Sperm bound to zona pellucida in hemizona assay demonstrate acrosome reaction when stained with T -6 antibody*

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1 FERTILITY AND STERILITY Copyright 199 The American Fertility Society Printed on acid-free paper in U.S.A. Sperm bound to zona pellucida in hemizona assay demonstrate acrosome reaction when stained with T -6 antibody* Charles C. Coddington, M.D.t:j: David L. Fulgham, B.A. t Nancy J. Alexander, Ph.D.t Deborah Johnson, M.A.t John C. Herr, Ph.D. II Gary D. Hodgen, Ph.D. t The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk; Portsmouth Naval Hospital, Portsmouth; and University of Virginia, Charlottesville, Virginia A monoclonal antibody, T-6, useful for detecting acrosome-reacted sperm based on an immunofluorescent assay, was employed to evaluate acrosomal status of human sperm that were tightly bound to hemisected human zonae pellucidae (hemizona assay). Over 9% of the bound sperm evaluated exhibited immunofluorescent patterns indicative of acrosome reaction. This staining method for evaluating the acrosomal status of sperm bound to the zona pellucida may enable definition of a group of male infertility patients heretofore not recognized. Fertil Steril 54:54, 199 Mature spermatozoa undergo changes allowing fertilization of the mature oocyte. Austin 1,2 and Chang3 separately coined the term capacitation to describe this sequence of physiological and biochemical alterations necessary before fertilization. Additional modifications during the process of egg penetration include the acrosome reaction and acquisition of "hyperactivated" motility.4,5 The acrosome develops as a product of the Golgi complex at the time of spermiogenesis and mi- Received January 8, 199; revised and accepted May 23, 199. * This work was supported in part by the Contraceptive Research and Development (CONRAD) Program, Eastern Virginia Medical School, under a Cooperative Agreement (DPE-244-A--663-) with the United States Agency for International Development (A.I.D.). The views expressed by the author(s) do not necessarily reflect the views of A.I.D. t The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical... School. :t: Portsmouth Naval Hospital. Reprint requests: Charles C. Coddington, M.D., The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 855 West Brambleton Avenue, Norfolk, Virginia II Department of Internal Medicine, University of Virginia. grates to the anterior region of the sperm head just above the nucleus and beneath the plasma membrane.6 During the acrosome reaction, the acrosome becomes fenestrated and forms vesicles on the sperm surface6 that are released from the sperm head, exposing the acrosomal enzymes and the inner acrosomal membrane.6-s The current dogma is that only mammalian sperm that have undergone acrosome reaction can penetrate the zona pellucida and fuse with the egg plasma membrane Sperm that are acrosomereacted may not be able to bind to the zona. Antibody T -6 with immunofluorescent staining interacts with antigens in the acrosome ofthe spermatozoa, giving a distinct pattern consisting of a "cap" in un reacted acrosomes or an equatorial bar in reacted acrosomes; thus, T -6 can be used to define acrosomal status.13,14 Other authors have tried unsuccessfully to establish a relationship between performance in the sperm penetration assay and sperm acrosomal status.15,16 In one of these reports, T -6 antibody with immunofluorescent staining was utilized. 16 We suggest that evaluation of the acrosomal status of sperm bound to zonae would answer this question. 54 Coddington et al. Hemizona assay and T-6 antibody

2 The hemizona assay (HZA)17 has been developed to compare the fertilization potential of spermatozoa from infertile patients with those from fertile control males. The HZA is performed by using nonliving human oocytes that have been disected using micromanipulation. This assay has confirmed that sperm from fertile men will attach better than sperm from infertile men.17 It is useful also in predicting fertility in in vitro fertilization patients.18 The zona pellucida may enhance the induction of the human acrosome reaction,12,17 but due to ethical issues and technical limitations of electron microscopy, little is known about the acrosome status of sperm tightly bound to the zona pellucida. To evaluate acrosomal status of sperm bound from fertile males to the zona pellucida, we employed the HZA as a functional zona surface and used T-6 monoclonal antibody and immunofluorescence to study dynamic changes of the acrosome. Semen Analysis MATERIALS AND METHODS A semen analysis was performed at the initial clinical evaluation using an automated, computerized system (Cell Soft Semen Analyzer; Labsoft Division of Cryo Resources Ltd., New York, NY). The semen characteristics were assessed within a Makler chamber (Zygotek System Inc., Springfield, MA). Details of these procedures have been previously described.17 Semen samples from four fertile males (two were used twice) who had fathered a child within the last 2 years were employed after informed consent was given. Oocyte Handling and Zona Cutting Human oocytes used in this study were obtained from surgically removed ovarian tissue after informed consent. The tissue was placed on ice after excision, immediately dissected,19 and minced in phosphate-buffered saline (PBS).2 The zona-intact eggs were stored in small plastic vials containing.5 ml of 1.5 M MgCl2 (Mallinkrodt Chemical Works, St. Louis, MO),.1 % polyvinylpyrrolidone, and 4 mm of Hepes Buffer, then stored at room temperature (23 C). Narishige micro manipulators (Narishige, Tokyo, Japan) mounted on a phase contrast inverted microscope (Nikon Diaphot, Garden City, NY) were used for cutting the oocytes. A detailed description of the cutting procedures has been pub- lished.17,18 Briefly, a Beaver microsharp blade (#757; Beaver, Denville, NJ) was attached to a Beaver chuck handle (#1312), and movement was controlled by a single micromanipulator. A 1- mm plastic Petri dish (Falcon #25382; Falcon Plastics, Oxnard, CA) served as the cutting chamber and was partially filled with any of several media (Ham's F-1; Gibco, Grand Island, NY; Biggers, Whitten, and Whittingham [BWW]; PBS), supplemented with.3% bovine serum albumin. One egg was transferred to the cutting dish and cut in half as the blade was slowly lowered (total magnification of 2X). The shrunken ooplasm inside each hemizona was removed by vigorous pipetting. Each matched hemizonae pair was placed separately in a 5,uL droplet of Ham's F-1 medium in a small Petri dish, covered with mineral oil (Fischer Scientific, Fair Lawn, NJ), and stored overnight atrc. Sperm Handling for the HZA An aliquot of semen (.5 ml) was diluted with 1 ml of Ham's F-1 culture medium, supplemented with 7.5% heat-inactivated human fetal cord serum. After centrifugation (5 minutes, 3 X g), the pelleted sperm were washed a second time. The second pellet was overlaid with.5 ml of F -1 medium and incubated (37 C, 5% CO2 in air) to achieve a swim-up separation. After 1 hour ofincubation, the sperm supernatant was removed and binding to the hemizonae tested.17,18 Hemizona Assay Protocol Hemizona assay tests were performed utilizing sperm from four fertile males. For each evaluation, the half ofthe zona incubated with the fertile control was studied. A 1,uL droplet ofthe sperm suspension (5, motile spermjml) was placed in a Petri dish under oil. The sperm and hemizonae were coincubated for 4 hours (37 C, 5% CO2 in air). Each hemizona was then removed and rinsed in Ham's F -1 medium by pipetting five times with a finely drawn glass pipette to dislodge loosely associated sperm.17 Sperm from the fertile men bound to the hemizonae were evaluated. Mouse Monoclonal Antihuman Sperm Antibody (T-6) Processing and Interpretation After sperm were evaluated in the HZA, further reaction was stopped with addition of 1,uL of 5% sodium azide and 1,uL of 1 mm phenylmethylsul- Coddington et a1. Hemizona assay and T-6 antibody 55

3 RESULTS Acrosome Reacted Pre-Acrosome Reacted Acrosome Intact Acrosome Mottled Bar Ac: ++, uniform ± ' patchy Eq: ++, uniform ++, uniform ++, uniform -BOfIIfJ~ Figure 1 Fluorescent patterns on hemizona bound human sperm after staining with T -6/FITC. AC, acrosomal region; Eq, equitorial region. The intensity is noted to be increased when "++" is present, decreased when "+" is present, and absent fluorescence is noted with "_". fonyl fluoride to the 1 ~L sperm-zona reaction droplet. The hemizonae were washed two times with BWW medium with.5% sodium azide and then placed into individual 2 ~L droplets under oil. The monoclonal antibody used, T-6 (Humagin, Charlottesville, VA), was purified from tissue culture. It reacts to an unknown antigen in the sperm acrosome; the type for this antibody is immunoglobulin (Ig)G1.14 To each droplet 2 to 4 ~L oft-6 (32 ~g protein/ml with.1 % Nonidet P-4; 1 to t final dilution) was added. The droplets were incubated for 2 hours at 23 C; the hemizonae were washed two times in BWW before placement into individual 2 ~L droplets under oil. To each droplet 2 to 4 ~L of fluorescein isothiocyanate (FITC) conjugated rabbit or goat IgG antimouse IgG (Cappel Laboratories, Cochranville, PA), IgM, and IgA (1 to t final dilution) were added. The droplets were incubated in the dark for! hour at 23 C. The hemizonae were washed four times and placed upon a microscope slide in a minimum volume. The droplet was surrounded with a glycerol antiquenching mounting solution; a cover slip (22 X 22 mm) was placed over the droplet and seated with a paraffin wax/vaseline mixture (1:11). The sperm bound to the hemizonae were evaluated with FITC fluorescence and phase contrast light microscopy at 2X to 8X. 56 Coddington et al. Hemizona assay and T-6 antibody Four immunofluorescent staining patterns of the sperm were recognized (Fig. 1). Some sperm had bound, undergone an acrosome reaction, and exhibited a bar pattern. 13,14 A second group had a full fluorescent "cap" (nonacrosome-reacted).13,14 A third group had a mottled fluorescent stain over the acrosome region (faint cap). Last, a fourth group of tightly bound sperm demonstrated no fluorescence. An example of fluorescent and nonfluorescent sperm tightly bound to the hemizona may be seen in Figure 2. A range of 51 to 128 tightly bound sperm were noted in the six hemizona specimens. Because the sperm bound to the curved surface could not be easily reoriented, on the average 4% could be scored for immunofluorescent patterns (Table 1, columns 1 and 2). An acrosome reaction was interpreted to have occurred when a bar pattern or unstained sperm were observed (Fig. 1). For the six specimens observed, there was a range of 5 to 39 sperm with a bar pattern. This finding indicated that the acrosomal reaction occurred in 71.3% ± 4% (mean ± SEM) of attached sperm (Table 1). Other attached sperm (range 2 to 12 sperm) did not stain with fluorescent antibody. These unstained but tightly bound sperm were interpreted to have undergone an acrosomal reaction 13 and represented Figure 2 Top: Sperm tightly bound to the hemizona as part ofhza. Bottom: Same hemizona with sperm bound and stained with T -6/FITC with a bar pattern noted at the point of and adjacent to (I). Tightly bound sperm with no immunofluorescence are seen in the lower portion of the hemizona.

4 Table 1 Evaluation by T -6 Fluorescent Antibody of Spermatozoa Tightly Bound to the Zona Pellucida a No. of Sample No. of sperm bound No. of sperm No. of sperm with No. of sperm with No. of sperm with sperm no. duringhza scored cap pattern b faint cap pattern b bar pattern' unstained' a 121 3b 92 4a 68 4b 51 a Please see text for scoring criteria. b Cap pattern, acrosome intact , Bar pattern or no immunofluorescent pattern, acrosome-reacted. 21.6% ± 3% (mean ± SEM) of the bound sperm (Table 1). Figure 2 demonstrates sperm bound with no fluorescence. Tightly bound sperm were also noted with faint or fenestrated acrosomes (range o to 3 sperm) and comprised 4.6% ± 2.6% (mean ± SEM) of the sperm on the zona. These sperm were interpreted as not having acrosome-reacted completely at the time of staining. DISCUSSION The use of T -6 monoclonal antibody to evaluate the status of the acrosome has been described by others,13,14,16 but not in the context of zona binding. We observed that 71.3% ± 4% ofthe bound sperm were acrosome-reacted, a finding supported by others who have shown that more sperm undergo the acrosome reaction when zona pellucida or its products are present. 19,21 This high percentage of zonabound sperm that have undergone acrosome reaction is in contrast to presented results from our laboratory in which the swim-up sperm incubated for 4 hours and exposed to the hemizona but did not bind showed 1% acrosome reaction.22 We suggest that an even higher number of zona-bound sperm actually reacted since T -6 is very specific and may not reflect all of the acrosomal changes. The acrosome of bound sperm was noted in various states from a cap pattern to no immunofluorescence (Fig. 1), suggesting a dynamic process. We stopped the progress with sodium azide and phenylmethylsulfonyl fluoride to better measure this changing process. The study duration of 5 to 6 hours included liquefaction, swim-up, and a 4-hour incubation with the zonae. Cross et al.19 found a larger number of sperm acrosome-reacted after 1 hour of incubation with a whole zona, but this result could have been due to their preparation time (6.5 to 9.5 hours) before incubation with the zonae. The sperm in our study had a shorter preparation time before zona exposure but a longer incubation time with the zonae. The tightly bound sperm that did not fluoresce may have undergone further acrosomal changes.23 These sperm were viable because they had been processed using a swim-up technique and became tightly bound to the zona (Fig. 2); dead sperm would not have bound tightly. Use of sodium azide and phenylmethylsulfonyl fluoride was intended to prevent autodigestive changes. These nonfluorescent sperm represent 21.6% ± 3% (mean ± SEM) of the sperm studied. Grouping the band pattern and no fluorescent sperm together, a large percentage (93%) ofthe sperm bound to the hemizona had undergone the acrosome reaction. The fact that variations occurred between individual zona pellucidae and their ability to tightly bind sperm has been noted previously and presented.23 The range of tightly bound sperm for these specimens is similar to that for fertile males (42 to 215, and 27 to 142).17,24,25 Some changes may have occurred because of our methods, such as use of sodium azide, dilution of T -6, or exposure of the antibody /FITC to the sperm. Also, there may be a population of sperm that do not retain the cytoplasm near the junction of the acrosome and the equatorial segment. Thus, by not having this fibrillar component, T-6 may not bind well to these sperm. However, our findings consistently demonstrate acrosome reaction in the six specimens from fertile males. We suggest that the 4% of bound sperm that we studied equally represented all sperm on the zona surface. The acrosome reaction on the zona can be studied using the T -6 antibody. This study further validated the usefulness of the HZA in elucidating the mechanisms of fertilization. The HZA may identify a group of male infertility patients previously undefined. Additional studies will focus on the dynamic transformation of the acrosome as the sperm penetrate the zona pellucida. Coddington et al. Hemizona assay and T-6 antibody 57

5 Acknowledgments. We thank Ms. Dara Willett Leary for her editorial contribution and manuscript preparation as well as editorial assistance from Charlotte Schrader, Ph.D. REFERENCES 1. Austin CR: Observations on the penetration of the sperm into the mammalian egg. Aust J Sci Res 4:581, Austin CR: The "capacitation" of the mammalian sperm. Nature 17:326, Chang MC: Fertilizing capacity of spermatozoa deposited into the fallopian tube. Nature 168:697, Yanagimachi R: The movement of golden hamster spermatozoa before and after capacitation. J Reprod Fertil 23:193, Moore HDM, Bedford JM: The interaction of mammalian gametes in the female. In Mechanism and Control of Animal Fertilization, Edited by JF Hartmann. New York, Academic Press, 1983, p Wasserman PM: Early events in mammalian fertilization. Ann Rev Cell Bioi 3:19, Yudin AI, Gottleib W, Meizel S: Ultrastructural studies of early events of the human sperm acrosome reaction as initiated by human follicular fluid. Gamete Res 2:11, Nagae T, Yanagimachi R, Srivastava PN, Yanigamachi MH: Acrosome reaction in human sperm. Fertil Steril 45: 71, Yanagimachi R, Noda YD: Ultrastructural changes in the hamster sperm head fertilization. J Ultrastruct Res 31:465, Yanagimachi R, Noda YD: Physiological changes in the post-nuclear cap region of mammalian spermatozoa: a necessary preliminary to the membrane fusion between sperm and egg cells. J Ultrastruct Res 31:486, Yanagimachi R: Specificity of sperm-egg interaction. In Immunobiology of Gametes, Edited by M Edidin, M Johnson. Cambridge, England, Cambridge University Press, 1977,p Yanagimachi R: Mechanisms for fertilization in mammals. In Fertilization and Embryonic Development In Vitro, Edited by L Mastroianni, Jr, JD Biggers. New York, Plenum, 1981, p Wolf DP, Boldt J, Byrd W, Bechtol K: Acrosomal status evaluation in human ejaculated sperm with monoclonal antibodies. Bioi Reprod 32:1157, Ochs D, WolfDP, Ochs RL: Intermediate filament proteins in human sperm heads. Exp Cell Res 167:495, Fukuda M, Cross NL, Cummings-Paulson L, Yee B: Corre- lation of acrosomal status and sperm performance in sperm penetration assay. Fertil Steril52:836, Scully NF, Johnson AJ, Bryant D, Woodward M, Lipshultz LI, Lamb DJ: T-6 antibody test of acrosome reaction does not correlate with the sperm penetration assay. (Abstr. - 79) 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 Burkman LJ, Coddington CC, Franken DR, Kruger TF, Rosenwaks Z, Hodgen GD: The hemizona assay: development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization po.tential. Fertil SteriI49:688, Oehninger S, Coddington CC, Scott R, Franken DR, Burkman LJ, Acosta AA, Hodgen GD: Hemizona assay: assessment of sperm dysfunction and prediction of in vitro fertilization outcome. Fertil Steril51:665, Cross NL, Morales P, Overstreet JW, Hanson FW: Induction of acrosome reaction by the human zona pellucida. Bioi Reprod 38:235, Overstreet JW, Hembree WC: Penetration of the zona pellucida of non-living human oocytes by human spermatozoa in vitro. Fertil Steril 27:815, Stock CE, Bates R, Lindsay KS, Edmonds DK, Fraser LR: Human oocyte-cumulus complexes stimulate the human acrosome reaction. J Reprod FertiI86:723, Fulgham DL, Johnson D, Coddington CC, Herr J, Alexander NJ, Hodgen GD: Human sperm acrosome reaction rate on zona pellucida: a time course study. (Abstr. -19) 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 Olson GE, Winfrey VP, Davenport GR: Characterization of matrix domains of the hamster acrosome. Bioi Reprod 39:1145, Oosthuizen WT, Coddington CC, Franken DF, Kruger TF, Cronje MS: Zona pellucida binding of spermatozoa from fertile men evaluated over a 3 month interval using the hemizona assay (HZA). [Abstr. -72] 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 Franken DR, Oehninger SC, Burkman LJ, Coddington CC, Kruger TF, Rosenwaks Z, Acosta AA, Hodgen GD: The hemizona assay (HZA): a predictor of human sperm fertilizing potential in in vitro fertilization (IVF) treatment. J In Vitro Fert Embryo Transfer 6:44, Coddington et ai. Hemizona assay and T-6 antibody