Human follicular fluid stimulates hyperactivated motility in human sperm*t

Transcription

1 FERTILITY AND STERILITY Copyright 1990 The American Fertility Society Printed on acid-free paper in U.S.A. Human follicular fluid stimulates hyperactivated motility in human sperm*t Michael T. Mbizvo, D.Phil.:j: Lani J. Burkman, Ph.D. Nancy J. Alexander, Ph.D. II The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia Since human follicular fluid (FF) is known to enhance the acrosome reaction and capacitation, we investigated whether hyperactivated motility is stimulated by FF. Follicular fluid-treated sperm exhibited a threefold increase in hyperactivation compared with the controls. The use of fetal cord serum in the medium, instead of bovine serum albumin, supported the same high levels of hyperactivation, although the peak occurred at 3 hours rather than 5 hours of capacitation. When sperm were treated with a steroid-rich fraction of the FF, hyperactivation was stimulated to the same degree as with whole FF. In contrast, no stimulation occurred when sperm were treated with a FF fraction stripped of steroids. The FF enhancement of hyperactivation in vitro could augment the fertilizing capacity of subfertile sperm samples, providing also a glimpse of possible in vivo events as sperm traverse the FF-laden cumulus oophorus. Fertil Steril54:708, 1990 During the past two decades, many studies have shown that capacitating sperm, near the time of fertilization, express a distinct motility pattern called hyperactivation. Sperm hyperactivation was first described in the golden hamster by Y anagimachi 1 and subsequently observed for a number of other species.2 Such hyperactivated motility is characterized by vigorous flagellar movements, marked lateral excursions ofthe sperm head, and a Received January 5, 1990; revised and accepted June 20, * 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 Presented in part at the 15th Annual Meeting of the American Andrology Society, Columbia, South Carolina, April6 to 9, :j: Present address: Monash University, Centre for Reproductive Biology, Clayton, Melbourne, Australia. Present address: Gibco Laboratories, Grand Island, New York. II Reprint requests: Nancy J. Alexander, Ph.D., Contraceptive Development Branch, Executive Plaza, North, 6130 Executive Boulevard, Suite 600, North Bethesda, Maryland nonprogressive trajectory.2 3 Hyperactivation is now viewed as a required step in the capacitation sequence. The very high power output during hyperactivation4 may facilitate sperm passage through the cumulus mass and/or the zona pellucida.3 In 1984, Burkman 5 and Mortimer et al. 6 described classical hyperactivated motility in human spermatozoa prepared for in vitro oocyte insemination. Subsequent studies have demonstrated that the incidence of hyperactivation is significantly correlated with sperm fertilizing capacity in vitro, 7-9 and sperm binding to the zona pellucida The initiation of this form of motility and the underlying cellular mechanisms are poorly understood. Treatment of spermatozoa with a variety of factors can stimulate hyperactivated motility. These include such physiological substances aspyruvate/2 cysteine, 5 taurine, 5 prolactin, 5 and serum/3 as well as other chemical factors (caffeine, 5 dibutyryl cyclic adenosine monophosphate, 5 14 calcium ionophore, increased osmolality, 9 10 and elevated ph 9 10 ). There is considerable evidence that human follicular fluid (FF) is a potent stimulator of human sperm capacitation and the acrosome reaction Mbizvo et al. FF affects sperm motion

2 1 I After ovulation, the ovum, cumulus oophorus, and accompanying FF are transported into the oviductal ampulla, the principal site of sperm/ egg interaction and fertilization. 2 In this study, we have investigated the in vitro effects of FF on human sperm hyperactivated motility, including a FF fraction devoid of steroids. MATERIALS AND METHODS Semen Collection and Processing Semen samples were obtained by masturbation from eight normal volunteers after 48 hours of sexual abstinence into sterile wide-mouth plastic containers provided by the laboratory. The specimens were allowed to liquefy at room temperature, after which a 0.5 ml aliquot was washed two times with equal volumes of Ham's F-10 medium (Gibco, Grand Island, NY). The medium was supplemented with either 3.5% (weight/vol) bovine serum albumin (BSA; Sigma Chemical Co., St. Louis, MO) or 7.5% (vol/vol) human fetal cord serum (heat-inactivated and filter-sterilized). The time at which sperm washing was initiated was defined as the beginning of capacitation. After the second centrifugation, the sperm pellet was gently overlayed with 600 p,l of the same medium and incubated (5% C0 2 in air) at 37oC for 1 hour. The upper portion of the sperm supernatant was collected and diluted with Ham's medium to a motile concentration of 2 to 3 X 10 6 /ml. Incubation was then continued for either 3 or 5 hours before exposure to FF or its fractions. Follicular Fluid Collection and Processing The contents of ovarian follicles were aspirated from women undergoing in vitro fertilization (IVF) at Norfolk, Virginia. 18 During this process, the follicular contents were always diluted approximately fourfold with Dulbecco's phosphate buffered saline (PBS; Gibco). After isolation of the oocyte and cumulus oophorus, residual diluted FF from follicles of mixed maturity was pooled and frozen at -70 C. For this study, the thawed FF was centrifuged (3,000 X g, 30 minutes) to remove cellular debris, then filter-sterilized (Millipore Corporation, Bedford, MA) and stored in aliquots at -20oC until further processing. Later, this FF was concentrated fourfold to approximate the volume present in the follicle before aspiration (whole FF). At this concentration the FF had fourfold higher PBS salt concentration. This concentration procedure utilized a Minitan Ultrafiltration System, type PTGC OMT 05 (Millipore). The total protein content of the resulting "whole" FF was determined using a spectrophotometer (Model MR 600; Dynatech Lab Inc, Alexandria, VA) against BSA standards. A portion of the FF was processed to remove unconjugated steroids and other low molecular weight substances. For this purpose, 1.0 ml of whole FF was diluted to 10 ml with PBS and extracted with a C 18 SEP-PAK column (Waters Associates, Milford, MA) The absorbed material was then eluted with 10 ml of methanol and evaporated to dryness under a stream of nitrogen. The dry material was resuspended in 1.0 ml of physiological saline with 0.1% BSA. Aliquots of the FF fractions and the whole FF were kept at -200C and brought to 37 C before their addition to capacitating sperm. The progesterone (P) and estradiol (E 2) concentrations in the whole FF, in the methanol extract ("steroid-rich"), and in the SEP-P AK eluent ("steroid-stripped") were determined by radioimmunoassay. The assay for P content utilized the Coata-Count Progesterone Kit (Diagnostic Products Corporation, Los Angeles, CA). Beta-estradiol concentrations were determined with the radioassay systems laboratory direct Estradiol-17 fj kit (ICN Diagnostic Division, Carson, CA). The concentration of total protein (mg per ml of the whole FF) was assessed for 1 ml of matching whole FF, and the two fractions derived from passage of 1 ml whole FF through the Sep-PAK column. For this purpose, the bicinchoninic acid Protein Assay Reagent (Pierce Chemical, Rockford, IL) was utilized. Computer-assisted Analysis of Hyperactivated Motility Sperm movement characteristics and hyperactivated motility were analyzed automatically using a Hamilton Thorn Motility Analyzer (model 2030, version 7.1; Hamilton-Thorn Research, Danvers, MA). The pertinent settings used during the Hamilton Thorn Motility analysis were: analysis duration of 0.67 seconds, minimum contrast is 7, minimum size is 6, "slow cells" are accepted as motile (slow gate is 5 p,m/s), magnification factor adjusted for 100 p,m, low size gate is 0.4, high size gate is 1.6, low intensity gate is 0.4, high intensity gate is 1.6. Before accumulating data, the technician made a daily check on a test field of sperm to confirm accurate identification of motile cells and nonmotile sperm cells versus debris. Mbizvo et al. FF affects sperm motion 709

3 ,.; "' 100 % HA 90 0 % Motile Cells ,--- () ~ ~ ~ ~ 1:2 1:5 1:10 1:20 1:40 1:60 c FF Dilutions Figure 1 The percentage of hyperactivated cells and percent motility as measured by computer-automated analysis at different dilutions offf. Based on recent work in this laboratory, 21 the automatic sort function was used to simultaneously identify the four patterns of hyperactivated movement (circling, thrashing, helical, and star-spin) that have been investigated for sometime. 22 Automatic hyperactivation discrimination was carried out using three simultaneous criteria: curvilinear velocity ~ 100 J.Lm/s, maximum amplitude for lateral head displacement ~ 7.5 J.Lm/s, and linearity :::; 65). After 3 hours and/or 5 hours of capacitation, an aliquot of each sperm sample was taken for FF stimulation of hyperactivated motility. In the first set of experiments, sperm were prepared in either of the two media, and whole FF was added to the sample at a dilution of 1:20. After addition of the FF, the sperm sample was incubated (37oC) for an additional hour before evaluating hyperactivated sperm motility. A second set of experiments tested the dose response for sperm prepared in Ham's medium with BSA using six dilutions of whole FF (1:2, 1:5, 1:10, 1:20, 1:40, and 1:60). The diluted FF was added after 5 hours of capacitation and hyperactivated sperm assessment was carried out 1 hour later. In the third group of eight experiments, sperm preincubated in Ham's-BSA were exposed to a steroid-rich fraction of the whole FF versus a fraction stripped of FF. Again, evaluation of hyperactivation was carried out 1 hour after addition of the fraction. The results were expressed as means ± SEM. Data were analyzed for significance of difference with analysis of variance, the t-test or x 2 fetal cord serum or BSA (Ham's-BSA). In early trials, FF in Ham's-fetal cord serum produced better stimulation after 3 hours compared with 5 hours (29.9% versus 16.1%). For all subsequent experiments utilizing Ham's-fetal cord serum, FF was tested after 3 hours only. In contrast, capacitation proceeded more slowly within the Ham's-BSA medium such that better FF stimulation was observed at the later, 5-hours time point (12.1% ± 2.4% after 3 hours versus 25.3% ± 2.0% after 5 hours, mean ± SEM, P < 0.001). Consequently, in all later experiments that utilized Ham's BSA, the effect of FF addition was evaluated after 5 hours only. Within eight experiments, exposure of sperm prepared in Ham's-fetal cord serum to whole FF after 3 hours of capacitation produced a mean incidence of hyperactivated sperm (25.0% ± 2.7%), which was significantly greater than the control value (12.4% ± 1.8%, P < 0.001). The same significant stimulation of hyperactivated motility was observed in the Ham's-BSA preparation when FF was added at 5 hours (n = 12, 25.3% ± 2.0% versus 8.0% ± 1.3% for the control, P < 0.001). The semen of several men was tested on 2 different days using this protocol. The sperm hyperactivation response to FF stimulation was quite reproducible within each man. Figure 1 illustrates the influence of various dilutions of whole FF on the incidence of hyperactivation. As the concentration of FF was increased, a larger proportion of the vigorously moving cells showed agglutination. Agglutinated cells are nonprogressive, thus accounting for the decline in percent motility and percent hyperactivated sperm with concentrations > 1:20. Whereas the control sperm exhibited a mean hyperactivation of 10.0% ± 0.6%, there was a maximal enhancement of hyperactivation when whole FF was added at dilutions of 1:10, 1:20, or 1:40. As given in Table 1, filtration of 1 ml of whole FF through the Sep-P AK cartridge produced an effective absorption of the steroid constituents. Table 1 Total Protein Concentration and Steroid Concentration in FF or FF Fractions Sample Protein Progesterone Estradiol RESULTS Significant stimulation of hyperactivated sperm motility was observed when FF was added to sperm in Ham's F-10 medium supplemented with either mgfml p.gfml WholeFF Whole FF, methanol extract Whole FF steroidstripped ngfml Mbizvo et al. FF affects sperm motion

4 Progesterone present in the eluent (steroid-free fraction) was negligible, whereas the concentration in the methanol (steroid-rich) fraction was 1,000X greater (6.9 J.Lg/mL). Likewise, there was an effective separation of E 2 between the two fractions. The protein contained in the eluent plus the methanol extract accounted for 99 mg of the original132 mg (recovery, 75%). Treatment of sperm with the steroid-rich FF fraction induced a stimulation in hyperactivation compared with the control that mimicked the use of whole FF (22.66% ± 2.31% and 8.1% ± 0.77%, respectively; P < 0.001). In sharp contrast, follicular fluid devoid of steroid activity did not enhance hyperactivation above these control levels (8.4% ± 0.76%, p > 0.05). DISCUSSION The results presented here demonstrate that human FF stimulates hyperactivated sperm motility. Follicular fluid, therefore, appears to act as a physiological stimulus for those events occurring near the time that capacitation is completed; these events include calcium influx, 19 hyperactivated sperm motility, and the acrosome reaction Since 1984, Burkman and colleagues12 have examined the presence of hyperactivated motility within human sperm suspensions during capacitation and its physiological significance. The incidence of hyperactivated sperm was significantly lower in subfertile specimens during in vitro capacitation5 and could be stimulated by the addition of several exogenous substances.5 In later studies, they showed that low levels of hyperactivation were associated with a decreased penetration rate in the hamster penetration assay9 and lower rates of human oocyte fertilization during IVF therapy.7 11 Most recently, it appears that a timely assessment of percent hyperactivation in sperm suspensions prepared for IVF inseminations may predict the fertilization outcome for individual patients. 8 During initial patient screening, assessment of hyperactivation incidence with and without FF treatment could suggest a critical sperm therapy to be used during assisted reproduction. These indications of a significant role played by hyperactivation during fertilization are consistent with the earlier literature, in which the presence of hyperactivated motility or the acrosome reaction indicated the completion of capacitation,2 and declining hyperactivated motility was temporally associated with the loss of zona-penetrating capacity.24 In 1981, Johnson and colleagues4 demon- strated that hyperactivated rabbit spermatozoa exhibit a 20-fold increase in power output; this increased energy would facilitate sperm passage through the highly viscoelastic cumulus matrix3 and probably the zona pellucida.3 4 The current data have indicated a significant stimulation of sperm hyperactivated motility after exposure to whole FF for 1 hour. When a nonsteroid fraction of FF was tested, there was no stimulation of hyperactivation. This finding supports the premise that steroids, in particular P in FF, are paramount for functional competence of sperm motion in vivo. Progesterone within FF causes an influx of extracellular Ca2 + by acting at the sperm cell surface level, 19 and furthermore, FF itself can stimulate the acrosome reaction in mammalian species, including man The amount of P measured in FF is similar to that found in cumulus cell culture. Thus, even if FF is diluted by tubal fluid, a high concentration might be expected near the egg and its vestments. 23 Follicular fluid contains other active substances that act at the end of the capacitating period. One factor that has acrosome reacting properties is not found in the lipid fraction but is associated, instead, with N-linked oligosaccharides or a glycoprotein or proteoglycan.14 Most likely, several different molecules are involved in capacitation and the acrosome reaction. It would seem reasonable that in vivo hyperactivation might occur at the level of the cumulus, with the acrosome reaction occurring at the time of sperm-zona binding. Work in our laboratory indicates that the acrosome reaction occurs at a high rate only in those sperm that are tightly bound to the zona pellucida compared with those sperm swimming adjacent to the zona.25 These free-swimming spermatozoa exhibited a low frequency of acrosome reactions, which was comparable with sperm never exposed to a zona pellucida. Out data suggest variability in the in vitro expression of hyperactivation that depends on the nature of the supporting protein (BSA versus fetal cord serum). The maximal incidence of hyperactivation was not different for the two protein supplements, although peak hyperactivation occurred sooner with fetal cord serum compared with BSA. This observation is of importance when evaluating sperm capacity for hyperactivated motility, as well as in preparation of sperm for artificial insemination. Our in vitro observation on the stimulation of sperm motility attributable to FF demonstrate that the FF microenvironment can affect sperm fertilizing potential. Follicular fluid appears to be one of Mbizvo et al. FF affects sperm motion 711

5 the capacitation factors present in the fluids of the female genital tract that can induce hyperactivated sperm motility and the acrosome reaction Hyperactivated motility may enhance the transit of the male gamete through the intercellular matrix between the cumulus cells and through the glycoprotein network of the zona pellucida. In addition, the hyperactivated movement of sperm, like the acrosome reaction, provides an accessible feature for studying the in vitro temporal events that culminate in fertilization. Studies have shown that intracellular calcium levels rise during capacitation, although the mechanisms controlling this phenomenon are poorly understood. Agonistic interactions between calcium channels and molecules in the immediate FF microenvironment may play a role. Acknowledgments. We gratefully acknowledge the technical assistance of Janice Hammond, B.S., and Pius Adoyo, M.Sc. Thanks are due to Ms. Patricia Hollis for typing the manuscript. REFERENCES 1. Yanagimachi R: The movement of golden hamster spermatozoa before and after capacitation. J Reprod Fertil 23:193, Yanagimachi R: Mammalian fertilization. In The Physiology of Reproduction, Edited bye Knobil, JD Neill, LV Ewing, CL Markert, GS Greenwald, DW Pfaff. New York, Raven Press Ltd, 1988, p Katz DF, Drobnis EZ, Overstreet JW: Factors regulating mammalian sperm migration through the female reproductive tract and oocyte vestments. Gamete Res 22:443, Johnson LL, Katz DF, Overstreet JW: The movement characteristics of rabbit spermatozoa before and after activation. Gamete Res 4:275, Burkman LJ: Characterization of hyperactivated motility by human spermatozoa during capacitation: comparison of fertile and oligospermic sperm populations. Arch Androl 13:153, Mortimer D, Courtot AM, Giovangrandi Y, Jeulin C, David G: Human sperm motility after migration into and incubation in synthetic media. 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J Exp Zool220:109, Fulgham DL, Johnson D, Coddington CC, Herr J, Alexander NJ, Hodgen DG: Human sperm acrosome reaction rate on zona pellucida: a time course study. (Abstr ) Presented at the 45th Annual Meeting of The American Fertility Society, San Francisco, California, November 13 to 16, Published by The American Fertility Society in the program supplement, 1989, p S Mbizvo et al. FF affects sperm motion