (E2) and progesterone (P). Early follicular phase serum (low E2 and low P) maintained motility and longevity. Sperm in E2-treated medium exhibited higher progressive velocity, linear motility, and longevity. In contrast, the percent motility decreased as sperm exhibited hyperactivated motility with P. Fertil Steril54:113, 1990 During sperm capacitation, changes in movement characteristics consistent with fertilization have been described, including the expression of hyperactivated motility in animal 1-3 and human 4,5 spermatozoa. The quality of sperm movement is an important consideration for fertilization; optimal sperm performance is a prerequisite for successful therapeutic insemination. Components in serum, including albumin, provide a milieu appropriate for sperm function in vitro. Sperm movement and for- Received November 20, 1989; revised and accepted March 1, * Supported by a grant of the Contraceptive Research and Development Program (CONRAD), Eastern Virginia Medical School, under a Cooperative Agreement (DPE-2644-A ) with the United States Agency for International Development (AID). The views expressed by the authors do not necessarily reflect the views of AID. t Recipient of a CONRAD Fellowship at the Jones Institute for Reproductive Medicine. :j: Reprint requests: Nancy J. Alexander, Ph.D., Director, Fundamental Research, The Jones Institute for Reproductive Medicine, Eastern Virginai Medical School, 855 W. Brambleton Avenue, Norfolk, Virginia ward progression are an interactive process between the sperm cell and the surrounding environment. The flagellar bend propagation and modifications of the axoneme are modulated by both physical and biochemical properties of the microenvironment. These changes in the kinetics of sperm cell movement are functionally significant during in vivo sperm transport and ovum penetration. In vivo, epithelial surfaces and secretions of the female tract modulate sperm motility. Sperm migration through cervical mucus or a buffered albumin solution in vitro is affected by the addition of steroids. 6 7 In fact, specific steroid binding sites have been reported to be present on human spermatozoa.s Recently, progesterone (P) and 17 a-hydroxyprogesterone have been shown to stimulate calcium influx in capacitated human sperm 9 and to initiate the acrosome reaction. to Such findings point to an important relationship between P and estradiol (E 2 ) levels in serum or capacitation medium and sperm longevity, as well as alterations in sperm movement characteristics. The effects of these steroids on sperm motion parameters are described. Vol. 54, No.1, July 1990 Mbizvo et al. Hormone effects on sperm motion 113

2 MATERIALS AND METHODS Subject Selection and Sample Collection Semen samples were obtained from healthy, normal volunteer men after 48 hours sexual abstinence. Semen was collected by masturbation into sterile, wide-mouth plastic containers provided by the laboratory. Samples were allowed to liquefy for 30 minutes at room temperature before processing. Blood was collected every 4 days from seven regularly cycling volunteer women who were not using hormonal contraception. Blood samples were processed to separate serum, which was aliquoted and frozen. Hormone levels were assayed by radioimmunoassay (RIA), using for E2, the direct 1251 Estradiol-17{3 kit (Radio System Laboratory, ICN Diagnostic Division, Los Angeles, CA); and for P, the Coat-a-Count Progesterone kit (Diagnostics Product Corporation, Los Angeles, CA). The remaining aliquots were used for incubation with sperm samples. Semen Sample Processing and Incubation After liquefaction, 0.5 ml of semen was underlayed in 2 ml of Biggers, Whitten, and Whittingham buffer, to which 0.35% bovine serum albumin (BSA) was added, and incubated at 37 C for 90 minutes. Washed swim-up sperm with ±80% motile cells were pooled, and the motile cell concentration brought to 70 X 10 6 /ml. On average, three donor specimens were pooled and the motile cell concentration adjusted before each evaluation. Biggers, Whitten, and Whittingham medium with 7.5% ofthe serum under study was mixed with 2 X 10 6 /ml sperm and allowed to incubate at 37 C under 5% CO2 + 95% air in 0.1 ml volumes in sterile tissue culture "U" well microplates (#3595, Costar; Cambridge, MA). Commercial E2 and 4- pregnene-3,20-dione (Sigma Chemical Company, St. Louis, MO) were added to Biggers, Whitten, and Whittingham medium to concentrations of 400 pg/ml and 12 ng/ml, respectively. The E2 and P were serially diluted to give five stepwise concentrations (E2, 104 to 8.6 pg/ml) and (P, 313 to 26 pg/ml). These were placed in separate microplate wells, to which at least 4 X 10 6 swim-up sperm were added and incubated in 115 ~L final volumes. Normal cycling women's sera (code named CH17 and T012) were run in addition to Biggers, Whitten, and Whittingham medium as serum control for E2 samples; serum T021 and T24 were serum controls for P samples. At least three pooled donor semen specimens were used per evaluation of five hormone concentrations plus controls and eight evaluations were made on each hormone concentration, from which mean values were derived. Assessment of Motility The motility patterns of capacitating sperm were analyzed immediately after mixing the sperm with serum or treated medium at time zero (T = 0), followed by 1-, 4-, and 24-hour time points. Analysis was performed in a 37 C prewarmed Makler Chamber (Sefi-Medical Instruments, Haifa, Israel), using a Nikon model Labphot microscope (Nikon Inc., Tokyo, Japan) with an X-10 reverse phase contrast objective. The microscope was interfaced with a Panasonic videocamera through a PL X-5 lens (Panasonic CCTV Model-WV-1410; Matsushita Communication Industrial Co., Tokyo, Japan). This was connected to two video monitors interfacing with a version 3.0 Cellsoft Computer Analyzer (Cellsoft Automated Semen Analyzer [CASA], Cellsoft; CRYO Resources Ltd., New York, NY). The CASA used digitized gray level images to identify sperm cells. The threshold gray level was adjusted before each evaluation to capture all motile cells, with the pixel scale set at ~m/pixel. The cell size (pixels) range was low 4 and high 25 analyzing 20 frames at 30 frames per second. The threshold velocity was set at 10 ~m/s, and maximum velocity at 150 ~m/s. The distribution of motility parameters of the analyzed cells, at increasing time intervals, was computed for subsequent analysis of variance (ANOVA). Due to variation in individual semen samples, and before statistical analysis, the data were transformed into difference units. Each serum or treated medium sample was analyzed and the T = 0 value was treated as the baseline. All subsequent determinations were subtracted from the T = 0 value to give a difference unit value. This transformation controlled for semen sample variation. ll The individual difference values for each sample classification (low E2/low P, high E2/low P, high E2/high P, low E2/high P) were analyzed by ANOVA with Duncan's test of ranges for independent significance. Statistical Analysis System software program was used to draw up general linear models and stepwise univariate linear regression. The paired t-test was employed for testing significance of difference at given hormone doses. Liquefied donor semen was washed twice in Ham's F-lO medium (Gibco, Grand Island, NY) 114 Mbizvo et al. Hormone effects on sperm motion Fertility and Sterility

3 Table 1 Mean Serum E2 and P Hormone Levels No. of serum Time E2/P samples E2 P of cycle" pg/ml ng/ml Low/low High/low High/high Low/high "Time of cycle is average number of days (cycle length 30 days) since menses. supplemented with 3.5% BSA. The sperm pellet was overlayed with Ham's F -10 medium containing either 7.5% fetal calf serum (FCS) or 3.5% BSA and incubated for 1 hour at 37 C in a 5% CO2 and 95% air atmosphere. Swim-up sperm were incubated for another 2 hours before adjusting the concentration to 2 X 10 6 /ml. After 3 hours, 58 ~L of sperm were added to a 58 ~L volume of varying concentrations of E2-treated media (E2, 104 to 86 pg/ml) or P-treated media (P, 313 to 26 pg/ml). Hyperactivation was assessed on these samples within 4 hours of the initiation of sperm wash and incubation under capacitating conditions. In addition, sperm were added to Ham's F-10 medium, without E2 or P, which were used as hyperactivated motility controls. Analysis of sperm for hyperactivation was performed using a Hamilton-Thorn Motility Analyzer, version 7.1 (Hamilton-Thorn Research, Danvers, MA). Samples were drawn into capillary tubes.200 ~m deep and placed onto a cannula chamber holder. The Hamilton-Thorn Motion Analyzer internal phase-contrast optics were adjusted to capture all motile cells at a frame acquisition rate of 30 Hz. Hyperactivated sperm were identified using the automatic sort function criteria. The preset critical thresholds (Hamilton Thorn Motion Analyzer, June 1989 Version 7 Operations Manual) for hyperactivated sperm were: curvilinear velocity ~ 100 ~m/s; linearity ~ 65%; lateral head displacement ~ 7.5 ~m; progressive velocity 0 to 290 ~m/s; path velocity 0 to 290 ~m/s; beat frequency 0 to 30 Hz. Analysis was performed at a magnification factor of 2.04 with main gates set at 7 minimum contrast, 6 minimum size, and 0.6 to 1.8 for low to high intensity gates. Data were analyzed by x2 and Fisher's exact test. RESULTS The distribution of serum E2 and P hormone levels in the cycling women used in the study is shown in Table 1. The time of cycle was the average number of days after the start of the last menstrual period. The low E 2/low P group exhibited increased progressive sperm motility, velocity, linearity, and longevity, as compared with high Edhigh P, high Edlow P, and low Edhigh P. Differences in movement characteristics were observed between sperm samples exposed to serum collected at various times throughout the menstrual cycle (Fig. 1). There was a significant difference in the mean percentage of motile sperm at 4 hours (P < 0.05) between samples incubated in low Edhigh P sera, compared with high Edlow P, high Edhigh P, and low E2/high P. The mean percentage motility at the initiation of evaluation, T = 0 was 80%. The low Edhigh P group had a lower percentage of motile sperm at 4 and 24 hours, as compared with the other groups. At 24 hours, the mean percentage of motile cells in low Edlow P and high E2/high P serum samples were significantly different (P < 0.05) as compared with high Edlow P and low E2/high P. The mean sperm velocity at different time intervals in EdP serum groups is shown in Figure 2. There was a decrease in mean sperm velocity after 4 hours. The mean velocity at T = 0 was 63 ~m/s. At 1 hour, the mean sperm velocity in low E2/low 2(x; so) (x; so) t. Motility ,--,---, o !:!!~!.~~.. ~~~p.~.~~!i:!!,.~.2... Low EzlLow Ez Figure 1 Effects of serum estrogen (E2) and progesterone (P) on sperm motility. Mean sperm motility is given in delta values which were mean (X) values of differences from T = 0 motility for each time points 1, 4, and 24 hours. Sperm motility (baseline X) at T = 0 was 80%. The delta (difference unit) values varia tion over time (2[X ± SD]) up to the 4-hour time point was within the ±15%. Mean delta values were derived from mea surements conducted at 0-, 1-,4-, and 24-hour time points for each of the following serum hormone concentrations (see Table 1 for values): low E2 and high P, high E2 and high P, high E2 and low P, low E2 and low P. Mean sperm motility in low E2/ low P and low E2/high P differed significantly (P < 0.05) at 4 hours. Although motility in all groups fell at the 24-hour measurement, sperm in low E2/low P sera had significantly higher (P < 0.05) mean sperm motility. Vol. 54, No.1, July 1990 Mbizvo et at. Hormone effects on sperm motion 115

4 20 2(X ± SO) 17 X 2(X±so) -17 l!. VeI.clty, ~.' \ :: \.!:!!~!.~~- ~~~p.~.~~!':~. ~~... Low E,/Low E2 2(X±so) 2.5 A BCF.~...;.;.;.:';;: '~~""" ''':'::'' '0, 'r:-::,' -4 \ -Ii \ ~!:!!~~.~~.. ~~~p.~.~~!i:~.~;2... Low Ezllow Ez Figure 2 Effects of sperm E2 and P levels on sperm mean velocity. Mean sperm velocity values at 1-,4-, and 24-hour time points were subtracted from the T = 0 value to give a difference unit value for each time point. Mean delta values were derived from the computed differences from each experiment. The delta values for the mean sperm velocity are significantly different (P < 0.05) between low E2/low P and high E2/high P sera, and high E2/low P and low E2/hig,h P sera at 1 hour. The velocity at T = 0 was 63 ± 17 J.Lm/s (2[X ± SD]). Sperm in low E2/low P, high E2/low P, and high E2/high P sera exhibited significantly higher (P < 0.05) mean velocity at 24 hours, compared with low E2/high P-supported sperm. P and high E2/high P serum was significantly higher (P < 0.05) as compared with that in high E2/ low P and low E2/high P serum. Low E2/high P serum-incubated sperm had a significantly lower velocity at 24 hours (P < 0.05), compared with samples in low E2/low P, high E2/low P, and high E2/ high P sera. Low E2/high P groups had a lower mean velocity at 24 hours than the other E2/P combinations. Sperm linearity (Fig. 3) decreased with time in all groups, with the low E2/high P group exhibiting 2(X± so) 1.2 2(X± SO) -1.2 l!. Linewity -2...:;::.:..::::... ~~._.-...;::: ';",. \... <.:: ~----,----, o !:!!~~-~~..!!!'~It!!!.P_. ~.~~~.~~... Low E,II.owE, Figure 3 Effects of serum E2 and P levels on sperm linearity. Sperm linearity decreased with time. At T = 0, the mean sperm linearity (2[X ± SD]) was 6.2 ± 1.2. Sperm in low E2/low P and high E2/high P exhibited significantly higher (P < 0.05) linearity at 24 hours, as compared with high E2/low P and low E2/high P sera-supported sperm. Linearity values were expressed as mean difference unit values from T = 0 for the time points 1,4, and 24 hours. Figure 4 Effects of serum E2 and P on sperm beat cross frequency. Sperm in high E2/low P sera exhibited a high beat cross frequency at 1- and 4-hour time points. At 24 hours, low E2/ high P sera sperm had significantly lower (P < 0.05) beat cross frequency, as compared with high E2/high P, high E2/low P, and low E2/low P. The mean sperm delta points beat cross frequency for high EJhigh P, high E2/low P, and low E2/low P were within the 2(X ± SD) T = 0 value at 1-, 4-, and 24-hour time points. The T = 0 beat cross frequency was 17 ± 2.5 Hz (mean ± 2[X ± SD]). Beat cross frequency was expressed in delta values which were mean difference value at 1, 4, and 24 hours from the mean T = 0 values. a lower mean linearity at 1 and 24 hours (P < 0.05), as compared with the high E2/high P, high E2/low P, and low E2/low P serum groups. The mean linearity at T = 0 was 6.2. Figure 4 outlines the beat cross frequency pattern over time with the addition of E2/P sera. With time, there was a decrease in beat cross frequency, with low E2/low P having the lowest beat cross frequency (1 hour) and high E2/low P showing the highest beat cross frequency. The mean beat cross frequency at T = 0 was 17Hz, and, at 24 hours, low E2/high P serum samples exhibited the lowest beat cross frequency (P < 0.05) as compared with the other serum levels. When capacitating sperm were incubated with E2-treated medium at various concentrations, higher mean percentage motility and longevity were observed, as compared with medium containing P and controls containing no steroid additions (Fig. 5). Both motility and longevity were increased at 104 pg/ml E2 concentrations, compared with the lower concentrations. At 4 hours, the 104 pg/ml E2-spiked media supported a significantly higher percentage of motile sperm (P < 0.004), compared with controls. The mean sperm motility ± SD at T = 0 was 80% ± 6.1 %. Higher P concentrations (313-, 157, 78 pg/ml) reduced the percentage of motile sperm motility, as compared with lower concentrations (52 and 26 pg/ml). The higher P concentration samples had lower percent 116 Mbizvo et al. Hormone effects on sperm motion Fertility and Sterility

5 10 o ~ -10 ~ ::E -20 <I -30.~~... ~ ~~~-.. - Control ~---~--~--~ 4 24 Figure 5 Effects of media containing E2 on sperm motility. Mean sperm progressive motility was expressed as the mean of difference unit values form T = 0 at 1, 4, and 24 hours. Sperm in media treated with E2 exhibited significantly higher mean sperm motility at 4-hour time point (P < 0.004), compared with sperm in control media. At T = 0, the sperm motility was 81 % ± 2.2% (mean ± SEM). Estradiol-treated media supported the highest motile sperm concentration at 24 hours. Sperm in serum controls (CHI7 and TOI2) had lower mean sperm motility at 1-, 4-, and 24-hour time points, as compared with E2-spiked media. motility at 1 and 4 hours as compared with serum and medium controls; however, sperm longevity at 24 hours did not decrease significantly below control medium levels at any of the P concentrations. The mean sperm motility ± SEM at T = 0 in P treated medium (313 pg/ml) was 80% ± 2.53%, and difference unit values at 1-, 4-, and 24-hour time points were -5.9, 3.9, and 20.7, respectively. The effect of P at 157 pg/ml was not significantly different from the control medium at 1 and 4 hours (P> 0.05). However, when sperm motility in high and low P concentrations (313 and 26 pg/ml) were compared at 24 hours, the high dose P sperm had significantly lower (P < 0.05) sperm motility. Figure 6 shows the effect of E2-treated medium (104 pg/ml) on sperm velocity over time. Mean sperm velocity was lower with the low dose E2 concentrations (26 to 9 pg/ml), as compared with the higher concentrations (104 to 52 pg/ml), (P < 0.05). There was a significantly greater effect on sperm velocity at 1 hour with 104 pg/ml E2 compared with 9 pg/ml E2 (P < 0.01) and control media (P < 0.01) at 4 hours. Mean sperm velocity at the 104 pg/ml dose was 51 ± 5.7 #Lm/s (mean ± SD).. Mean sperm velocity at 1 hour in all P-spiked medium samples was higher than that of sperm in control Biggers, Whitten, and Whittingham medium, irrespective of concentration variation. Sperm velocity remained slightly, but not significantly, higher at 4 hours with P, as compared with the control, especially at ~52 pg/ml concentra- tions. At 24 hours, all P samples had lower mean sperm velocity compared with control medium. Sperm velocity in the control sera was also significantly lower (P < 0.05) at 24 hours. The T = 0 sperm velocity at 104 pg/ml P sample concentration was 51 ± 2.49 #Lm/s (mean ± SEM) and the difference unit values were 4.2, 2.07, and at 1-,4-, and 24-hour time points, respectively. Sperm linearity varied with time, depending on whether E2 or P was added to medium. E2-treated medium maintained constant sperm linear movement at 1-, 4-, and 24-hour intervals at ~52 pg/ml concentration. At the lower concentration (:::::;;26 pg/ml), linearity decreased with time. Sperm linearity at T = 0 (104 pg/ml ofe2) was 4.5% ± 0.25% (mean ± SEM) and difference unit values for 1-, 4-, and 24-hour time points were 0.21, 0.16 and -0.39, respectively. The mean sperm linearity in control media remained lower at the three time intervals. The lower E2 concentration (:::::;;17.4 pg/ml) resulted in significantly lower (P < 0.01) sperm linearity, as compared with the high dose (104 pg/ml). The mean sperm linearity in P-spiked media was decreased within the I-hour time interval at a P concentration of ~52 pg/ml and remained lower at the 4-hour interval at both high and low concentrations, compared with E2-spiked media-supported sperm. At 24 hours, P-medium-supported sperm exhibited higher, but not significant, linearity. The mean sperm linearity at T = 0 (P = 104 pg/ml) was 3.9% ± 0.5% (mean ± SD) with 1-,4-, and 24-hour difference unit values of -0.51, and -1.1, respectively ;., 0 j :> -10 <l -20 ~""'--"-"'"./ ~ ~ \."......,\...,... '....~!... ~ ~!~-.. ~ ~---~--~--~ 4 24 Figure 6 Effects of medium containing E2 on sperm velocity. Delta values for sperm velocity in E2-treated media were obtained by subtracting time points 1, 4, and 24 hour mean data values form T = 0 mean value. Increased sperm velocity was seen with E2 at 1-, 4-, and 24-hour time points. The velocity at T = 0 was ± 2.01 (mean ± SEM). Sperm velocity at 1 and 4 hours was significantly higher in E 2-treated media (P < 0.01), as compared with control media. Vol. 54, No.1, July 1990 Mbizvo et al. Hormone effects on sperm motion 117

6 Table 2 Nontransformed Data of Sperm Motion Parameters Over Time Based on a Single Evaluation of Pooled Sperm in Medium Under Capacitating Conditions Variables over time (hours) Sperm parameters Sample Motility (%) E Velocity (p.m/s) Linearity BCFa(Hz) ALH,b(p.m) Motility (%) P Velocity (p.m/s) Linearity BCF(Hz) ALH(p.m) Motility (%) Control Velocity (p.m/s) Linearity BCF(Hz) ALH(p.m) a Beat cross frequency. b Lateral head displacement amplitude. The mean beat cross frequency of sperm in E2- spiked media decreased with time; difference unit mean values were 0.1 at 1 hour, at 4 hours, and at 24 hours with 104 pg/ml E2. The decrease in beat cross frequency at both 1, 4, and 24 hours with various E2 doses was comparable with that in control sera and control media. At the 104 pg/ml E2 concentration, the mean beat cross frequency was 17.3 ± 0.7 Hz (mean ± SD). Progesterone-treated media sperm maintained a beat cross frequency at the various time intervals that was comparable with control media (P > 0.05). The beat cross frequency was, however, higher with all concentrations of P, as compared with serum or media controls. At 313 pg/ml P, the mean beat cross frequency was 16.6 ± 1.6 Hz (mean ± SD) (difference mean values being: P = 1.0, control = 0.5 at 1 hour; P = 0.9, control 1.2 at 4 hours; P = -2.5, control = -4.2 at 24 hours). The amplitude of lateral head displacement increased with time up to 4 hours and was lower at 24 hours in all E2-containing media. This increase and subsequent decrease was also observed with control media samples, although, at 24 hours, control media sperm exhibited slightly higher lateral head displacement. The mean ± SD lateral head displacement at 104 pg/ml of E2 was 3.0 ± 0.4 f.lm/s at T = 0 (delta mean differences for E2 being 0.31, 0.62, and 0.16 at 1, 4, and 24 hours, respectively, and 0.17, 0.25 and 0.44 for control at similar time points). Media containing P-supported sperm caused lateral head displacement that was higher than that of sperm in control medium at 1 and 4 hours at all P concentrations, but at 24 hours the control medium sperm sample exhibited significantly higher lateral head displacement than medium containing P at all concentrations (P < 0.05). The mean lateral head displacement (±SD) at T = 0 for 313 pg/ml ofp 4 was 2.4 ± 0.6 f.lm (mean differences for P being 0.2,0.3,0.1 at 1-, 4-, and 24-hour time points, respectively, and 0.2,0.3 and 0.4 for controls at similar time points). Table 2 outlines sperm parameter data drawn from a single evaluation of pooled sperm before obtaining delta difference values and mean values for subsequent comparative analysis. Sperm were incubated in capacitating conditions before P and E2 addition for hyperactivated motility analysis. Sperm samples incubated in medium containing P (313 pg/ml) exhibited enhanced hyperactivated sperm motility (Fig. 7). When the P concentration was increased, there was marked sperm agglutination in some samples. Use of FCS instead of BSA resulted in higher sperm hyperactivated motility in the steroid P-treated medium. Also, when some sperm samples were observed after 6 hours under c ~ > +l () til "- CD Q. >. J: all i p e P4 A. B. 0 ~..,~ N.S. e E2 Figure 7 Effects of media containing P and E2 on sperm hyperactivated motility. The percentage of hyperactivated sperm cells was increased significantly (P < 0.001) in media treated with P, as compared with controls (C). Sperm in E2-treated media did not show significant hyperactivated motility difference with sperm in control media. Mean values for percentage hyperactivated motility were obtained from the ratio of hyperactivated motility sperm and total motile sperm cells derived from five experiments. The mean hyperactivated motility for controls was 6.6%. Each measurement was taken using sperm at 2 X 10 6 /ml concentration. 118 Mbizvo et al. Hormone effects on sperm motion Fertility and Sterility

7 capacitating conditions (with 1 hour exposure to P), some sperm samples had become agglutinated, whereas some of the remainder still exhibited nonprogressive hyperactivated sperm movement. Samples treated with the higher concentration of P exhibited hyperactivated sperm motility of up to twofold higher than control medium (P < 0.01) after 4 hours under capacitating conditions. Medium containing E2 did not stimulate hyperactivated motility above the controls. DISCUSSION Understanding the fundamental mechanisms regulating human sperm function and ovum interactions is important in the development of techniques designed to improve the functional competence of human spermatozoa and fertilization. Not much is known about the conditions or factors directly controlling capacitation of spermatozoa within the female genital tract. The demonstrated effects of steroids on sperm function during capacitation should have significance' not only during assisted reproduction, but also in the regulation of fertility. The binding of steroids to human spermatozoa has previously been described in semen samples,12 and further evidence indicates that spermagglutinins, present in the serum of some women, are not antibodies but steroids bound to ~-lipoproteinsp It is therefore conceivable that steroids in serum exert a direct effect on spermatozoal motion parameters. Results from the present study document the effects of both E and P levels on capacitating sperm longevity and movement characteristics, including hyperactivated motility. The data show that the quality of sperm motion is affected by the levels of P and E2 in women's sera at different time phases of the menstrual cycle. In serum with a combination of low E2 and low P, optimal support of sperm in terms of motion parameters and longevity was demonstrated. When sperm were incubated with medium containing E2 alone, sperm progressive motility, velocity, linear movement, and longevity were maintained. Thus, this effect was demonstrated with both endogenous serum and exogenous E2 levels. In capacitating media, P alone did not support progressive sperm motility and longevity, but enhanced hyperactivated sperm motility. Higher P doses resulted in sperm agglutination in some samples. Optimal sperm function was observed when serum P levels were lower. During therapeutic insemination, sperm are sometimes supported in vitro with serum collected from the wife regardless of the time phase of her menstrual cycle. The present findings, however, suggest that use of early follicular phase serum could enhance fertilization by maintaining sperm progressive movement and longevity while sperm migrate to the site of fertilization. Capacitating sperm must also survive long enough to undergo the acrosome reaction at the right time and place. The expression of hyperactivated motility, which Katz and coworkers14 have defined as "the completion of capacitation or expression of its final phases," is required during sperm penetration of the ovum.15 If hyperactivated motility were induced too early, a large proportion of sperm might be beyond their peak before fully traversing anatomicallocations to the site of fertilization. The expression of hyperactivation adds a yet new dimension to sperm evaluation. The present findings indicate a specific steroid effect. Progesterone was seen, experimentally, to promote sperm hyperactivated motility. This action of P is consistent with studies by Meizel16 on the effect of P on the acrosome reaction. Progesterone stimulates intracellular Ca2 + uptake with resultant changes in sperm movement. Extracellular calcium ion influx stimulates membrane bound adenylate cyclase, resulting in an increased intracellular concentration of adenosine 3',5'-monophosphate, which, together with Ca2 +, are important regulators of sperm movement. Maintaining low intracellular Ca2 + concentration is important initially for sperm survival and protects spermatozoa from premature acrosome reaction.15 Extracellular calcium is required during development of hyperactivated motility in mouse sperm incubated in vitroy In fact, the initiation and maintenance of hyperactivated motility, the acrosome reaction, penetration of acrosome-reacted spermatozoa into the zona pellucida, fusion of spermatozoa with eggs, and development of pronuclear eggs into two-cell embryos,15 all require extracellular Ca2 +. Hyperactivated sperm movement in high ionic strength media can be prevented18 by removing or chelating Ca2 + or replacing Ca 2 + with Ba2 +. In a separate study, we have observed increased hyperactivated sperm motility with human follicular fluid,19 whose active steroid-rich fraction caused an immediate increase in cytosolic calcium (Ca2 +) in capacitating human sperm. 9 Estradiol does not promote the acrosome reaction but stimulates motility of human sperm in semen, 7 and P does not initiate the acrosome reaction Vol. 54, No.1, July 1990 Mbizvo et al. Hormone effects on sperm motion 119

8 of uncapacitated sperm. 16 The present findings demonstrate similar E2 effects on capacitating sperm, whereas P action could be confined to capacitated sperm. The mechanism of P action is probably mediated by a P receptor resident in the plasma membrane of sperm. Binding of P to this receptor then activates a Ca 2 + channel in the plasma membrane or inhibits the plasma membrane Ca 2 + adenosine triphosphatase pump. High Ca 2 + concentrations are known to inhibit hyperactivated motility, even though Ca 2 + provides the primary signal for the expression of hyperactivated motility and the acrosome reaction. The capacitation process, which prepares the sperm to respond to this signal, clearly involves an intricate interplay of ions with enzyme and steroid modulation. The effect of the steroids on sperm function was more marked at higher concentrations in buffered media containing BSA and in lower concentrations in serum. This could be due to the presence of other cofactors in serum, such as corticosteroid-binding globulin and serum albumin. Sperm hyperactivation is increasingly recognized as an important biological marker during capacitation, which could assist sperm penetration of the cumulus and zona pellucida. 14,15 Both in vitro and in vivo observations link hyperactivation with the process of capacitation and fertilization, whereas fertilization failure, in vitro, has been correlated with nonhyperactivated sperm. 20 The data presented here indicate that E2 and P hormone levels effect recognizable changes in the pattern of sperm movement and longevity at the time of capacitation. This information should be useful for further studies in the in vivo and in vitro stimulation and inhibition of sperm motion, as well as guide the timing for use of female sera in supporting sperm function. Acknowledgments. 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