Induction of the human sperm acrosome reaction by human oocytes*

FERTILITY AND STERILITY Copyright C> 1988 The American Fertility Society Vol. 50, No.6, December 1988 Printed in U.S.A. Induction of the human sperm acrosome reaction by human oocytes* Christopher J. De Jonge, M.S.t:j: Richard G. Rawlins, Ph.D.tll Lourens J. D. Zaneveld, D.V.M., Ph.D.t Rush University, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois The acrosome reaction of human spermatozoa incubated in the presence or absence of vested human oocytes was investigated. All gametes were obtained from human in vitro fertilization (IVF) cases. Spermatozoa were collected after incubation in insemination medium only and following removal of the oocytes from insemination medium during the IVF procedure. After 16 hours of incubation 18.5% of the spermatozoa in insemination medium alone were acrosome-reacted compared to 31.5% for spermatozoa incubated in medium containing oocytes. The acrosome reaction of spermatozoa incubated with fertilized or unfertilized oocytes was also investigated. The percentage of acrosome reaction did not differ (P> 0.05) between the two groups (29.7% in the fertilized cases versus 30.7% in the unfertilized cases). Completion of oocyte nuclear maturation did not affect the proportion of acrosome-reacted spermatozoa observed with unfertilized eggs. A similar (P > 0.05) percentage of acrosome reacted spermatozoa were observed regardless of whether the unfertilized oocytes had (29%) or had not (35%) reached metaphase II. These findings indicate the acrosome reaction of human spermatozoa is enhanced in the presence of vested human oocytes. Furthermore, there is no apparent correlation between the percentage of the population of spermatozoa that acrosome react in the medium and the potential of an oocyte for fertilization. Fertil Steril50:949, 1988 Penetration of a spermatozoon through vestments of the oocyte is accomplished only after it has undergone fusion and vesiculation of the plasma and outer acrosomal membranes, i.e., the acrosome reaction. The acrosome reaction can be induced by both physiologic and artificial stimuli. Recent investigations have shown that human follicular fluid and the outer vestments of the human oocyte, the granulosa cells, cumulus oophorus, and Received June 20, 1988; revised and accepted August 16, 1988. * Supported by the National Institutes of Health grant HD 19555. t Department of Obstetrics and Gynecology. :j: Department of Physiology. Reprint requests: Christopher J. De Jonge, M.S., Ob/Gyn Research, Rush-Presbyterian-St. Luke's Medical Center, 1653 West Congress Parkway, Chicago, Illinois 60612-3864. II Department of Anatomy. ~ Department of Biochemistry. zona pellucida can induce the human sperm acrosome reaction. 1-4 These findings are of particular interest to in vitro fertilization (IVF) programs because, excluding follicular fluid, three of the four biologic components are present that may stimulate the acrosome reaction and facilitate fertilization. Given that the acrosome reaction is an essential part of the fertilization process and, thus, of IVF outcome, several questions can be raised: (1) Is there a difference in acrosomal status between spermatozoa incubated in IVF insemination medium only and insemination medium-containing oocytes? (2) Does the percentage of spermatozoa that undergo the acrosome reaction correlate with oocyte maturation or fertilization outcome? (3) Does the status of the oocyte after insemination and the percentage of acrosome-reacted spermatozoa provide the reason(s) why an IVF procedure Vol. 50, No.6, December 1988 De Jonge et at. Human oocyte-induced sperm acrosome reaction 949

Figure 1 FRACTIDN A -EGGS 16 HOUR INCUBATION WASHED EJACULATE 1 wash twice by centrifugation SPERM PELLET LOWER SPERM LAYER FRACTION B IN INSEMINATION IN INSEMINATION MEDIUM DETERMINE SPERM MOTILITY AND ACROSOMAL STATUS Sperm treatment protocol. +EGGS did or did not succeed. A preliminary communication on this topic has been presented previously. 5 MATERIALS AND METHODS Induction of Ovulation for IVF The following protocol was used for the induction of ovulation. 6 Female patients received 50 mg clomiphene citrate (CC) (Serophene, Serono, Randolph, MA) orally for 5 days in combination with 75 to 150 IV human menopausal gonadotropin (hmg) (Pergonal, Serono, Randolph, MA) intramuscularly (1M) from cycle day 2 through day 8. Oocyte maturation was induced by administration of 10,000 IV human chorionic gonadotropin (hcg) (Profasi, Serono, Randolph, MA) 1M on day 10 and oocytes were aspirated from the follicles 34 to 36 hours later by laparoscopy. Oocytes were collected and washed (2 to 3X) in Dulbecco's phosphatebuffered saline (Gibco, Grand Island, NY), warmed to 37 C, and immediately transferred to 1.0 ml of insemination medium: Ham's F-I0 (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated, filtered (0.2 Jlm cellulose acetate filter unit), day 21 maternal serum. Oocytes were incubated under 5.0% CO 2 in humidified air at 3TC for 6 hours before insemination with spermatozoa at 40 to 42 hours after hcg. Sperm Preparation for IVF Semen samples were collected by masturbation 4 hours before insemination (Fig. 1). Samples were divided into 250 Jll aliquots in 15-ml centrifuge tubes (Corning Glass Works, Corning, NY) and washed twice by addition of 500 Jll of insemination medium followed by centrifugation at 200 X g for 10 minutes. After removal of the second supernatent the sperm pellet was overlain with 500 Jll of insemination medium, and allowed to swim-up for 1 hour under 5.0% CO 2 in air at 37 C. 7 The upper 250 Jll (A fraction) was then separated from the lower 250 Jll (B fraction), pooled, and counted. Both A and B fractions were incubated at 37 C; however, only the B fraction was used for insemination of the oocytes (B fraction spermatozoa contain a greater proportion of Y chromosome-bearing spermatozoa and its use for control of genetic sex in IVF was being tested concurrently with the present investigation 7 ). The B fraction was separated into two portions: one was used to inseminate the 00- cytes (50,000 to 100,000 spermatozoa/oocyte) and the other was incubated identically but without 00- cytes. Each oocyte was incubated in a separate culture dish. After 16 hours inseminated oocytes were examined for signs of fertilization: the presence of a second polar body and two pronuclei. Spermatozoa were also assessed for motility at that time. Fertilized and unfertilized eggs were transferred into fresh medium and the original medium containing spermatozoa exposed to oocytes was collected for acrosomal evaluation. Control sperm samples (both A and B fractions) incubated under identical conditions but without oocytes were also evaluated for acrosomal status. The maturational status of all unfertilized oocytes was determined by lacmoid staining of nuclear chromatin and evaluation of chromosomal configuration under XI000 magnification. Sperm Acrosomal Evaluation Because a high percentage (>75%) of the incubated spermatozoa retained motility (reflecting maintenance of viability), a modification 5 of the triple stain technique by Talbot and Chacon 8 was employed to assess acrosomal status. Spermatozoa were fixed in 3 % glutaraldehyde for 30 minutes, then washed twice by centrifugation at 1000 X g for 3 minutes in distilled water. The sperm pellet was resuspended in 50 Jll of distilled water, pipetted onto a microscope slide, and smears prepared. The air-dried slides were then incubated (37 C) in Bismark brown (0.8%) for 10 minutes. After two rinses (ten passes each) in distilled water, the slides were 950 De Jonge et al. Human oocyte-induced sperm acrosome reaction Fertility and Sterility

Table 1 Acrosomal Status of Human Spermatozoa Incubated in IVF Insemination Medium Only and IVF Insemination Medium Containing Oocytes Medium only Medium + eggs % Acrosome reaction a,b.c 18.5 ± 15.6 31.5 ± 18.9 a Values represent the mean ± SD (n = 40) and are significantly different from each other (P < 0.001). b Total no. of patients, 40; total no. of eggs, 203. If a patient had more than one incubate (oocyte), the data for all incubates ofthat patient were averaged. c No difference was present in sperm motility between control and oocyte exposed. incubated (22 C) in Rose bengal (0.8%) for 20 to 25 minutes. Slides were washed, passed through an alcohol dehydration series (50%, 95%, and 100% EtOH) and cleared in xylene for 10 minutes. Double-stained (vital stain omitted) spermatozoa (100/ slide, two replicates) from control (no oocytes) and experimental (oocyte-exposed) dishes were then examined by light microscopy under oil immersion (X1000) to determine the acrosomal status. Statistical Analysis For each IVF case (n = 40) the mean and SD were calculated for spermatozoa incubated in insemination medium alone (A and B fractions) and insemination medium-containing oocytes (B fraction only). Grand means were calculated for spermatozoa incubated in insemination medium alone. For comparisons between spermatozoa incubated in insemination medium versus insemination medium plus oocytes, the mean and SD for spermatozoa incubated in insemination medium plus 00- cytes (n = 203) were calculated for each patient and a grand mean was determined. Comparisons for oocyte status reflect pooled means and SD. Statistical significance was determined by analysis of variance followed by Student's t-test analysis. Spermatozoa isolated from the A and B fractions after the swim-up procedure differed in their chromosome content 7 and were compared for the percent acrosome reaction after incubation for 16 hours in insemination medium alone. Spermatozoa from the two fractions were similar (P> 0.05) in the percentage that acrosome reacted after incubation (n = 40); 18.7% acrosome reacted in the A fraction versus 18.5% in the B fraction. Subsequent experiments were performed only with spermatozoa isolated from the B fraction of the swim-up procedure. Spermatozoa incubated in insemination medium alone were compared to spermatozoa incubated in insemination medium-containing oocytes for the percent acrosome reaction. The data are shown in Table 1. Approximately a 2-fold greater percentage of acrosome-reacted spermatozoa was seen in the oocyte-exposed group as compared to oocyte-free medium (P < 0.001). Patients having multiple 00- cytes, in which some fertilized and some did not, were also analyzed. No difference was observed between the two groups per patient in the percentage that acrosome-reacted (P> 0.05). For these same patients the percent acrosome reaction ranged from 9% to 63% (27 ± 17) for spermatozoa in which the oocytes became fertilized and 7% to 60% (27 ± 18) for unfertilized oocytes. The maturational status of fertilized and unfertilized oocytes after incubation with spermatozoa was also examined as a possible factor that might influence the acrosome reaction. Subsequently, maturation and fertilization were tested against the percentage of spermatozoa that acrosome-reacted in the medium (Table 2). The percent acrosome reaction of spermatozoa in incubates that resulted in fertilized oocytes was essentially the same as those that failed fertilization. Additionally, those oocytes that were not fertilized, whether due to oocyte inactivation (failure to reach metaphase II following germinal vesicle breakdown) or the lack of sperm penetration in mature oocytes (metaphase II), stimulated the acrosome reaction to the same degree; the percent acrosome reaction was also not different from that of fertilized eggs. DISCUSSION A number of investigations have examined the influence of human follicular fluid,1,2 cumulus RESULTS Table 2 Percent Acrosome Reaction in Relationship to Oocyte Status After Incubation Oocyte status Fertilized Unfertilized (total) Unfertilized with polar body Unfertilized no polar body Total no. ofoocytes 124 79 40 39 % Acrosome reactiona,b 29.7 ± 20.2 30.7 ± 20.9 28.8± 21.8 34.8 ± 23.5 a Values are mean ± SD. b No significant differences between values (P> 0.05). Vol. 50, No.6, December 1988 De Jonge et al. Human oocyte-induced sperm acrosome reaction 951

cells2,4,9 and zona pellucidae3,lo,1l on the human sperm acrosome reaction. Depending on the media used for capacitation (e.g., serum albumin or fetal cord serum), follicular fluid induced between a 26% to 46% acrosome reaction in human spermatozoa. 1,2 Cumulus cells from human oocytes also induced an acrosome reaction (26% to 43%) similar to that of follicular fluid. 2 Siiteri et al. 4 washed human cumulus oophorus fragments and demonstrated that the wash-medium was able to induce an acrosome reaction, and that the percent acrosome reaction decreased (42%, 23%, and 13%) with increased washes (1, 2, and 3 washes). Additionally, these investigators 4 showed that the medium used to incubate (24 hours) cumulus cells also induced an acrosome reaction (46%). The percent acrosome reaction seen in spermatozoa obtained from the IVF incubation medium-containing oocytes in the present investigation is within the range reported by investigations that utilized cumulus cells as acrosome reaction inducers. Recently, Cross et al. 3 demonstrated an induction of the human sperm acrosome reaction by nonviable, human zona pellucidae (46% acrosome reaction), and disaggregated zona pellucidae (24% acrosome reaction). The statistical average for their data is similar to the percent acrosome reaction obtained for spermatozoa incubated in the presence of oocytes in the current system. In contrast to the present investigation, Plachot et al. 9 showed no difference in the percent acrosome reaction observed when spermatozoa were incubated in the presence (7%) or absence (8.2%) of human cumulus-oocyte complexes. These differences cannot be explained but may be due to the low percentage of acrosome reaction seen in their system. These investigators 9 also concluded that there is no correlation between the fertilization rate of mature human oocytes and the percentage of acrosome-reacted living spermatozoa. However, they did not investigate if the maturation stage of the oocyte affected the acrosome reaction. The follicular components that are capable of inducing an acrosome reaction in capacitated spermatozoa include follicular fluid, granulosa cells, cumulus cells, and the zona pellucida. Because the 00- cytes were washed before insemination, all of these components except the fluid were present in vitro during the IVF procedure. However, it is possible that some fluid components were present because they may have adhered to the oocyte vestments. Spermatozoa incubated under the present conditions and subsequently assayed for acrosomal status (in particular those that lost the acrosome) were free-swimming, i.e., not attached to or enmeshed in either cumulus or zona pellucida. The difference seen in the percentage of acrosome-reacted spermatozoa incubated in medium alone and medium -containing oocytes implies that one or more of several events occurs during IVF to induce an acrosome reaction: (1) the spermatozoa contact the cumulus, acrosome-react, and swim away; (2) the sperm penetrate the cumulus mass to contact the zona, then acrosome react, and swim back out; (3) a factor(s) may diffuse out from one or more of the oocyte vestments to induce the acrosome reaction. Because a high percentage of spermatozoa incubated in the absence of oocytes are not acrosome-reacted (as determined by double stain) and retain their motility (>70%), they can potentially serve as a reservoir for second insemination, instead of collecting a fresh specimen, if the initial attempt at fertilization fails. There is little or no information regarding the percentage of the sperm population that undergoes the acrosome reaction in relationship to IVF outcome. The data presented here show that no differences exist in the percentage of acrosome-reacted spermatozoa whether the oocyte fertilized or not, or whether the unfertilized oocyte remained immature (arrested in metaphase I) or completed nuclear maturation (metaphase II). Even if incubates from the same patient were observed, no correlation with fertilization was seen. Thus, there is no correlation between the percentage ofthe sperm population that undergoes the acrosome reaction and the outcome of IVF (i.e., the greater the percent acrosome reaction, the greater the chance of fertilization). In conclusion, cellular components that induce the acrosome reaction appear to be associated with all oocytes, independent of their maturity and capacity for fertilization. Acknowledgments. The authors thank Ms. Barbara Hartrampf for manuscript preparation. REFERENCES 1. Suarez SS, Wolf DP, Meizel S: Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid. Gamete Res 14:107, 1986 2. Tesarik J: Composition of acrosome reaction-inducing activities of human cumulus oophorus, follicular fluid and ionophore A23187 in human sperm populations of proven fertilizing ability in vitro. J Reprod Fertil 74:383,1985 3. Cross NL, Morales P, Overstreet JW, Hanson FW: Induc- 952 De Jonge et al. Human oocyte-induced sperm acrosome reaction Fertility and Sterility

tion of acrosome reactions by the human zona pellucida. Bioi Reprod 38:235, 1988 4. Siiteri JE, Dandekar P, Meizel S: Human sperm acrosome reaction-initiating activity associated with the human cumulus oophorus and mural granulosa cells. J Exp Zool 246: 71, 1988 5. De Jonge C, Rawlins RG, Zaneveld LJD: Induction of the human sperm acrosome reaction by human oocytes (Abstr. 88). J AndroI9:P46, 1988 6. Radwanska E, Basuray R, Rawlins R, Binor Z, Rana N, Henig I, Dmowski WP: In vitro fertilization program at Rush Presbyterian-St. Luke's Medical Center, Chicago, Illinois. J In Vitro Fert Embryo Transfer 3:265, 1986 7. Rawlins RG, Sachdeva S, Radwanska E, Binor Z, Rana N, Dmowski WP: Human sex preselection and in vitro fertilization (IVF): fraction separation of sperm enhances probability of conceiving male offspring (Abstr. 156). Presented at the Society for Gynecological Investigation, 1988, p 137 8. Talbot P, Chacon RS: A triple-stain technique for evaluating normal acrosome reactions of human sperm. J Exp Zool 215:201, 1981 9. Plachot M, Mandelbaum J, Junca A-M: Acrosome reaction of human sperm used for in vitro fertilization. Fertil Steril 42:418, 1984 10. Sathananthan AH, Trounson AO, Wood C, Leeton JF: Ultrastructural observations on the penetration of human sperm into the zona pellucida of the human egg in vitro. J Androl 3:356, 1982 11. Singer SL, Lambert H, Overstreet JW, Hanson FW, Yanagimachi R: The kinetics of human sperm binding to the human zona pellucida and zona-free hamster oocyte in vitro. Gamete Res 12:29, 1985 Vol. 50, No.6, December 1988 De Jonge et al. Human oocyte-induced sperm acrosome reaction 953