Human sperm capacitation in gelatin-fortified medium

FERTILITY AND STERILITY

Vol. 52, No.6, December 1989

Copyright" 1989 The American Fertility Society

Printed on acid-free paper in U.S.A.

Human sperm capacitation in gelatin-fortified medium*

Serdia O. Mack, Ph.D.t:!: Joseph S. Tash, Ph.D.§ Don P. Wolf, Ph.D.tll'lf Oregon Regional Primate Research Center, Beaverton, Oregon, Oregon Health Sciences University, Portland, Oregon, and Baylor College of Medicine, Houston, Texas

Human sperm capacitation can be achieved in vitro in a variety of simple and complex media using serum albumin as the protein component. 1 However, serum products can act as vectors for infectious diseases. The present study was designed to determine whether or not medium supplemented with gelatin, rather than serum albumin, could maintain human sperm motility in vitro and support capacitation as assessed by the zona-free hamster egg penetration assay. This protein is relatively inexpensive, and the risk of infectious disease transmission is reduced, supporting its potential use in homologous or donor inseminations involving washed sperm. MATERIALS AND METHODS

Semen samples, collected by masturbation and allowed to liquefy for 0.5 to 1.0 hour, were divided into two equal volumes and washed twice with 3 Received February 24, 1989; revised and accepted June 28, 1989. * This study, publication no. 1671 of the Oregon Regional Primate Research Center, was supported by grants HD-22996 and RR-OOI63 from the National Institutes of Health, Bethesda, Maryland. t Division of Reproductive Biology and Behavior, Oregon Regional Primate Research Center. Present address: University of Texas Health Science Center at Dallas, Green Center for Reproductive Biology, Dallas, Texas. § Department of Cell Biology, Baylor College of Medicine. II Department of Obstetrics and Gynecology, Oregon Health Sciences University. 'If Reprint requests: Don P. Wolf, Ph.D., Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, Oregon 97006.

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volumes of Ham's F-10 containing 2% human serum albumin (HSA [Fraction V; Sigma, St. Louis, MOD or 2% gelatin (Difco Laboratories, Detroit, MI) by centrifugation at 200 X g for 7 minutes. The final pellet was overlaid with 1 mL of fresh culture medium containing 2% HSA or 2% gelatin and incubated at 37°C in 5% CO2 in air for 24 hours. Aliquots were removed at 0.5 hour of incubation for manual determinations of concentration (with a hemacytometer) and motility, acrosomal status by indirect immunofluorescence, 2 and quantitative motility assessment by digital image analysis (DIA) using the CellSoft system at 30 Hz sampling frequency2,3 (Cryoresources, New York, NY). The supernatant (swim-up) was removed carefully from the pellet at 6 hours of incubation and placed in a separate tube for incubation and sampling at 6 and 24 hours. For the sperm penetration assay (SPA), semen samples were processed as described above using BWW (Biggers, Whitten, and Whittingham') medium containing either BSA or gelatin. After overnight preincubation of washed samples at 37°C with 5% CO2 in air, the swim-up fractions were harvested. Manual counts and motilities were done and 200-JLL drops, containing 5 X 106 motile sperm/mL, were covered with warmed, CO2-equilibrated silicon oil (200 fluid; Dow-Corning, Midland, MI). Hamster eggs were rendered zona-free by sequential exposure to hyaluronidase and trypsin in BSA- (1%) containing medium. Eggs were randomly but equally divided into two groups and washed three to five times with BWW-2% BSA or BWW -2% gelatin before addition to the sperm drops. After a 3-hour incubation at 37°C in 5% CO 2 in air, eggs were washed three times in BWW, Fertility and Sterility

mounted on glass microscope slides, flattened with coverslips, and scored for sperm penetration. A minimum of nine eggs were scored per sample. Statistical analyses were performed on a microcomputer with the multivariate general linear hypothesis module of Systat (Version 3.0; Systat, Inc., Evanston, IL). Sperm motility and acrosomal status data were analyzed as a paired design to account for protein effects based on individual donor response. Sperm penetration data were also analyzed by a paired t-test. A significance level of 0.05 was used and differences between means were detected by pairwise comparisons.

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RESULTS

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The percentages of motile sperm in HSA- and gelatin-containing medium were similar (P> 0.05) at the 0.5-hour (80% HSA, 84% ± 4.9% gelatin [least square mean ± SEl), 6-hour (85.3% HSA, 85% gelatin ± 2.9%), and the 24-hour (64.3% HSA, 64.8% gelatin ± 9.0%) time points. However, the concentrations of motile sperm were greater for sperm in the HSA-supplemented medium at the 0.5-hour (23 versus 12.5 X 106 /mL; P < 0.05), the 6-hour (30 versus 17 X 106 /mL), and 24-hour (22 versus 16 X 106/mL) time points (P > 0.05). Comparisons of DIA means for curvilinear and straight-line velocity, linearity, maximum and mean amplitudes of lateral head displacement, and beat cross frequency showed no differences (P > 0.05) between sperm incubated in medium containing HSA or gelatin at any of the time points studied (Fig. 1). Similarly, the mean levels of hyperactivated sperm 2 in HSA or in gelatin were statistically identical at 7.3% and 6.3% ± 1.4%,6.3% and 5.7% ± 0.8%, and 0.8% and 0.3% ± 0.5% at 0.5, 6, and 24 hours, respectively. The percentages of acrosome-intact cells were similar (P > 0.05) for sperm incubated in HSA- (84.1%, 88.1%, and 88.3% for 0.5, 6, and 24 hours, respectively) or gelatin-fortified medium (83.0%, 90.0%, and 87.4% for 0.5, 6, and 24 hours, respectively) and did not change with incubation length. Five different donors were tested in the SPA. The percentage of eggs penetrated in BSA-containing medium ranged from 8.4% to 67% as compared with a range of 10% to 65% for gelatin-containing medium. Least square means (SEM) for the percentage of eggs penetrated of 27.7% for BSA and 30% (9.9%) for gelatin were similar (P> 0.05). The mean number of penetrations per egg, 0.4 (0.1), was the same (P> 0.05) for the two media. Vol. 52, No.6, December 1989

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Figure 1 Comparisons of DIA motility parameters for sperm incubated in culture medium containing HSA or gelatin for 0.5, 5, and 24 hours. Bars denote SEM; slashed bars, gelatin; solid bars, albumin.

DISCUSSION

The experiments described in this study demonstrate that gelatin is an effective substitute for albumin with regard to long-term retention of sperm motility, including hyperactivation, acrosomal status, and fertility potential acquisition. However, the concentrations of motile sperm obtained in the swim-up fractions were lower, significantly so at 0.5 hour, with gelatin. This could reflect gel formation and/or a denser sperm pellet on centrifugation of gelatin-containing medium at room temperature. Gel formation may be decreased by altering the gelatin concentration, temperature, or by using modified gelatin products. 5 With the exception of gel formation, gelatin and serum albumin share similar physical properties, including isoelectric points of 4.7 to 4.9, water solubility, and tendency to form colloidal solutions. Based on the present findings, a clinical trial seems warranted in which gelatin is substituted for serum albumin in in vitro fertilization or therapeutic insemination with washed cells. Gelatin solutions have been used in intravenous therapy in man for over one-half century and appear to be physiologically inert. 5 The cost, as compared with albumin, is low, as is the risk of infectious disease transmission, making this protein an attractive alternative. Mack et al.

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SUMMARY

Gelatin was shown to be an effective substitute for serum albumin in human sperm capacitation. No protein-dependent differences were seen with regard to long-term retention of sperm motility, including hyperactivation, in acrosomal status or in the acquisition of fertility potential. Gelatin may serve as a cost-effective substitute for serum albumin, which carries minimal risk of infectious disease transmission.

Acknowledgment. The authors acknowledge the secretarial assistance of Ms. Patsy Kimzey.

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REFERENCES 1. Boldt J, WolfDP: Sperm capacitation. In Human In Vitro Fertilization and Embryo Transfer, Edited by DP Wolf, MM Quigley. New York, Plenum Press, 1984, p 171 2. Robertson L, Wolf DP, Tash JS: Temporal changes in motility parameters related to acrosomal status: identification and characterization of populations of hyperactivated human sperm. BioI Reprod 39:797, 1988 3. Mack SO, Wolf DP, Tash JS: Quantitation of specific parameters of motility in large numbers of human sperm by digital image processing. BioI Reprod 38:270, 1988 4. Biggers JD, Whitten WK, Whittingham DG: The culture of mouse embryos in vitro. In Methods in Mammalian Embryology, Edited by JC Daniel, Jr. San Francisco, WH Freeman and Company, 1971, p 86 5. Saddler JM, Horsey PJ: The new generation gelatins. A review of their history, manufacture and properties. Anaesthesia 42:998, 1987

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