Effects of human follicular fluid on spermatozoa that have been cocultured with human oviductal cells

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1 FERTILITY AND STERILITY VOL. 72, NO. 6, DECEMBER 1999 Copyright 1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Effects of human follicular fluid on spermatozoa that have been cocultured with human oviductal cells Yuan-qing Yao, M.D.,* Pak-chung Ho, F.R.C.O.G., and William Shu-biu Yeung, Ph.D. Tangdu Hospital, Xian; University of Hong Kong, Queen Mary Hospital, Hong Kong, China Objective: To investigate the sequential effects of human oviductal cells and human follicular fluid (hff) on various sperm functions. Design: Laboratory experimental study. Setting: University gynecology unit. Patient(s): Fallopian tubes were from patients undergoing tubal ligation or hysterectomy. Semen was from men attending the subfertility clinics. Intervention(s): Spermatozoa were treated with [1] 6 hours in Earle s balanced salt solution (EBSS-BSA; control); [2] 5 hours in EBSS-BSA and 1 hour with hff (hff); [3] 5 hours with oviductal cells and 1 hour in EBSS-BSA (coculture); and [4] 5 hours with oviductal cells and 1 hour with hff (sequential). Main Outcome Measure(s): Motility, acrosome reaction, zona binding, and oocyte fusion. Result(s): Groups II and III spermatozoa had similar motility and were better than that of group I. Group IV displayed higher motility parameters than the other groups. Human follicular fluid induced acrosome reaction. The incidence of acrosome reaction in group IV was significantly lower than that in group II. Group III did not affect the acrosome reaction. Spermatozoa in groups II IV had lower zona binding capacity than those in group I. Human follicular fluid stimulated oocyte penetration, whereas oviductal cells suppressed this effect of hff. Conclusion(s): Oviductal cells maintained the fertilizing capacity of spermatozoa, whereas hff facilitated the fertilization process of oviductal spermatozoa. (Fertil Steril 1999;72: by American Society for Reproductive Medicine.) Key Words: Oviductal cells, follicular fluid, sperm functions, coculture Received April 19, 1999; revised and accepted July 13, Supported by grant 337/043/0012 from the University of Hong Kong. Reprint requests: W. S. B. Yeung, Ph.D., Department of Obstetrics and Gynaecology, University of Hong Kong, Queen Mary Hospital, Hong Kong, China (FAX: ; wsbyeung@hkucc.hku.hk). * Department of Obstetrics and Gynaecology, Tangdu Hospital. Department of Obstetrics and Gynaecology, University of Hong Kong /99/$20.00 PII S (99) Before ovulation, spermatozoa in the oviduct interact with the oviductal epithelium. We recently showed that the oviductal cells maintained the fertilization capacity of the cocultured spermatozoa (1). After ovulation, follicular fluid together with the oocyte-cumulus mass are transferred to the oviduct. The spermatozoa residing in the oviduct are then exposed to the incoming follicular fluid. Although follicular fluid is known to affect various sperm functions (2), the response of the oviductal spermatozoa to follicular fluid is unknown. There is some evidence suggesting that the follicular fluid and oviductal cells act coherently to promote normal fertilization. Follicular fluid contains progesterone that increases the incidence of polyspermic fertilization (3). The presence of oviductal cells significantly reduces polyspermy but does not influence the success of fertilization (4). Oviductal cells decrease sperm-oocyte fusion (1). On the other hand, human follicular fluid (hff) stimulates the sperm-oocyte penetration (2). The relationship between follicular fluid and oviductal cells during fertilization is not at all clear. We present here the first report on the sequential effects of oviductal cells and follicular fluid on the functional status of the spermatozoa using in vitro coculture model. MATERIALS AND METHODS Culture of Human Oviductal Cells This study was approved by the University Ethics Committee, the University of Hong Kong. Human oviductal cells were cultured in 1079

2 vitro as previously described (5). Fallopian tubes were collected during tubal ligation or hysterectomy due to uterine leiomyoma. The inner epithelium of the oviduct was minced and digested in 0.05% (wt/vol) trypsin/0.53 mm ethylenediaminetetraacetic acid (EDTA) solution (GIBCO, Grand Island, NY) at 37 C in a shaking incubator (Julabo, Schwarzwald, Germany) for approximately 45 minutes. The enzyme was washed away by centrifugation with phosphate-buffered saline (PBS). The harvested cells were cultured in serum supplemented Dulbecco s minimum essential medium (DMEM)/F-12 (sdmem/f12) at 37 C under an atmosphere of 5% CO 2 in air. After confluent growth, the cells were trypsinized, resuspended in sdmem/f-12 supplemented with 10% dimethylsulphoxide (DMSO) (Sigma, St. Louis, MO), and cryopreserved in liquid nitrogen until use. The frozen oviductal cells used in this study came from more than one woman. Immunocytochemical staining with antibody against cytokeratin showed that 85% of the cultured cells were positively stained, suggesting most of them were epithelial in nature. Collection of hff Human follicular fluid was collected during oocyte retrieval from women attending assisted reproduction program in the Queen Mary Hospital, Hong Kong. Human menopausal gonadotropin and hcg were used for ovulation induction in these women. Only hff without blood contamination and from follicles containing an oocyte was collected. Two or three milliliters of hff from each sample was centrifuged at 600 g for 10 minutes to remove cellular debris and filtered with a 0.2- m filter, before being stored at 20 C until used. The hff pool was made by mixing 2 ml of hff from each of 10 individual samples of hff. Semen Collection, Basic Evaluation, and Processing All semen samples were collected by masturbation from men visiting the subfertility clinics of Queen Mary Hospital. Only semen samples with normal semen parameters according to World Health Organization (WHO) criteria (6) were used for this study. Spermatozoa were separated from seminal plasma by two-step Percoll density centrifugation (45% and 90%; Pharmacia, Uppsala, Sweden) as previously described (7). The resulting sperm pellet was washed and resuspended in Earle s balanced salt solution supplemented with bovine serum albumin (EBSS-BSA). Effect on Sperm Motility Sperm motility was evaluated by the Hobson Sperm Tracker System as described previously (8). Sperm suspension was adjusted to a concentration of spermatozoa/ml. Then 6 L of the sperm suspension was transferred to a prewarmed Cell-VU disposable semen analysis chamber (Fertility Technologies, Inc., Natick, MA) with chamber depth of 30 m. Motility analysis was performed on a warmed microscope stage at 37 C. The fields were selected randomly during evaluation. Five hundred spermatozoa were analyzed in each specimen. The following seven parameters were determined: [1] curvilinear velocity (VCL, m/s), [2] straight line velocity (VSL, m/s), [3] average path velocity (VAP, m/s), [4] linearity (LIN, VSL/VCL), [5] amplitude of lateral head displacement (ALH, m), [6] beat cross frequency (BCF, Hz), and [7] percentage of sperm exhibiting hyperactivation (HA). Spermatozoa agreeing with the criteria LIN of 65, VCL of 100 m/s, ALH of 7 m were classified as hyperactivated spermatozoa. Effect on Acrosome Reaction Sperm acrosomal status was determined by fluorescein isothiocyanate-labeled Pisum sativum lectin (FITC-PSA; Sigma) as described by Cross et al. (9). In brief, 45 L of sperm suspension were incubated with 5 L of PBS containing 0.001% (wt/vol) Hoechst (bisbenzimide; Sigma) at 37 C under 5% CO 2 in air for 10 minutes. Excessive Hoechst was removed by centrifugation through a solution of 2% (wt/vol) polyvinylpyrrolidone-40 (PVP-40; Sigma) in PBS at 900 g for 5 minutes. The washed spermatozoa were fixed in 300 L of 95% ethanol at 4 C for 30 minutes, dried on a glass slide, and stained with 30 L of 0.01% (wt/vol) FITC-PSA in PBS for 10 minutes. The acrosomal status and viability of 100 spermatozoa were determined under a fluorescence microscope with 1,000 magnification. Hoechst staining was observed with a filter set consisting of an excitation filter G365, a chromatic beam splitter FT395, and a barrier filter LP420. The filter set for FITC-PSA staining consisted of an excitation filter BP , a chromatic beam splitter FT510, and a barrier filter LP520. Spermatozoa with a strong Hoechst staining (Hoechst staining positive) were considered dead spermatozoa. The acrosomal status of spermatozoa was classified according to the lectin staining into [1] intact acrosome: complete staining of acrosome, [2] reacting acrosome: Hoechst staining negative, partial or patchy staining of acrosome, [3] reacted acrosome: Hoechst staining negative, complete staining of the equatorial segment only or no staining of the whole sperm head. The proportions of intact, reacting, and reacted acrosome were expressed as percentages of the respective patterns in the total number of spermatozoa counted. Effect on the Zona Pellucida Binding Capacity of Spermatozoa Hemizona assay (HZA) was performed as described by Burkman et al. (10) with modifications (11). Unfertilized oocytes from the assisted reproduction program of Queen Mary Hospital were used. Oocytes were bisected into two hemizona by a beaver Micro-Sharp blade (Becton Dickinson AcuteCare, Franklin Lakes, NJ) connected to a micromanipulator. The ooplasm inside each hemizonae was dislodged by pipetting the hemizonae through a micropipette Yao et al. Follicular fluid and oviductal sperms Vol. 72, No. 6, December 1999

3 During HZA, one hemizonae was placed in a 100- L droplet of EBSS-BSA containing motile spermatozoa that had been pretreated according to the experimental design. Matching hemizonae was placed in another 100- L droplet of EBSS-BSA containing motile control spermatozoa. After 4 hours of incubation at 37 C under 5% CO 2 in air, the hemizona were transferred to fresh EBSS- BSA and were gently rinsed through a micropipette to remove loosely attached spermatozoa. The hemizonae with the bound spermatozoa were observed under a Nikon inverted microscope with the image projected onto a monitor through a video camera. The tightly bound spermatozoa on the outer surface of the zona were marked on a transparency attached to the monitor. The number of marked spermatozoa was counted. The index of HZA (HZI) was calculated as: number of spermatozoa bound in test droplet HZI 100 number of spermatozoa bound in control droplet Effect on Oocyte Fusion Capacity of Spermatozoa The oocyte fusion capacity of human spermatozoa was evaluated by zona-free hamster oocyte penetration test as described by the WHO (6). Pregnant mare serum gonadotropin and hcg were injected intraperitoneally 48 hours apart to induce superovulation in golden hamsters. Hamsters were killed 18 hours after hcg injection. Zona-free oocytes were obtained from these animals by successive treatment of the cumulus mass in hyaluronidase (0.1% in Biggers, Whitten, and Whittingham [BWW]) for 5 minutes and in trypsin (0.1% in BWW) for 1 2 minutes. Twenty to 30 zona-free oocytes were incubated in a 100- L BWW droplet containing motile spermatozoa/ml at 37 C under 5% CO 2 in air for 3 hours. The spermatozoa tested were derived from sequential incubation experiments with human oviductal cells and hff. After incubation, oocytes were washed free of loosely bound spermatozoa with BWW, fixed in a mixture of ethanol and acetic acid (vol/vol 3:1), transferred to microscopic slides, and stained with acetolacmoid solution. Sperm penetration of oocytes was indicated under phase contrast microscopy ( 400) by the presence of a decondensed sperm head (swollen heads) with an adjacent or closely associated sperm tail within the cytoplasm. The percentage of sperm penetration was calculated by dividing the number of oocytes with penetrated spermatozoa by the total number of oocytes inseminated. Penetration index was determined by dividing the total number of spermatozoa penetrated by the number of oocytes inseminated. Sequential Incubation of Spermatozoa With Human Oviductal Cells and hff Percoll-processed spermatozoa were divided into four groups: group I: spermatozoa incubated in EBSS-BSA for 6 hours (control); group II: spermatozoa incubated in EBSS- BSA for 5 hours and then treated with 25% pooled hff for 1 hour (hff treatment); group III: spermatozoa cocultured with human oviductal cells for 5 hours and then incubated in EBSS-BSA for 1 hour (coculture); and group IV: spermatozoa cocultured with human oviductal cells for 5 hours and subsequently incubated with 25% hff for 1 hour (sequential treatment). All the incubations were done at 37 C and 5% CO 2 in air. In groups III and IV, the cocultured spermatozoa did not bind to the oviductal cells. After treatment, the spermatozoa were washed with fresh EBSS-BSA before they were incubated in EBSS-BSA and hff, respectively. At the end of the incubation, all the spermatozoa were washed with fresh EBSS-BSA by centrifugation at 300 g. Their functional statuses were then evaluated as determined by lectin staining. Data Analysis Data were expressed as means SEM, unless otherwise indicated. One-way repeated measures ANOVA was used to compare sperm motility parameters, acrosomal status, and HZI of spermatozoa in different groups. Paired Student s t-test was used to compare the number of bound spermatozoa between matching hemizona. Wilcoxon signed-rank test was used to compare the penetration rate and penetration index between hff treatment and sequential treatment. RESULTS Effect on Sperm Motility Table 1 shows the motility of spermatozoa after different treatment. Spermatozoa in group IV (sequential treatment) had significantly higher motility parameters (VCL, VSL, VAP, BCF, ALH, and HA) than the control spermatozoa in group I (P.05). Group IV spermatozoa also had significantly higher BCF and HA than the cocultured spermatozoa (group III) and higher VCL than hff-treated spermatozoa (group II). Except for LIN, all the motility parameters of spermatozoa in groups II and III were significantly higher than that of the control spermatozoa. No difference in motility was found between the cocultured spermatozoa (group III) and hff-treated spermatozoa (group II), except that BCF of group II was significantly higher than that of group III. Effect on Acrosome Reaction The effects of different methods of treatment on acrosome reaction are shown in Table 2. Compared with spermatozoa without hff treatment (control or cocultured spermatozoa), those after hff treatment, i.e., groups II or IV, had significantly higher incidence of acrosome reaction (P.05). However, hff-induced acrosome reaction in sequentially treated spermatozoa (group IV) was significantly lower than that in spermatozoa treated with hff alone (group II). No difference in spontaneous acrosome reaction was found between the cocultured spermatozoa (group III) and the control spermatozoa (group I). FERTILITY & STERILITY 1081

4 TABLE 1 Sequential effect of coculture and human follicular fluid treatment on sperm motility. Group (n 10) Sperm motility parameters VCL VSL VAP LIN BCF ALH HA I * * * * * * II * * * * * III * IV * * Note: Group I control; group II human follicular fluid treatment; group III coculture with oviductal cells; group IV sequential treatment with oviductal cells and human follicular fluid. ALH amplitude of lateral head displacement ( m), BCF beat cross frequency (Hz), HA hyperactivation, LIN linearity (VSL/VCL), VAP average path velocity ( m/s), VCL curvilinear velocity ( m/s), VSL straight velocity ( m/s). * P.05 between the same * within the same column, determined by one-way repeated measures analysis of variance. Yao. Human follicular fluid. Fertil Steril Effect on Zona Binding Capacity of Spermatozoa The HZAs were performed between group I spermatozoa (control) with the other groups of spermatozoa with use of matching hemizona. The number of bound spermatozoa in group I (control, ) was significantly higher (P.01) than that in group II ( , n 10). Group III spermatozoa also had a significantly lower zona binding capacity (P.01) than that of the control (control vs. group III , n 10). The number of bound spermatozoa in group IV was This was significantly lower (P.05) than that of the corresponding control ( , n 10). No significant difference in HZI was found between groups II ( ), III ( ), and IV ( ). TABLE 2 Sequential effect of coculture and human follicular fluid treatment on acrosome reaction. Group (n 10) Intact acrosome Reacting acrosome Reacted acrosome I * * II * * III IV * * Note: Group I control; group II hff treatment; group III coculture with oviductal cells; group IV sequential treatment. Intact acrosome: complete staining of acrosome; reacting acrosome: Hoechst staining negative, partial, or patchy staining of acrosome; reacted acrosome: Hoechst staining negative, complete staining of the equatorial segment only, or no staining of the sperm head. * P.05 between the same * within the same column, one-way repeated measures analysis of variance. Yao. Human follicular fluid. Fertil Steril Effect on Oocyte Fusion Capacity of Spermatozoa No penetration was observed in groups I and group III. Group II spermatozoa had a penetration rate of 34.3 (median). This was significantly higher than that of group IV (median 27.0, P , Wilcoxon signed-rank test). Similarly, the penetration index of group II (median 0.470) was significantly higher than that of group IV (median 0.295, P , Wilcoxon signed-rank test). DISCUSSION Compared with the control spermatozoa (group I), the present study demonstrates that 1 hour of hff treatment stimulates various motility parameters of capacitated spermatozoa. The effects of follicular fluid on sperm motility in published reports are conflicting. Earlier studies showed a negative effect, whereas most subsequent studies demonstrated a stimulatory influence of hff on sperm motility (2). The response of spermatozoa to hff treatment is affected by the capacitation status of the treated spermatozoa. Our unpublished data show that 25% hff affects only BCF of the uncapacitated spermatozoa, whereas hff at the same concentration stimulates most of the motility parameters of the capacitated spermatozoa. This finds support from Kulin and coworkers study (12) that 25% hff enhanced HA of preincubated spermatozoa, but not those without preincubation. The mechanism by which capacitation affects sperm motility is unknown. Our previous data showed that oviductal cells maintained the motility of spermatozoa in vitro (7). The present study confirms this finding: the sperm velocities (VCL, VSL, VAP, BCF, ALH, and HA) were higher in group III than in group I (control). Similar to groups II and III, sequential treatment (group IV) resulted in spermatozoa having higher velocities (VCL, VSL, VAP, BCF, ALH, and HA) than the control spermatozoa (group I). Compared with the cocultured spermatozoa (group III), sequentially treated spermatozoa (group 1082 Yao et al. Follicular fluid and oviductal sperms Vol. 72, No. 6, December 1999

5 IV) had higher BCF, HA, suggesting that hff further enhanced sperm motility of the cocultured spermatozoa. Most of the motility parameters of the sequentially treated spermatozoa are higher than that of the hff-treated spermatozoa, although only the difference of VCL reaches statistical significance. There are two possible explanations for this observation. First, hff may be more efficient in enhancing motility of the cocultured spermatozoa than that of the spermatozoa preincubated in EBSS-BSA. Second, it is known that the motility of spermatozoa is maintained by the oviductal cells, whereas that of the control spermatozoa decreases continuously with increase in the duration of incubation (7). Thus, hff may have similar efficiency in improving motility in both types of spermatozoa, and the observed difference is just a reflection of the differences of the two groups of spermatozoa after the first 5 hours of incubation. The relative contribution of the two possibilities in the present study remains to be determined. It is known that hff induces acrosome reaction (13) and that the human oviductal cells do not have an effect on the spontaneous acrosome reaction in vitro (1, 14). The present study supports these observations. Human oviductal cells may stabilize the acrosome membrane of spermatozoa (14) via an unknown mechanism. Our recent data also showed that human oviductal cells inhibit calcium ionophore-induced acrosome reaction, suggesting that oviductal cells may reduce the influx of Ca 2 (1). This is in agreement with the study in the horse, in which both the oviductal epithelial cells and their conditioned medium reduce the intracellular Ca 2 concentration of the treated spermatozoa (15). Previous studies also found that spermatozoa flushing out from the oviduct of various animals had intact acrosome (16). However, Ellington (17) found that horse oviductal cells induced acrosome reaction in vitro. It is not clear whether this discrepancy is due to species specificity or differences in experimental conditions. This study further shows that hff significantly increases the spontaneous acrosome reaction of the cocultured spermatozoa. Probably because of the membrane stabilization effect of oviductal cells on spermatozoa (1), the effect of sequential treatment (group IV) on acrosome reaction is less when compared with that of hff treatment alone (group II). Zhu et al. (18) concluded that human oviductal fluid delayed hff-induced acrosome reaction. The present results are in line with the conclusion of Zhu et al. (18) and provide the first evidence that human oviductal cells are involved in delaying hff-induced acrosome reaction. In Zhu and coworkers study (18), 1 and 3 hours of hff treatment failed to induce acrosome reaction of oviductal fluid-treated spermatozoa. In the present study, hff induced acrosome reaction of the cocultured spermatozoa after 1 hour of treatment. The discrepancy may be due to the acrosome-stabilizing effect of the limited number of oviductal cells in the culture well of this study is not as strong as that of the oviductal fluid that is produced by enormous number of oviductal cells in vivo. Both the present and our previous studies show that human oviductal cell (1) and follicular fluid (8) inhibit the zona binding capacity of spermatozoa. Thus, it is not surprising that sequential treatment inhibits sperm binding to the zona pellucida. Because there is no difference in HZI among all the groups, there seems to be no cumulative effect in treating spermatozoa successively with oviductal cells and hff. Two glycoproteins with molecular weight of 32 and 192 kda that inhibit the zona binding capacity of spermatozoa have recently been isolated from hff (19). The 32-kDa glycoprotein has a number of characteristics similar to that of glycodelin-a (8, 19). Human oviductal cells also produce glycodelin-a (20). Therefore, it is possible that glycodelin also contributes to the spermatozoa-zona binding inhibitory activity of oviductal cells. Oocyte penetration is observed after hff treatment in groups II and IV in the present study. These results are comparable with other reports (21, 22). It is interesting to note that hff treatment of cocultured spermatozoa (group IV) has significantly lower penetration rate than those preincubated in EBSS-BSA (group II). This is in line with our previous data showing that the oocyte fusion capacity of spermatozoa was decreased after coculture with human oviductal cells (1). For some unknown reason, Bastias and coworkers (23) were unable to demonstrate a similar effect. The mechanism by which oviductal cells attenuate the effect of hff on sperm-oocyte fusion is unclear. This may be due to the suppression effect of oviductal cells on hff-induced acrosome reaction (Table 2), and acrosome reaction is a prerequisite of sperm-oocyte fusion. It has also been suggested that hff may stimulate sperm-oocyte fusion by an acrosome reaction-independent mechanism (21) via some sperm proteins that are involved in the fusion process (16). The effect of oviductal cells on this latter mechanism is unknown. It is well known that capacitation significantly influence sperm-oocyte fusion in vitro (24). In this study, no penetration is observed in groups I and III. Although our preliminary data using chlortetracycline staining show that coculture enhances sperm capacitation and that this effect starts to level off in about 3 hours, 6 hours of treatment in this study are probably still insufficient to capacitate enough spermatozoa to allow penetration to be detected. On the other hand, hff (groups II and IV) promotes capacitation and allows penetration to occur after 1 hour of treatment. Indeed, hff stimulates capacitation of the treated spermatozoa within 1 hour (Yao YQ, Yeung WSB, unpublished data). When coitus occurs before ovulation, spermatozoa travel to and reside in the oviduct waiting for the arrival of the oocyte. During this period, the oviductal cells maintain the fertilization potential of the spermatozoa by maintaining sperm motility and stabilizing the acrosome membrane (1, 7). After ovulation, hff reaches the oviduct together with FERTILITY & STERILITY 1083

6 the oocyte cumulus complex. The present sequential study demonstrates that hff activates the dormant oviductal spermatozoa by stimulating the motility, acrosome reaction, and sperm-oocyte fusion of the oviductal spermatozoa. Sperm motility, especially HA, may be crucial for the penetration of the spermatozoa through the zona pellucida (16). The hff-induced acrosome reaction may serve to select an optimal population of fertilizing spermatozoa (25). Enzymes released from the acrosome also facilitate the passage of the spermatozoa through the cumulus mass (16). Thus, the oviductal cells and hff play different roles for fertilization. The former maintains the fertilizing capacity of the spermatozoa, whereas the latter facilitates the fertilization process. When coitus occurs after ovulation, spermatozoa will be activated by the hff already in the oviduct to fertilize the oocyte. The maintenance function of the oviductal cells as described when coitus occurs before ovulation would probably be of lower importance. There is, however, one inexplicable observation in this study. It is the spermatozoa-zona binding inhibitory activity of hff. In two small studies, Morales et al. (26) and Huyser et al. (27) reported variable effects of hff on HZA results, ranging from stimulation to inhibition. Recently, we conclusively demonstrated that hff inhibited spermatozoa-zona binding (8, 28). In pigs, follicular fluid also inhibits the zona binding capacity of spermatozoa (29). The inhibitory effect of follicular fluid on spermatozoa-zona binding is dose dependent in both human and pig (8, 29). In the human, the inhibitory activity is due to the presence of at least two glycoproteins in hff (19). One of them is immunologically similar to glycodelin-a (Chiu PCN, Leung M, Yeung WSB, unpublished observation). The physiological role of the inhibitory effect of hff on sperm binding to the zona is unclear. In the pig, follicular fluid inhibits sperm zona binding and reduces the incidence of polyspermic fertilization (29). It is also possible that this inhibitory effect of hff is a process to select spermatozoa with stronger zona binding capacity. On the other hand, hff may not have the observed inhibitory activity in vivo because cumulus cells contain glycosidases (30), and deglycosylation is known to modify the activity of glycoproteins. Acknowledgments: The authors thank the whole staff of our IVF laboratory for their skillful technical assistance. References 1. Yao YQ, Ho PC, Yeung WSB. The effects of human oviductal cell coculture on various functional parameters of human spermatozoa. Fertil Steril 1999;71: Hong CY, Chao HT, Lee SL, Wei YH. Modification of human sperm function by human follicular fluid: a review. Int J Androl 1993;16: Hunter RH. Local action of progesterone leading to polyspermic fertilization in pigs. J Reprod Fertil 1972;31: Nagai T, Moor RM. Effect of oviduct cells on the incidence of polyspermy in pig eggs fertilized in vitro. Mol Reprod Dev 1990;26: Yeung WS, Ho PC, Lau EY, Chan STH. Improved development of human embryos in vitro by a human oviductal cell co-culture system. Hum Reprod 1992;7: World Health Organization. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 3rd ed. New York: Cambridge University Press, 1992; Yeung WS, Ng VK, Lau EY, Ho PC. Human oviductal cells and their conditioned medium maintain the motility and hyperactivation of human spermatozoa in vitro. Hum Reprod 1994;9: Yao YQ, Yeung WS, Ho PC. Human follicular fluid inhibits the binding of human spermatozoa to zona pellucia in vitro. Hum Reprod 1996;11: Cross NL, Morales P, Overstreet JW, Hanson FW. Two simple methods for detecting acrosome-reacted human sperm. Gamete Res 1986;15: Burkman LJ, Coddington CC, Franken DR, Krugen TF, Rosenwaks Z, Hogen GD. The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential. Fertil Steril 1988;49: Yao YQ, Yeung WS, Ho PC. The factors affecting sperm binding to the zona pellucida in the hemizona binding assay. Hum Reprod 1996;11: Kulin S, Bastiaans BA, Hollanders HM, Janssen HJ, Goverde HJ. Human serum and follicular fluid stimulate hyperactivation of human spermatozoa after preincubation. Fertil Steril 1994;62: De Jonge CJ, Barratt CL, Radwanska E, Cooke ID. The acrosome reaction-inducing effect of human follicular and oviductal fluid. J Androl 1993;14: Morales P, Palma V, Salgado AM, Villalon M. Sperm interaction with human oviductal cells in vitro. Hum Reprod 1996;11: Dobrinski I, Smith TT, Suarez SS, Ball BA. Membrane contact with oviductal epithelium modulates the intracellular calcium concentration of equine spermatozoa in vitro. Biol Reprod 1997;56: Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill JD, eds. The physiology of reproduction. 2nd ed. Vol. 1. New York: Raven Press, 1994: Ellington JE, Ball BA, Yang X. Binding of stallion spermatozoa to the equine zona pellucida after coculture with oviductal epithelial cells. J Reprod Fertil 1993;98: Zhu J, Barratt CL, Lippes J, Pacey AA, Cooke ID. The sequential effects of human cervical mucus, oviductal fluid, and follicular fluid on sperm function. Fertil Steril 1994;61: Yao YQ, Chiu CNP, Ip SM, Ho PC, Yeung WSB. Glycoproteins present in human follicular fluid that inhibit the zona binding capacity of spermatozoa. Hum Reprod 1998;13: Laird SM, Hill CJ, Warren MA, Tuckerman EM, Li TC. The production of placental protein 14 by human uterine tubal epithelial cells in culture. Hum Reprod 1995;10: Fukuda M, Cross NL, Cummings-Paulson L, Yee B. Correlation of acrosomal status and sperm performance in the sperm penetration assay. Fertil Steril 1989;52: McClure RD, Tom RA, Dandekar PV. Optimizing the sperm penetration assay with human follicular fluid. Fertil Steril 1990;53: Bastias MC, Kamijo H, Osteen KG. Assessment of human sperm functional changes after in-vitro coincubation with cells retrieved from the human female reproductive tract. Hum Reprod 1993;8: Chang YS, Lee JY, Moon SY, Kim JG, Pang MG, Shin CJ. Factors affecting penetration of zona-free hamster ova. Arch Androl 1990;25: Kopf GS, Gerton GL. The mammalian sperm acrosome and the acrosome reaction. In: Wassarman PG, ed. Elements of mammalian fertilization. Vol. 1. Boca Raton (FL): CRC Press, 1991: Morales P, Vigil P, Franken DR, Kaskar K, Coetzee K, Kruger TF. Sperm-oocyte interaction: studies on the kinetics of zona pellucida binding and acrosome reaction of human spermatozoa. Andrologia 1994;26: Huyser C, Fourie FR, Moolman H. The influence of sera, follicular fluids and seminal plasma on human sperm-zona pellucida binding. Hum Reprod 1997;12: Qiao J, Yeung WSB, Yao YQ, Ho PC. The effects of follicular fluid from patients with different indications for IVF treatment on the binding of human spermatozoa to the zona pellucida. Hum Reprod 1998; 13: Funahashi H, Day BN. Effects of follicular fluid at fertilization in vitro on sperm penetration in pig oocytes. J Reprod Fertil 1993;99: Gwatkin RB, Andersen OF. Effect of glycosidase inhibitors on the capacitation of hamster spermatozoa by cumulus cells in vitro. J Reprod Fertil 1973;35: Yao et al. Follicular fluid and oviductal sperms Vol. 72, No. 6, December 1999

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