The effect of albumi~ gradients and human serum on the longevity and fertilizing capacity of human spermatozoa in the hamster ova penetration assay*

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1 FERTn.1TY AND STERIL1TY Copyright c 1982 The American Fertility Society Vol. 38, No.2, August 1982 Printed in U.SA. The effect of albumi~ gradients and human serum on the longevity and fertilizing capacity of human spermatozoa in the hamster ova penetration assay* Zvi Binor, M.D.t:J: Ramaa Rao, M.D.t Hans van der Yen, M.D. Antonio Scommegna, M.D.t Michael Reese Hospital and Medical Center and Pritzker School of Medicine, University of Chicago, Chicago, Illinois The motility and the capability to penetrate zona-free hamster eggs of Y -enriched or washed sperm were evaluated after protracted in vitro incubation with and without the addition of preheated human serum. The addition of 50% preheated serum decreased the motility loss over time for both the washed and Y -enriched sperm. Such motility loss was decreased by 25% and 27% at 20 hours, by 31% and 39% at 30 hours, and by 30% and 40% at 40 hours of incubation for the washed and Y -enriched sperm, respectively. The washed and Y-enriched sperm suspensions with and without addition of preheated human serum achieved 100% penetration rate after 2 hours of preincubation. However, when scored by the sperm per egg ratio, washed sperm achieved 1.2 ± 0.2 (mean ± standard error [SE]) sperm per egg, while the Y enriched sperm achieved 3.0 ± 0.4 sperm per egg. After 24 hours of incubation, penetration by washed sperm decreased to a mean of 62.8%. The Y-enriched sperm penetrated a mean of 18% of the ova. Addition of preheated serum increased the egg penetrating capacity of washed sperm at 24 hours but failed to improve the Y enriched spermatozoa. This study suggests that for optimum conception rates, precise ovulation timing is crucial when Y -enriched fractions are used for insemination. Fertil Steril38:222, 1982 Sperm capacitation and acrosome reaction can be induced in vitro in various mammalian species by the addition of the heat-stable fraction of human serum.!' 2 Bovine serum albumin (BSA) and Received August 24, 1981; revised and accepted April 26, *Supported by the Medical Research Institute Council and Michael Reese Hospital and Medical Center. tdivision of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology. :f:reprint requests: Zvi Binor, M.D.,'Michael Reese Hospital and Medical Center, Department of Obstetrics and Gynecology, 29th Street and Ellis Avenue, Chicago, Illinois Present address: Department of Physiology and Biophysics, University of Illinois at the Medical Center, Chicago, Illinois human serum albumin (hsa) are capable of replacing the preheated serum in the capacitation process. Chemically defined media containing albumin have been employed for fertilization of various mammalian eggs. 3-5 Concentration gradients of hsa (10% to 20%) have been used to obtain a fraction of human semen enriched with Y spermatozoa. The fraction obtained after separation is characterized by a significant increase in sperm motility and progressive drive, along with a substantial decrease in the total count and in the percentage of abnormal forms. However, when the Y -enriched sperm fraction was used in artificial homologous insemination (AIH) for male preselection, a rather low (27%) total conception rate was achieved. 6 It 222 Binor et ai. Hamster ova penetration and X-Y sperm separation Fertility and Sterility

2 SEMINAL PLASMA SEPARATION SPERM APPLIED TO HSA COLUMNS Y-ENRICHED SPERM RECOVERED FROM 20% HSA LAYER + RESUSPENSIONIN EJACULATE ~~. " MOD. BWW /' MOD. BWW MOD. BWW ONLY +20% HUMAN +lk>%human ~ SERUM SERUM ~ ~ \ SPERM WASHING + RESUSPENSION IN I + \ MOD. BWW MOD. BWW MOD. BWW ONLY +20%HUMAN +50% HUMAN SERUM SERUM SPERM INCUBATION AND OET RMINATION OF % MOTILITY EVERY 10 HOURS AND SPERM I EGG PENETRATION ASSAY AFTER 2,24 and 48 HOURS OF INCUBATION Figure 1 Experimental protocol used to determine the percentage of sperm motility and sperm/egg penetration rates in Y-enriched and washed sperm, with and without addition of 20% or 50% preheated human serum. was our intention to evaluate the capacity of spermatozoa processed through the albumin gradient columns to penetrate the zona-free hamster ova. Preheated human serum was found to contain a "motility factor" that stimulates the motility of hamster epididymal spermatozoa in vitro. 7 Thus, we evaluated the effect of preheated human serum on the fertilizing capacity and motility of human spermatozoa during prolonged in vitro culture. MATERIALS AND METHODS PREPARATION OF SPERM SUSPENSIONS Fifteen ejaculates from ten fertile donors were each divided into two portions following liquefaction. One portion of the ejaculate was diluted in four fold volumes of modified Biggers, Whitten and Whittingham (BWW) medium containing 3.5% hsa (Fraction V, Sigma Chemical Company, St. Louis, MO). The increased concentration of serum albumin suggested by Overstreet et al. 8 was used after we confirmed that capacitation is more rapid in the 3.5% hsa medium, allowing for a shorter incubation time. The diluted ejaculate was centrifuged at 600 x g for 5 minutes. The sperm pellet was isolated and resuspended, and the "washing" with modified BWW medium (Fig. 1) was repeated three times. Another portion of ejaculate underwent X-Y sperm separation as previously described by our laboratory.b The method is a modification of a technique described by Ericsson et al. 9 (Fig. 1): Following liquefaction, the semen was diluted 1:1 with Tyrode's solution and centrifuged at 600 x g for 10 minutes. The supernatant was discarded, and the sperm pellet was resuspended in Tyrode's solution (Difco Laboratories, Detroit, MI) to contain approximately 50 to 60 X lob sperm/ml. Aliquots of 0.5 ml were then layered on isolation columns. Each column contained two discrete layers ofhsa. The lower layer consisted of 0.5 ml of 20% hsa, and the upper layer consisted of 1 ml of 10% hsa. After 60 minutes, the upper 0.5-ml layer was removed. Thirty minutes later, the 20% hsa layer was collected and centrifuged at 600 x g for 10 minutes. The supernatant was removed, and the sperm pellet was resuspended in modified BWW. Fifteen to twenty percent of the sperm originally present in the ejaculate were recovered with enhanced motility. At the onset and conclusion of the separation process, we obtained and stained sperm smears with quinacrine dihydrochloride to determine the percentage of sperm with a fluorescent Y body. Sperm separation resulted in Y sperm enrichment as assessed by Y body fluorescent staining. In the fresh ejaculate the percentage of sperm stained by Y fluorescence ranged between 34% to 58% (mean of 41 %). After Y -enrichment, sperm with Y fluorescence ranged between 62% to 74% (a mean of 68%), which resulted in an average increase of 27% Y fluorescence. The final sperm suspension of both portions of the ejaculate (the washed sperm and the albumin separated) was divided into three aliquots (Fig. 1). The first was diluted in culture medium containing 3.5% hsa, and the other two with culture medium containing an additional 20% or 50% serum preheated at 57 C for 30 minutes, respectively. After adjusting to a concentration of 20 to 30 x lob/ml, the sperm were incubated at 37 C in 5% CO 2 in air. Sperm motility was assessed at 10-hour intervals by microscopic determinations.lo PREPARATION OF ZONA-FREE HAMSTER OOCYTES AND PENETRATION Tubal oocytes were recovered from superovulated mature Golden hamsters and were sus- Vol. 38, No.2, August 1982 Binor et ai. Hamster ova penetration and X-Y sperm separation 223

3 eo 60 nme (hours) Figure 2 Percentage of spenn motility following albumin gradients (10% to 20%), separation, and in'vitro incubation with or without human serum; Each point represents the mean ± SE of 15 detenninations. ----, Y-enriched spenn (BWW medium alone); 0--0, Y -enriched spenn with 20% serum in medium; 0-0, Y -enriched sperm with 50% serum in medium. pended in BWW medium containing bovine testes hyaluronidase type 1-S (1 mg/ml) (Sigma Chemical Company) and bovine pancreatic trypsin Type 1 crystallized two times (1 mg/ml), resulting in the removal of both the cumulus and the zona pellucida. The zona-free hamster oocytes were then placed in insemination dishes containing 0.2 ml of fresh culture medium under silicone oil (Dow-Corning 200 dielectric fluid; dimethylpolysiloxane; Accumetric, Elizabethtown, KY) and mixed with 20 to 40 j.ll of sperm suspension. Sperm preincubation varied between 2 and 48 hours. It had been found, that a final sperm concentration of 3 to 5 X 10 6 motile sperm per milliliter was optimal. 11 Following 5 hours of sperm-egg incubation, the eggs were removed, from the insemination dishes, washed, fixed, stained with aceto-lacmoid, and examined microscopically for evidence of sperm penetration. Eggs were considered penetrated when a swollen sperm head or a male pronucleus and a corresponding sperm tail were found within the ooplasma. The sperm incubated with BWW showed the greatest decrease in motility (Fig. 2). After 40 hours of culture, sperm suspended in culture medium alone showed a motility of only 8.6%, while the sperm suspension containing 20% and 50% serum maintained 46% and 52% motility, respectively. Following 50 hours of incubation, the sperm suspension in medium alone was immobile; conversely, following the same length of incubation in presence of the serum, a 30% motility was retained. No difference was noted when either 20% or 50% human serum was added. Similar results were noted in the washed sperm fractions (Fig. 3). Initially, a mean motility value of 76% was recorded. After 40 hours ofincubation, the sperm motility had fallen to 6.5%, while spenn with added:serum maintained a 27% and 37% motility with 20% and 50% human serum,' respectively. Addition of preheated human serum increased the sperm motility by 15 to 20 hours in both the Y -enriched and washed sperm suspensions. When viewed at given time intervals of incubation, the addition of 50% preheated serum to the medium increased the mean sperm motility for both the washed and Y ~enriched sperm by 25% and 27% at 20 hours, by 31% and 39% at 30 hours, and by 30% and 40% at 40 hours of incubation, respectively (P < by randomization test).12 The rate of motility loss was the same for the washed and the Y -enriched sperm suspensions. 100 SPERM MOTILITY STUDIES RESULTS At the beginning of the incubation, the Y enriched sperm fraction had a motility of 95%. However, the motility decreased at different rates, depending on whether heat-treated serum or BWW was added' to the incubation medium nme(hours) Figure 3 Percentage of spenn motility following seminal plasma washing and in vitro incubation with or without human serum. Each point represents the. mean, ± SE of 15 detenninations. - _, washed spenn; D- - -0, washed spenn with 20% serum; , washed spenn with 50% serum. 224 Binor et ai. Hamster ova penetration and X-Y sperm separation Fertility and Sterility

4 Table L Sperm Penetration into Zona-Free Hamster Ova a Sperm population A. Washed sperm B. Y-enriched sperm C. Washed sperm + 50% serum D. Y-enriched sperm + 50% serum % Motilityb 76 ± ± ± ± Hours preincubation Penetration 100% ( ) 100% ( ) 100% (116/116) 100% ( ) Sperm/eggb 1.2 ± 0.2c 3.0 ± O.4 c 1.7 ± ± 0.3 % Motility 41.8 ± ± ± ± Hours preincubation Penetration Sperm/egg" 62.8% (65/104)" % (18/100)" 0.2 ± % (112/112) 1.3 ± % (251104) 0.2 ± 0.04 aone hundred to 120 eggs were inseminated with each of the sperm suspensions in five separate experiments. Inseminati~n performed with 48-hour-preincubated washed sperm plus 50% human serum or with Y-enriched sperm plus 50% serum resulted m no egg penetration. byalues are mean ± SE. ca significant difference for penetration rate by paired t-test (P < 0.05) was found between A and B groups after 2 hours and 24 hours of preincubation. IN VITRO SPERMIEGG PENETRATION After 2 hours, 24 hours, and 48 hours of preincubation, the above sperm suspensions were tested in the hamster ova penetration assay. Five separate experiments were performed with the use of a total of 100 to 120 eggs for each sperm fraction (Table 1). Both the washed and Y -enriched sperm suspensions, with and without addition of 50% preheated human serum in the culture medium, achieved 100% fertilization rate after 2 hours of preincubation. However, when scored by the number of sperm that penetrated each egg, the washed sperm averaged 1.2 ± 0.2 sperm/egg {mean ± standard error [SED, while the Y enriched sperm had a significantly increased ratio of 3.0 ± 0.4 sperm/egg (paired t-test; P < 0.05). Addition of heat-treated human serum had no effect in both groups. After 24 hours of incubation, the number of eggs penetrated by washed sperm decreased to a mean of 62.8%. At the same time, Y-enriched sperm achieved a mean penetration rate of 18% while still maintaining motility of 61.8%. The paired t-test for the penetration rate between the two sperm fractions showed a significant difference (P < 0.05). The addition of 50% preheated serum enhanced the fertilizing capacity of 24-hour washed sperm to 100% but failed to improve the fertilizing capacity of Y-enriched spermatozoa, which had a 24% penetration rate. Insemination performed with preincubated for 48 hours washed sperm plus 50% human serum or with Y-enriched sperm plus 50% serum resulted in no egg penetration. DISCUSSION Concentration gradients of hsa, using 10% to 20% solutions, have been used for Y-enrichment of sperm suspensions employed for sex preselection. 6 The same technique has been suggested for the treatment of male infertility13 and for improvement of sperm recovery following cryopreservation. 14 BSA and hsa in concentrations not exceeding 3% to 5% have been added to defined media to induce sperm capacitation and acrosome reaction in various mammalian species. The effect of a high concentration (10% to 20%) of albumin in the in vitro fertilizing capacity of human spermatozoa has not been evaluated; however, insemination of Y-enriched sperm has resulted in low conception rates.6, 13 Our study indicates that the exposure of human spermatozoa to the high concentration of albumin (10% to 20%) enhances its in vitro egg penetration capacity {expressed by sperm/egg ratio) perhaps by a more efficient acrosome reaction, as was shown in other mammalian spermatozoa. However, this initial effect is not long-lasting, and after 24 hours the fertilizing capacity of albuminseparated sperm relative to control sperm is significantly decreased in spite of normal sperm motility. This loss of fertilizing capacity is irreversible and is not restored by addition of heat-treated human serum to the culture medium. The egg penetration capacity of both the washed and Y -enriched spermatozoa is reduced approximately 24 hours before any significant decrease in sperm motility. Thus, the hamster ova penetration test maybe more sensitive than motility assessment in predicting the fertilizing capacity of human spermatozoa. 15 Since the fertilizing potential of 10% to 20% albumin-exposed spermatozoa may be time limited, precise ovulation timing is crucial when Y enriched sperm fractions are used for AIH in sex preselection. The low conception rate reported by Dmowski et al. 13 may be explained by the de- Yol. 38, No.2, August 1982 Binor et al. Hamster ova penetration and X-Y sperm separation 225

5 creased fertilizing capacity of the sperm after 24 hours. Since this study took place, ovulation in women undergoing insemination for sex preselection has been timed by daily hormonal assay. This has increased the conception rate in treat~d couples from a prior rate of 27% to the present rate of 40%. Sperm motility studies are in agreement with prior studies that indicated no difference in motility between X and Y spermatozoa. 16 Sperm motility was enhanced by the addition of 20% and 50% heat-treated human serum to the culture medium. The improvement of sperm motility noted with the addition of human serum may indicate that the modified BWW medium is deficient in some energy sources for the spermatozoa. A factor that stimulates spermatozoa motility (SMF) in the hamster had been demonstrated in blood serum. 7, 17 Meizel et al. 18 and Mrsny et ap9 correlated this motility-stimulating activity to the concentration of j3-amino acids taurine and hypotaurine. These two amino acids also share some physicochemical properties with SMF, since they are both dialyzable and heat-stable, and have a low molecular weight. 20 Whether these j3-amino acids are capable of stimulating the motility of the human spermatozoa in a manner similar to preheated serum is now being investigated. The sperm samples used in this study were obtained from healthy fertile donors. Whether preheated human serum can similarly increase sperm motility in infertile males is also the subject of current investigation. REFERENCES 1. Bavister BD, Morton DB: Separation of human serum components capable of inducing the acrosome reaction in hamster spermatozoa. J Reprod Fertil 40:495, Rogers BJ: Mammalian sperm capacitation and fertilization in vitro: a critique of methodology. Gamete Research 1:165, Hoppe PC, Whitten WK: An albumin requirement for fertilization of mouse eggs in vitro. J Reprod Fertil 39:433, Miyamoto H, Chang MC: The importance of serum albumin and metabolic intermediates for capacitation of spermatozoa and fertilization of mouse eggs in vitro. J Reprod Fertil 32:193, Davis BK: Influence of serum albumin on the fertilizing ability in vitro of rat spermatozoa. Proc Soc Exp BioI Med 151:240, Dmowski WP, Gaynor L, Rao R, Lawrence M, Scommegna A: Use of albumin gradients for X and Y sperm separation and clinical experience with male sex preselection. Fertil Steril 31:52, Yanagimachi R: In vitro capacitation of Golden hamster spermatozoa by homologous and heterologous blood sera. BioI Reprod 3:147, Overstreet JW, Yanagimachi R, Katz DF, Hayashi K, Hanson FW: Penetration of human spermatozoa into the human zona pellucida and the zona-free hamster egg: a study of fertil donors and infertile patients. Fertil Steril 33:534, Ericsson RJ, Langevin CN, Nishino M: Isolation offractions rich in human Y sperm. Nature 246:421, Zaneveld L, Polakoski K: Collection and physical examination of the ejaculate. In Techniques of Human Andrology, Edited by ESE Hafez. New York, Elsevier-North Holland, 1977, p Binor Z, Sokolaski JE, WolfDP: Penetration of the zonafree hamster egg by human sperm. Fertil Steril 33:321, Edgington ES: Independent t-test with systematic and random data permutation. In Randomization Tests, New York, Marcel-Dekker, 1980, p Dmowski WP, Gaynor L, Lawrence M, Rao R, Scommegna A: Artificial insemination homologous with oligospermic semen separated on albumin columns. Fertil Steril31:58, Glaub JC, Mills RN, Katz DF: Improved motility recovery of human spermatozoa after freeze preservation via a new approach. Fertil Steril 27:1283, Karp LE, Williamson RA, Moore DE, Shy KK, Plymate SR, Smith WD: Sperm penetration assay: useful test in evaluation of male fertility. Obstet Gynecol 57:620, Downing DC, Black DL: Equality in survival of X and Y chromosome-bearing human spermatozoa. Fertil Steril 27:1191, Bavister BD: Properties of the sperm motility stimulating component derived from human serum. J Reprod Fertil 43:363, Meizel S, Lui CW, Working PK, Mrsny RJ: Taurine and hypotaurine: their effects on motility, capacitation and acrosome reaction of hamster sperm in-vitro and their presence in sperm and reproductive tract fluids of several mammals. Develop Growth and Differ 22:483, Mrsny RJ, Waxman L, Meizel S: Taurine maintains and stimulates motility of hamster sperm during capacitation in-vitro. J Exp Zool 210:123, Bavister BD, Yanagimachi R: The effects of sperm extract and energy sources on the motility and acrosome reaction of hamster sperm in-vitro. BioI Reprod 16:228, Binor et al. Hamster ova penetration and X-Y sperm separation Fertility and Sterility