Vol. 58, No.5, November 1992
FERTILITY AND STERILITY
Printed on acid-free paper in U.S.A.
Copyright 0 1992 The American Fertility Society
A microwell sperm penetration assay
Richard A. Bronson, M.D.*t Luciia OuIa, B.A.* Susan K. Bronson, M.P.H.:!: Health Sciences Center, State University of New York, Stony Brook, New York
This report describes the use of a new test configuration of the sperm penetration assay (SPA) in which lO-ILL wells of Tersaki tissue-typing plates are used to study the interaction of very small numbers of sperm with single zona-free hamster eggs. By minimizing gamete requirements, this technique facilitates the systematic study of sperm-egg interactions over a wide range of varying culture conditions and may also be useful in cases with severe semen abnormalities, when recovery of a large population of motile spermatozoa is difficult. MATERIALS AND METHODS Preparation of Gametes
Female golden hamsters, 4 to 6 weeks old, were superovulated with gonadotropins, and eggs were harvested and treated with hyaluronidase (H3884; Sigma Chemical Co, St. Louis, MO) and trypsin (T8003; Sigma Chemical Co), as previously described (1). Spermatozoa were recovered by swim-up from ejaculates of known fertile donors or men from infertile couples as previously described (2). In summary, 1.0-mL aliquots of liquefied semen were injected under 2.5 mL of Biggers, Whitten, and Whittingham (BWW) medium containing 5 mg/mL human serum albumin (HSA; Sigma A-951l, Lot No. 127F9320) in Falcon 2001 tubes (Becton-Dickson, Lincoln Park, NJ), in a slant rack. After 1 hour
Received April 7, 1992; revised and accepted July 24,1992.
* Department of Obstetrics and Gynecology.
t Reprint requests: Richard A. Bronson, M.D., Department of Obstetrics and Gynecology, Health Sciences Center, State University of New York, Stony Brook, New York 11794-8091. :I: Department of Pathology. 1078
Bronson et aI.
Techniques and instrumentation
incubation at 37°C in 5% CO 2 in air, the top 2.0 mL of each tube was combined in a Falcon 2095 tubes and centrifuged for 8 minutes at 300 X g. Aliquots of 5 X 106 sperm, resuspended in 1 mL of BWW/ HSA 30 mg/mL, were centrifuged 5 minutes at 300 X g and resuspended in 250 ILL of BWW /HSA 30 ng/mL. These spermatozoa were capacitated by overnight incubation at 37°C in 5% C0 2 in air. Standard Assay
Two hundred-microliter droplets of BWW/HSA 30 mg/mL containing motile, capacitated sperm at concentrations ranging from 0.5 to 5.0 X 106 were prepared, and zona-free eggs were added. After a 3hour incubation at 37°C in 5% CO 2 and air, eggs were recovered, washed, and whole mounts prepared. Eggs obtained from the microwell assay were scored concurrently, at comparable times as those in the standard assay. Thereafter, spermatozoa within the standard and microwell assay droplets were resuspended by thorough mixing and aliquots placed within a Makler chamber to determine the actual concentration of spermatozoa present. Microwell Assay
Terasaki tissue-typing plates (60 well, Falcon 3034; Becton-Dickson, Lincoln Park, NJ) consist of rectangular disposable microtest plates containing 10 rows of 6 flat bottom wells each of 10-ILL volume. Nine microliters of BWW/HSA 30 mg/mL, equilibrated with 5% CO 2 in air at 37°C, were placed in each microwell, and the loaded plates were returned to the incubator for an additional 5- to 10-minute equilibration. Sperm concentrations were made up from the 20 X 106 sperm/mL stock of overnight capacitated sperm, so that 1 to 3 ILL of sperm, when Fertility and Sterility
Table 1
Comparison of Results of Standard and Microwell SPAs at Varying Sperm Concentrations * Microwell assay
Standard assay
Concentration t
Percent of eggs penetrated
No. of penetrating sperm per egg
No. of adherent sperm per egg
Percent of eggs penetrated
No. of penetrating sperm per egg
No. of adherent sperm per egg
5 2 1
91.3 ± 4.1 (8) t 88.4 ± 9.1 (3) 82.8 ± 8.1 (3)
3.4 ± 0.42 2.9 ± 0.41 1.4 ± 0.07
14.2 ± 1.8 10.6 ± 2.5 4.6 ± 0.6
91.8 ± 3.5 (8) 87.5 ± 12.5 (2) 27.2 ± 6.9 (3)
3.5 ± 0.5 2.1 ± 0.6 0.3 ± 0.1
17.1 ± 3.1 3.3 ± 2.5 2.0 ± 0.8
* Spermatozoa from a known fertile donor were used from ejaculates on several different days after swim-up and overnight capacitation in BWW /HSA 30 mg/mL. All parameters in the micro well and standard assays were significantly different (P < 0.05), by Wilcoxon's two-sample rank test, when eggs were inseminated at 1 X 10 6 motile sperm/mL.
added to the wells with an Eppendorf Ultramicropipette (Bio-Rad, Richmond, CA), resulted in the motile concentrations used in these experiments. After preliminary equilibration and after addition of sperm, one or two zona-free hamster eggs were added to each microwell. Thereafter, microwells were each overlaid with mineral oil (Squibb, Princeton, NJ) to prevent evaporation. The mineral oil was added dropwise, using a tuberculin syringe and 25-gauge needle, and the well was observed under a stereoscope to confirm that it was completely covered with oil. Failure to do so resulted in loss of sperm motility during incubation. One mL of mineral oil and 1 mL of BWW had been placed in a Falcon 2995 tube, vigorously shaken, and then incubated overnight against 5% CO 2 in air at 37°C. Using the rectangular configuration of the microwells, each row could be loaded with sperm at a single concentration so that a single plate possessed rows with different concentrations. Enumeration of Oolemmal Adherent and Penetrating Spermatozoa
In both assays, eggs were recovered from sperm suspension, after a 3-hour incubation at 37°C, 5% CO 2 in air, and washed by serial passage through three 75-IlL microdrops of BWW/HSA, 5 mg/mL in Falcon dishes. Eggs were stained by 5- to 15-second exposure to 1 mM of acridine orange-3% dimethyl sulfoxide in BWW/HSA 5 mg/mL, washed and whole mounts prepared under 22- by 22-mm coverslips (2). Under epifiuorescence illumination (480 nm excitation filter and 520 nm barrier filter), the unexpanded sperm heads adherent to the oolemma appeared bright green or orange against the dark field and could be counted at 400X. Penetrating spermatozoa exhibited expanded, green heads within Vol. 58, No.5, November 1992
t Motile sperm X
10 6 /mL.
t Values are means ± SEM; values in parentheses are numbers of different ejaculates studied. For each experiment, 8 to 20 eggs were inseminated.
the orange background of the ooplasm. A modification of the procedure of Wolf and Hamada (3) was used to quantitate sperm binding to the oolemma. RESULTS
When the microwell and standard assays were compared using a single fertile donor studied on several days, there was no difference in the percentage of eggs penetrated, the number of penetrating sperm per egg, nor the number of oolemmal adherent sperm per egg between assays, at sperm concentrations of 5 and 2 X 106/mL. However, at 1 X 106 motile sperm/mL, significantly fewer eggs were penetrated, and diminished numbers of penetrating and adherent sperm were noted in the standard assay, when compared with the microwell assay (Table 1). These differences were not because of pipetting errors associated with the transfer of small volumes of sperm suspension. When the actual sperm concentration within the insemination droplets were determined, these were 5.9 ± 1.8 and 5.1 ± 1.0 X 106 , 2.5 ± 0.36 and 1.6 ± 0.36 X 106 , and 0.9 ± 0.48 and 1.0 ± 0.39 (mean ± sri) for the standard assay and microwell assays at theoretical sperm concentrations of 5, 2, and 1 X 106 /mL, based on dilutions of the stock capacitated sperm suspension. In the second series of experiments, ejaculates of nine men from infertile couples were studied in standard and microwell assays run concurrently. Because no significant difference in penetration frequencies had been noted in the initial experiments using fertile donor sperm at 5 X 106 motile cells/mL, this concentration was chosen for the comparison. Although the percentage of eggs penetrated and number of penetrating sperm per egg varied widely for different men, these parameters did not differ between the Bronson et al.
Techniques and instrumentation
1079
Table 2
Comparison of Results of Standard and Microwell SPAs in Consecutive Men From Infertile Couples * Standard assay
Microwell assay Percent of eggs penetrated
Patient no. 1 2 3 4 5 6 7 8 9
100 40 100 90 50 46 100 38.5 38.5
(9)t (10) (10) (10) (10) (12) (13) (13) (12)
No. of penetrating sperm/egg 4.6 0.4 3.6 3.0 0.5 0.83 4.5 0.42 0.42
± ± ± ± ± ± ± ± ±
0.7+ 0.2 0.5 0.9 0.2 0.2 0.7 0.1 0.1
Percent of eggs penetrated 100 27.7 100 94.7 73.9 66.7 93.3 33.3 42.9
No. of penetrating sperm/egg
(20) (18) (20) (19) (23) (15) (15) (15) (14)
6.2 0.33 4.8 2.8 1.74 1.1
5.2 0.47 0.50
± ± ± ± ± ± ± ± ±
0.82 0.14 0.45 0.37 0.31 0.25 0.81 0.19 0.17
* There was no significant difference between percent of eggs penetrated nor number of penetrating sperm/egg when comparing results of the microwell versus standard assays, by Wilcoxon two-sample rank test. In the nine ejaculates studied, sperm concentration ranged from 28 to 339 X 10 6/mL and sperm motility from 32% to 73%. t Values in parentheses are number of eggs inseminated. + Values are means ± SEM.
standard and microwell assays for each individual man (Table 2). DISCUSSION
The microwell configuration used in these experiments offers two advantages over current assays. Through the employment of very small volumes (10 ~L), highly effective sperm concentrations can be achieved with very low absolute sperm numbers. Hence, although 1 X 106 motile cells must be used to achieve a 5 X 106 motile sperm concentration in a 200-~L drop, only 50,000 sperm (lOO-fold less) need to be added to a lO-~L well. Indeed, the percentage of eggs penetrated in the microwell assay did not drop significantly as motile sperm concentration was lowered to one 1 X 106/mL (10,000 motile sperm in 10 ~L). In addition, the microwell configuration allows variation in the experimental conditions, such as sperm concentration, while minimizing gamete requirements. Either individual wells or individual rows of wells can be used, allowing the investigator to vary the number of replicates and variables within the experiment. This can be particularly important
1080
Bronson et a1.
Techniques and instrumentation
in studying the effects of factors that promote sperm fertilizing ability, such as progesterone, follicular fluid, or high osmotic strength media on sperm function from a particular individual. SUMMARY
Egg penetration rates in a modified SPA using microwells in tissue typing plates were comparable with those in a standard assay. This technique allows sperm penetrating ability to be determined using single zona-free hamster eggs and as few as 10,000 spermatozoa.
Key Words: Sperm penetration assay, sperm penetration, fertilization. REFERENCES 1. Rogers BJ, Van Campen H, Veno M, Lambert H, Bronson R, Hale R. Analysis of human spermatozoal fertilizing ability using zona-free ova. Fertil SterilI979;32:664-70. 2. Bronson RA, Cooper G, Rosenfeld DL. Ability of antibodybound sperm to penetrate zona-free hamster ova in vitro. Fertil Steril 1981;36:778-83. 3. WolfDP, Hamada M. Sperm binding to the mouse eggplasmalemma. Bioi Reprod 1979;21:205-11.
Fertility and Sterility
FERTILITY AND STERILITY
Printed on acid-free paper in U.S.A.
Copyright 0 1992 The American Fertility Society
A microwell sperm penetration assay
Richard A. Bronson, M.D.*t Luciia OuIa, B.A.* Susan K. Bronson, M.P.H.:!: Health Sciences Center, State University of New York, Stony Brook, New York
This report describes the use of a new test configuration of the sperm penetration assay (SPA) in which lO-ILL wells of Tersaki tissue-typing plates are used to study the interaction of very small numbers of sperm with single zona-free hamster eggs. By minimizing gamete requirements, this technique facilitates the systematic study of sperm-egg interactions over a wide range of varying culture conditions and may also be useful in cases with severe semen abnormalities, when recovery of a large population of motile spermatozoa is difficult. MATERIALS AND METHODS Preparation of Gametes
Female golden hamsters, 4 to 6 weeks old, were superovulated with gonadotropins, and eggs were harvested and treated with hyaluronidase (H3884; Sigma Chemical Co, St. Louis, MO) and trypsin (T8003; Sigma Chemical Co), as previously described (1). Spermatozoa were recovered by swim-up from ejaculates of known fertile donors or men from infertile couples as previously described (2). In summary, 1.0-mL aliquots of liquefied semen were injected under 2.5 mL of Biggers, Whitten, and Whittingham (BWW) medium containing 5 mg/mL human serum albumin (HSA; Sigma A-951l, Lot No. 127F9320) in Falcon 2001 tubes (Becton-Dickson, Lincoln Park, NJ), in a slant rack. After 1 hour
Received April 7, 1992; revised and accepted July 24,1992.
* Department of Obstetrics and Gynecology.
t Reprint requests: Richard A. Bronson, M.D., Department of Obstetrics and Gynecology, Health Sciences Center, State University of New York, Stony Brook, New York 11794-8091. :I: Department of Pathology. 1078
Bronson et aI.
Techniques and instrumentation
incubation at 37°C in 5% CO 2 in air, the top 2.0 mL of each tube was combined in a Falcon 2095 tubes and centrifuged for 8 minutes at 300 X g. Aliquots of 5 X 106 sperm, resuspended in 1 mL of BWW/ HSA 30 mg/mL, were centrifuged 5 minutes at 300 X g and resuspended in 250 ILL of BWW /HSA 30 ng/mL. These spermatozoa were capacitated by overnight incubation at 37°C in 5% C0 2 in air. Standard Assay
Two hundred-microliter droplets of BWW/HSA 30 mg/mL containing motile, capacitated sperm at concentrations ranging from 0.5 to 5.0 X 106 were prepared, and zona-free eggs were added. After a 3hour incubation at 37°C in 5% CO 2 and air, eggs were recovered, washed, and whole mounts prepared. Eggs obtained from the microwell assay were scored concurrently, at comparable times as those in the standard assay. Thereafter, spermatozoa within the standard and microwell assay droplets were resuspended by thorough mixing and aliquots placed within a Makler chamber to determine the actual concentration of spermatozoa present. Microwell Assay
Terasaki tissue-typing plates (60 well, Falcon 3034; Becton-Dickson, Lincoln Park, NJ) consist of rectangular disposable microtest plates containing 10 rows of 6 flat bottom wells each of 10-ILL volume. Nine microliters of BWW/HSA 30 mg/mL, equilibrated with 5% CO 2 in air at 37°C, were placed in each microwell, and the loaded plates were returned to the incubator for an additional 5- to 10-minute equilibration. Sperm concentrations were made up from the 20 X 106 sperm/mL stock of overnight capacitated sperm, so that 1 to 3 ILL of sperm, when Fertility and Sterility
Table 1
Comparison of Results of Standard and Microwell SPAs at Varying Sperm Concentrations * Microwell assay
Standard assay
Concentration t
Percent of eggs penetrated
No. of penetrating sperm per egg
No. of adherent sperm per egg
Percent of eggs penetrated
No. of penetrating sperm per egg
No. of adherent sperm per egg
5 2 1
91.3 ± 4.1 (8) t 88.4 ± 9.1 (3) 82.8 ± 8.1 (3)
3.4 ± 0.42 2.9 ± 0.41 1.4 ± 0.07
14.2 ± 1.8 10.6 ± 2.5 4.6 ± 0.6
91.8 ± 3.5 (8) 87.5 ± 12.5 (2) 27.2 ± 6.9 (3)
3.5 ± 0.5 2.1 ± 0.6 0.3 ± 0.1
17.1 ± 3.1 3.3 ± 2.5 2.0 ± 0.8
* Spermatozoa from a known fertile donor were used from ejaculates on several different days after swim-up and overnight capacitation in BWW /HSA 30 mg/mL. All parameters in the micro well and standard assays were significantly different (P < 0.05), by Wilcoxon's two-sample rank test, when eggs were inseminated at 1 X 10 6 motile sperm/mL.
added to the wells with an Eppendorf Ultramicropipette (Bio-Rad, Richmond, CA), resulted in the motile concentrations used in these experiments. After preliminary equilibration and after addition of sperm, one or two zona-free hamster eggs were added to each microwell. Thereafter, microwells were each overlaid with mineral oil (Squibb, Princeton, NJ) to prevent evaporation. The mineral oil was added dropwise, using a tuberculin syringe and 25-gauge needle, and the well was observed under a stereoscope to confirm that it was completely covered with oil. Failure to do so resulted in loss of sperm motility during incubation. One mL of mineral oil and 1 mL of BWW had been placed in a Falcon 2995 tube, vigorously shaken, and then incubated overnight against 5% CO 2 in air at 37°C. Using the rectangular configuration of the microwells, each row could be loaded with sperm at a single concentration so that a single plate possessed rows with different concentrations. Enumeration of Oolemmal Adherent and Penetrating Spermatozoa
In both assays, eggs were recovered from sperm suspension, after a 3-hour incubation at 37°C, 5% CO 2 in air, and washed by serial passage through three 75-IlL microdrops of BWW/HSA, 5 mg/mL in Falcon dishes. Eggs were stained by 5- to 15-second exposure to 1 mM of acridine orange-3% dimethyl sulfoxide in BWW/HSA 5 mg/mL, washed and whole mounts prepared under 22- by 22-mm coverslips (2). Under epifiuorescence illumination (480 nm excitation filter and 520 nm barrier filter), the unexpanded sperm heads adherent to the oolemma appeared bright green or orange against the dark field and could be counted at 400X. Penetrating spermatozoa exhibited expanded, green heads within Vol. 58, No.5, November 1992
t Motile sperm X
10 6 /mL.
t Values are means ± SEM; values in parentheses are numbers of different ejaculates studied. For each experiment, 8 to 20 eggs were inseminated.
the orange background of the ooplasm. A modification of the procedure of Wolf and Hamada (3) was used to quantitate sperm binding to the oolemma. RESULTS
When the microwell and standard assays were compared using a single fertile donor studied on several days, there was no difference in the percentage of eggs penetrated, the number of penetrating sperm per egg, nor the number of oolemmal adherent sperm per egg between assays, at sperm concentrations of 5 and 2 X 106/mL. However, at 1 X 106 motile sperm/mL, significantly fewer eggs were penetrated, and diminished numbers of penetrating and adherent sperm were noted in the standard assay, when compared with the microwell assay (Table 1). These differences were not because of pipetting errors associated with the transfer of small volumes of sperm suspension. When the actual sperm concentration within the insemination droplets were determined, these were 5.9 ± 1.8 and 5.1 ± 1.0 X 106 , 2.5 ± 0.36 and 1.6 ± 0.36 X 106 , and 0.9 ± 0.48 and 1.0 ± 0.39 (mean ± sri) for the standard assay and microwell assays at theoretical sperm concentrations of 5, 2, and 1 X 106 /mL, based on dilutions of the stock capacitated sperm suspension. In the second series of experiments, ejaculates of nine men from infertile couples were studied in standard and microwell assays run concurrently. Because no significant difference in penetration frequencies had been noted in the initial experiments using fertile donor sperm at 5 X 106 motile cells/mL, this concentration was chosen for the comparison. Although the percentage of eggs penetrated and number of penetrating sperm per egg varied widely for different men, these parameters did not differ between the Bronson et al.
Techniques and instrumentation
1079
Table 2
Comparison of Results of Standard and Microwell SPAs in Consecutive Men From Infertile Couples * Standard assay
Microwell assay Percent of eggs penetrated
Patient no. 1 2 3 4 5 6 7 8 9
100 40 100 90 50 46 100 38.5 38.5
(9)t (10) (10) (10) (10) (12) (13) (13) (12)
No. of penetrating sperm/egg 4.6 0.4 3.6 3.0 0.5 0.83 4.5 0.42 0.42
± ± ± ± ± ± ± ± ±
0.7+ 0.2 0.5 0.9 0.2 0.2 0.7 0.1 0.1
Percent of eggs penetrated 100 27.7 100 94.7 73.9 66.7 93.3 33.3 42.9
No. of penetrating sperm/egg
(20) (18) (20) (19) (23) (15) (15) (15) (14)
6.2 0.33 4.8 2.8 1.74 1.1
5.2 0.47 0.50
± ± ± ± ± ± ± ± ±
0.82 0.14 0.45 0.37 0.31 0.25 0.81 0.19 0.17
* There was no significant difference between percent of eggs penetrated nor number of penetrating sperm/egg when comparing results of the microwell versus standard assays, by Wilcoxon two-sample rank test. In the nine ejaculates studied, sperm concentration ranged from 28 to 339 X 10 6/mL and sperm motility from 32% to 73%. t Values in parentheses are number of eggs inseminated. + Values are means ± SEM.
standard and microwell assays for each individual man (Table 2). DISCUSSION
The microwell configuration used in these experiments offers two advantages over current assays. Through the employment of very small volumes (10 ~L), highly effective sperm concentrations can be achieved with very low absolute sperm numbers. Hence, although 1 X 106 motile cells must be used to achieve a 5 X 106 motile sperm concentration in a 200-~L drop, only 50,000 sperm (lOO-fold less) need to be added to a lO-~L well. Indeed, the percentage of eggs penetrated in the microwell assay did not drop significantly as motile sperm concentration was lowered to one 1 X 106/mL (10,000 motile sperm in 10 ~L). In addition, the microwell configuration allows variation in the experimental conditions, such as sperm concentration, while minimizing gamete requirements. Either individual wells or individual rows of wells can be used, allowing the investigator to vary the number of replicates and variables within the experiment. This can be particularly important
1080
Bronson et a1.
Techniques and instrumentation
in studying the effects of factors that promote sperm fertilizing ability, such as progesterone, follicular fluid, or high osmotic strength media on sperm function from a particular individual. SUMMARY
Egg penetration rates in a modified SPA using microwells in tissue typing plates were comparable with those in a standard assay. This technique allows sperm penetrating ability to be determined using single zona-free hamster eggs and as few as 10,000 spermatozoa.
Key Words: Sperm penetration assay, sperm penetration, fertilization. REFERENCES 1. Rogers BJ, Van Campen H, Veno M, Lambert H, Bronson R, Hale R. Analysis of human spermatozoal fertilizing ability using zona-free ova. Fertil SterilI979;32:664-70. 2. Bronson RA, Cooper G, Rosenfeld DL. Ability of antibodybound sperm to penetrate zona-free hamster ova in vitro. Fertil Steril 1981;36:778-83. 3. WolfDP, Hamada M. Sperm binding to the mouse eggplasmalemma. Bioi Reprod 1979;21:205-11.
Fertility and Sterility
