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Isolation of a Spermatozoal Immobilization Factor from Escherichia Coli Filtrates*
FERTILITY AND STERILITY Copyright 1977 The American Fertility Society
Vol. 28, No.2, February 1977 Printed in U.s.A.
ISOLATION OF A SPERMATOZOAL IMMOBILIZATION FACTOR FROM ESCHERICHIA COLI FILTRATES*
JOHN D. PAULSON, M.D.t KENNETH L. POLAKOSKI, PH.D. Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110
A low-molecular weight spermatozoal immobilization factor (SIF) has been isolated from Escherichia coli cultures. This factor is stable to heating, freezing, and lyophilization, and immobilizes but does not kill spermatozoa. The concentration of spermatozoa influenced the effectiveness of the SIF in immobilizing spermatozoa, indicating that bacterial infections could have a greater influence on the oligospermic ejaculate than on a normal specimen. SIF is a stable factor and binds to spermatozoa via a reversible mechanism.
Bacterial infections have long been recognized as having an association with infertility.1-3 It is also known that bacteria are capable of agglutinating and immobilizing spermatozoa. 2-5 Antibiotic treatment of spermicidal bacteria in women,6 men,1 and the ejaculate itself8 has resolved the infections and resulted in pregnancies in many of these previously infertile couples. Occasionally, some infertility problems have not been associated with frank infections but have presented as subclinical asymptomatic prostatitis in males whose initial spermatozoal motility diminished over a several-hour time interval9 ; antibiotic treatment of these males resulted in loss of the seminal infection and in several pregnancies in their partners. We have undertaken an investigation into the mechanism of how bacteria immobilize spermatozoa. This report describes the preliminary characterization of a spermatozoal immobilization factor (SIF) isolated from Escherichia coli filtrates.
was kept on trypticase-soy-agar slanted medium at 4° C. Before being used for inoculation, the cultures were placed on sheep blood agar plates (St. Louis Biological Labs, St. Louis, Mo.). Organisms from a single colony were transferred to a growth medium consisting of trypticase-soy broth (Lot 11768, Baltimore Biological Laboratories, Cockeysville, Md.) in Baker's solution lO (pH 7.85) and incubated at 37° C for 18 hours. An l8-hour culture grew approximately 3.2 x 108/m l organisms as quantitated by duplicate pour-plate dilutions. The organisms were removed by passing the culture medium through a 0.22-JLm Millipore filter with negative pressure. The SIF was assayed by incubating the filtrate with an equal volume of a spermatozoal solution. After incubation at 37° C for specified time intervals, aliquots were removed for microscopic observation utilizing a double-blind technique, and the percentage of spermatozoa demonstrating forward motility was recorded. As control for all experiments, a sterile growth medium was used which was processed in a manner identical with that for the SIF preparation. In several experiments, additional control media consisting of Baker's solution and normal saline were used. The pH for all experiments was within the physiologic range of spermatozoa (7.0 to 7.5). The ejaculates were collected by masturbation; after liquefaction, the ejaculates were filtered through
MATERIALS AND METHODS
A culture of E. coli was obtained from the Center for Disease Control in Atlanta, Ga., and Accepted September 16, 1976. *Supported in part by a grant from The Rockefeller Foundation. tPresent address and address for reprint requests: 4701 Kenmore Avenue, Alexandria, Va. 22304.
182
Vol. 28, No.2
ISOLATION OF SPERMATOZOAL IMMOBILIZATION FACTOR
183
a glass wool column ll , 12 which removed a majority of the debris and dead and agglutinated spermatozoa and resulted in a population of highly motile spermatozoa. Ejaculates which were not subjected to glass wool filtration yielded similar results. The concentration of spermatozoa in the ejaculate was determined by the use of a hemocytometer, and then adjusted with Baker's solution to a final sperm density of 20 x 106 /ml, except where noted. In selected experiments, the percentage of dead spermatozoa was determined by the eosin Y staining technique. 13 Dialysis. was performed on the E. coli filtrate and control filtrate versus a lOa-fold excess volume of distilled water. The solutions inside and outside the dialysis tubing were frozen, lyophilized, and reconstituted to 1 ml with distilled water. They were then added to equal volumes of the spermatozoal suspension, as previously described. The E. coli solution and the control buffer were each filtered through a UM05 Amicon filter, and the filter was rinsed with several washes of water. The solution which passed through the filter, that which was retained, and the control solution were then analyzed for the SIF. The effects of spermatozoal concentrations on the potency of the SIF were determined by adding an equal volume of SIF to the spermatozoal solutions to yield final spermatozoal concentrations of 100 x 106 /ml, 50 x 106 /ml, and 20 x 106 /ml. The motility of the spermatozoa was then analyzed over a 4-hour time interval. An experiment was designed to analyze the reversibility of the action of the SIF. After a 21f2hour incubation, both the suspensions containing the SIF and the control filtrate samples were analyzed microscopically and centrifuged at 40 to 60 x g for 20 minutes, and the spermatozoa were resuspended in spermatozoa-free seminal plasma from vasectomized donors. After centrifugation and res us pension of the spermatozoa, the motilities were again analyzed. Aliquots of the SIF were heated, then frozen, lyophilized, and resuspended in an attempt to characterize the stability of the SIF; they were then added to suspensions of spermatozoa, and motility determinations were performed as previously described.
FIG. 1. The effects of the Escherichia coli solution (___ ), the SIF (_ _ ), the control filtrate (o----Q), and Baker's solution (11.-11.) on the forward motility of spermatozoa.
RESULTS
FIG. 2. After dialysis and reconstitution of the Escherichia coli filtrate and the control filtrate, the solutions inside the dialysis tubing (E. coli filtrate, .--.; control filtrate, 11.--11.), the solutions outside the dialysis tubing (E. coli filtrate, ___ ; control filtrate, 0 - 0 ) , and the original filtrates (E. coli, e--e; control, 0 - - 0 ) were added to spermatozoal suspensions to assess effects on motility.
Figure 1 demonstrates the effects of the E. coli solution and the SIF on the progressive motility
60
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2 TIME (Hours)
3
4
50
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::;;:
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3
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TIME (Hours)
184
PAULSON AND POLAKOSKI
February 1977 70
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FIG. 3. The effects of the SIF in the Escherichia coli filtrate on the motility of solutions containing 100 x 10· spermatozoa/ml (.6r.-4) , 50 x 10 6 spermatozoa/ml ( _ _ ), and 20x 106 spermatozoa/ml (___ ). Like concentrations of spermatozoa in control filtrates are represented by open symbols (6-6, 100 x 106/ml; 0----0, 50 x !06/ml; 0 - 0 , 20 x 106/ml).
of the spermatozoa and compares them with those of the normal controls. After 4 hours, the forward motility of the spermatozoa in the controls was 55%, whereas the motility of the spermatozoa incubated with either the E. coli organisms or the SIF was less than 5%. The percentage of dead spermatozoa in the control solutions and in solutions containing the SIF remained between 9 and 12%; however, 80% of the spermatozoa in a suspension containing the E. coli organisms were dead after 4 hours. The results obtained from dialysis, lyophilization, and reconstitution of the filtrates and their addition to spermatozoal suspensions are shown in Figure 2. Only the E. coli solution which passed through the dialysis bag contained the SIF. After filtration of the E. coli solution and the control buffer through the UM05 Amicon filter, the solution which passed through the filter, that which was retained, and the control solution were analyzed for the presence of SIF. Neither the retarded E. coli filtrate solution nor the controls had an observable effect on spermatozoal motility. However, theE. coli filtrate solution which passed
TIME
2 (Hours)
3
FIG. 4. Solution containing 20 x 106 spermatozoa/ml and SIF (e--e) and control filtrate (0--0) were incubated for 21h hours. The arrow indicates when the samples were centrifuged for 20 minutes and resuspended in seminal plasma from vasectomized donors.
through the membrane immobilized the spermatozoa in a fashion comparable with that of the original filtrate. The effect of the SIF on several concentrations of spermatozoa was compared with the controls, and the results are shown in Figure 3. With increasing concentrations of spermatozoa, there was an inverse effect on the loss of spermatozoal motility over the 4-hour time interval, indicating that the effectiveness of SIF in immobilization is dependent upon the concentration of the spermatozoa. Figure 4 demonstrates the reversible effect of sperm-free seminal plasma on the SIF-treated spermatozoal suspension. The SIF was further characterized as it remained active after being. heated to 90° C for 10 minutes. It was also stable to freezing, lyophilization, and reconstitution. DISCUSSION
This report confirms the immobilization of spermatozoa by E. coli and demonstrates a factor (SIF), apparently excreted by E. coli, which immobilizes spermatozoa. The E. coli organisms
Vol. 28, No.2
ISOLATION OF SPERMATOZOAL IMMOBILIZATION FACTOR
and the SIF had similar immobilization capacities. Initially, theE. coli immobilized spermatozoa and with time also had a spermicidal effect, whereas the SIF almost totally immobilized the spermatozoa after 4 hours while it did not affect the livedead ratio compared with the controls. The concentration of spermatozoa influenced the effectiveness of the SIF, indicating that bacterial infections could have a greater influence on the oligospermic ejaculate than on the normal specimen. This finding would appear to be valid for both male and female reproductive tract infections. In fact, it may be even more significant in female infections, for the spermatozoa which reach the cervical os of the female would be drastically reduced in number compared with the total in the ejaculate. We were able to stimulate spermatozoal motility by removing the seminal plasma and resuspending the spermatozoa in normal seminal plasma, indicating that this mechanism is reversible. The SIF is a stable compound, because boiling, freezing, thawing, and lyophilization had no observable effect on the ability of this factor to immobilize spermatozoa. The factor passed through the dialysis tubing and through a 500 molecular weight cutoff membrane, indicating that it is a relatively small component. Acknowledgments. The authors wish to thank Ms. Betsy Duhn and Ms. Faye Zvibleman for their help in typing the manuscript.
185
REFERENCES 1. Matthews CS, Buxton CL: Bacteriology of the cervix in cases of infertility. Fertil Steril 2:45, 1951 2. Rosenthal L: Spermagglutination by bacteria. Proc Soc Exp BioI Med 28:827,1931 3. Wu DH: Bacteriological studies on the semen, and experimental studies on the influences of antibiotics, crystalline penicillin potassium, dihydrostreptomycin sulfate and terramycin hydrochloride upon the semen. Kei-O Med J 34:509, 1957 4. Teague NS, Boyarsky S, Glenn JF: Interference of human spermatozoa motility by Escherichia coli. Fertil Steril 22:281, 1971 5. DelPorto GB, Derrick FC Jr, Bannister ER: Bacterial effect on sperm motility. Urology 5:638, 1975 6. Buxton CL, Wong MB: Spermicidal bacteria in the cervix as a cause of sterility. Am J Obstet Gynecol 64:628, 1952 7. Quesada EM, Dukes CD, Deen GH, Franklin RR: Genital infection and sperm agglutinating antibodies in infertile men. J Urol 99:106, 1968 8. Wu DH: Personal communication 9. Dahlberg B: Asymptomatic bacteriospermia and male infertility. Presented at the International Conference of Andrology, Wayne State University, Detroit, Mich, April 26, 1975 10. Keller DW, Polakoski KL: L-Arginine stimulation of human sperm motility in vitro. BioI Reprod 13:154, 1975 11. Paulson JD, Polakoski KL: Removal of extraneous material from the ejaculate. Presented at the First International Congress of Andrology, Barcelona, Spain, July 1976 12. Paulson JD, Polakoski KL: Preparation of human semen by glass wool columns. In Techniques in Human Andrology, Edited by ESE Hafez. New York, Elsevier Publishing Co. In press 13. Eliasson R, Treichl L: Supravital staining of human spermatozoa. Fertil Steril 22:134, 1972
Vol. 28, No.2, February 1977 Printed in U.s.A.
ISOLATION OF A SPERMATOZOAL IMMOBILIZATION FACTOR FROM ESCHERICHIA COLI FILTRATES*
JOHN D. PAULSON, M.D.t KENNETH L. POLAKOSKI, PH.D. Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110
A low-molecular weight spermatozoal immobilization factor (SIF) has been isolated from Escherichia coli cultures. This factor is stable to heating, freezing, and lyophilization, and immobilizes but does not kill spermatozoa. The concentration of spermatozoa influenced the effectiveness of the SIF in immobilizing spermatozoa, indicating that bacterial infections could have a greater influence on the oligospermic ejaculate than on a normal specimen. SIF is a stable factor and binds to spermatozoa via a reversible mechanism.
Bacterial infections have long been recognized as having an association with infertility.1-3 It is also known that bacteria are capable of agglutinating and immobilizing spermatozoa. 2-5 Antibiotic treatment of spermicidal bacteria in women,6 men,1 and the ejaculate itself8 has resolved the infections and resulted in pregnancies in many of these previously infertile couples. Occasionally, some infertility problems have not been associated with frank infections but have presented as subclinical asymptomatic prostatitis in males whose initial spermatozoal motility diminished over a several-hour time interval9 ; antibiotic treatment of these males resulted in loss of the seminal infection and in several pregnancies in their partners. We have undertaken an investigation into the mechanism of how bacteria immobilize spermatozoa. This report describes the preliminary characterization of a spermatozoal immobilization factor (SIF) isolated from Escherichia coli filtrates.
was kept on trypticase-soy-agar slanted medium at 4° C. Before being used for inoculation, the cultures were placed on sheep blood agar plates (St. Louis Biological Labs, St. Louis, Mo.). Organisms from a single colony were transferred to a growth medium consisting of trypticase-soy broth (Lot 11768, Baltimore Biological Laboratories, Cockeysville, Md.) in Baker's solution lO (pH 7.85) and incubated at 37° C for 18 hours. An l8-hour culture grew approximately 3.2 x 108/m l organisms as quantitated by duplicate pour-plate dilutions. The organisms were removed by passing the culture medium through a 0.22-JLm Millipore filter with negative pressure. The SIF was assayed by incubating the filtrate with an equal volume of a spermatozoal solution. After incubation at 37° C for specified time intervals, aliquots were removed for microscopic observation utilizing a double-blind technique, and the percentage of spermatozoa demonstrating forward motility was recorded. As control for all experiments, a sterile growth medium was used which was processed in a manner identical with that for the SIF preparation. In several experiments, additional control media consisting of Baker's solution and normal saline were used. The pH for all experiments was within the physiologic range of spermatozoa (7.0 to 7.5). The ejaculates were collected by masturbation; after liquefaction, the ejaculates were filtered through
MATERIALS AND METHODS
A culture of E. coli was obtained from the Center for Disease Control in Atlanta, Ga., and Accepted September 16, 1976. *Supported in part by a grant from The Rockefeller Foundation. tPresent address and address for reprint requests: 4701 Kenmore Avenue, Alexandria, Va. 22304.
182
Vol. 28, No.2
ISOLATION OF SPERMATOZOAL IMMOBILIZATION FACTOR
183
a glass wool column ll , 12 which removed a majority of the debris and dead and agglutinated spermatozoa and resulted in a population of highly motile spermatozoa. Ejaculates which were not subjected to glass wool filtration yielded similar results. The concentration of spermatozoa in the ejaculate was determined by the use of a hemocytometer, and then adjusted with Baker's solution to a final sperm density of 20 x 106 /ml, except where noted. In selected experiments, the percentage of dead spermatozoa was determined by the eosin Y staining technique. 13 Dialysis. was performed on the E. coli filtrate and control filtrate versus a lOa-fold excess volume of distilled water. The solutions inside and outside the dialysis tubing were frozen, lyophilized, and reconstituted to 1 ml with distilled water. They were then added to equal volumes of the spermatozoal suspension, as previously described. The E. coli solution and the control buffer were each filtered through a UM05 Amicon filter, and the filter was rinsed with several washes of water. The solution which passed through the filter, that which was retained, and the control solution were then analyzed for the SIF. The effects of spermatozoal concentrations on the potency of the SIF were determined by adding an equal volume of SIF to the spermatozoal solutions to yield final spermatozoal concentrations of 100 x 106 /ml, 50 x 106 /ml, and 20 x 106 /ml. The motility of the spermatozoa was then analyzed over a 4-hour time interval. An experiment was designed to analyze the reversibility of the action of the SIF. After a 21f2hour incubation, both the suspensions containing the SIF and the control filtrate samples were analyzed microscopically and centrifuged at 40 to 60 x g for 20 minutes, and the spermatozoa were resuspended in spermatozoa-free seminal plasma from vasectomized donors. After centrifugation and res us pension of the spermatozoa, the motilities were again analyzed. Aliquots of the SIF were heated, then frozen, lyophilized, and resuspended in an attempt to characterize the stability of the SIF; they were then added to suspensions of spermatozoa, and motility determinations were performed as previously described.
FIG. 1. The effects of the Escherichia coli solution (___ ), the SIF (_ _ ), the control filtrate (o----Q), and Baker's solution (11.-11.) on the forward motility of spermatozoa.
RESULTS
FIG. 2. After dialysis and reconstitution of the Escherichia coli filtrate and the control filtrate, the solutions inside the dialysis tubing (E. coli filtrate, .--.; control filtrate, 11.--11.), the solutions outside the dialysis tubing (E. coli filtrate, ___ ; control filtrate, 0 - 0 ) , and the original filtrates (E. coli, e--e; control, 0 - - 0 ) were added to spermatozoal suspensions to assess effects on motility.
Figure 1 demonstrates the effects of the E. coli solution and the SIF on the progressive motility
60
50
40 >-
f-
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030 ::;;:
2 TIME (Hours)
3
4
50
40 >-
I::: ::! f0
::;;:
30
* 20
10
2
3
4
TIME (Hours)
184
PAULSON AND POLAKOSKI
February 1977 70
r
60
,
, ,, , ,,, , ,,, , ,,, ,
50
40 >f-
>-
:::::i
f-
i=
---.l
0
f-
~
~
~
o
30
20
10
t 4 TI ME (Hours)
FIG. 3. The effects of the SIF in the Escherichia coli filtrate on the motility of solutions containing 100 x 10· spermatozoa/ml (.6r.-4) , 50 x 10 6 spermatozoa/ml ( _ _ ), and 20x 106 spermatozoa/ml (___ ). Like concentrations of spermatozoa in control filtrates are represented by open symbols (6-6, 100 x 106/ml; 0----0, 50 x !06/ml; 0 - 0 , 20 x 106/ml).
of the spermatozoa and compares them with those of the normal controls. After 4 hours, the forward motility of the spermatozoa in the controls was 55%, whereas the motility of the spermatozoa incubated with either the E. coli organisms or the SIF was less than 5%. The percentage of dead spermatozoa in the control solutions and in solutions containing the SIF remained between 9 and 12%; however, 80% of the spermatozoa in a suspension containing the E. coli organisms were dead after 4 hours. The results obtained from dialysis, lyophilization, and reconstitution of the filtrates and their addition to spermatozoal suspensions are shown in Figure 2. Only the E. coli solution which passed through the dialysis bag contained the SIF. After filtration of the E. coli solution and the control buffer through the UM05 Amicon filter, the solution which passed through the filter, that which was retained, and the control solution were analyzed for the presence of SIF. Neither the retarded E. coli filtrate solution nor the controls had an observable effect on spermatozoal motility. However, theE. coli filtrate solution which passed
TIME
2 (Hours)
3
FIG. 4. Solution containing 20 x 106 spermatozoa/ml and SIF (e--e) and control filtrate (0--0) were incubated for 21h hours. The arrow indicates when the samples were centrifuged for 20 minutes and resuspended in seminal plasma from vasectomized donors.
through the membrane immobilized the spermatozoa in a fashion comparable with that of the original filtrate. The effect of the SIF on several concentrations of spermatozoa was compared with the controls, and the results are shown in Figure 3. With increasing concentrations of spermatozoa, there was an inverse effect on the loss of spermatozoal motility over the 4-hour time interval, indicating that the effectiveness of SIF in immobilization is dependent upon the concentration of the spermatozoa. Figure 4 demonstrates the reversible effect of sperm-free seminal plasma on the SIF-treated spermatozoal suspension. The SIF was further characterized as it remained active after being. heated to 90° C for 10 minutes. It was also stable to freezing, lyophilization, and reconstitution. DISCUSSION
This report confirms the immobilization of spermatozoa by E. coli and demonstrates a factor (SIF), apparently excreted by E. coli, which immobilizes spermatozoa. The E. coli organisms
Vol. 28, No.2
ISOLATION OF SPERMATOZOAL IMMOBILIZATION FACTOR
and the SIF had similar immobilization capacities. Initially, theE. coli immobilized spermatozoa and with time also had a spermicidal effect, whereas the SIF almost totally immobilized the spermatozoa after 4 hours while it did not affect the livedead ratio compared with the controls. The concentration of spermatozoa influenced the effectiveness of the SIF, indicating that bacterial infections could have a greater influence on the oligospermic ejaculate than on the normal specimen. This finding would appear to be valid for both male and female reproductive tract infections. In fact, it may be even more significant in female infections, for the spermatozoa which reach the cervical os of the female would be drastically reduced in number compared with the total in the ejaculate. We were able to stimulate spermatozoal motility by removing the seminal plasma and resuspending the spermatozoa in normal seminal plasma, indicating that this mechanism is reversible. The SIF is a stable compound, because boiling, freezing, thawing, and lyophilization had no observable effect on the ability of this factor to immobilize spermatozoa. The factor passed through the dialysis tubing and through a 500 molecular weight cutoff membrane, indicating that it is a relatively small component. Acknowledgments. The authors wish to thank Ms. Betsy Duhn and Ms. Faye Zvibleman for their help in typing the manuscript.
185
REFERENCES 1. Matthews CS, Buxton CL: Bacteriology of the cervix in cases of infertility. Fertil Steril 2:45, 1951 2. Rosenthal L: Spermagglutination by bacteria. Proc Soc Exp BioI Med 28:827,1931 3. Wu DH: Bacteriological studies on the semen, and experimental studies on the influences of antibiotics, crystalline penicillin potassium, dihydrostreptomycin sulfate and terramycin hydrochloride upon the semen. Kei-O Med J 34:509, 1957 4. Teague NS, Boyarsky S, Glenn JF: Interference of human spermatozoa motility by Escherichia coli. Fertil Steril 22:281, 1971 5. DelPorto GB, Derrick FC Jr, Bannister ER: Bacterial effect on sperm motility. Urology 5:638, 1975 6. Buxton CL, Wong MB: Spermicidal bacteria in the cervix as a cause of sterility. Am J Obstet Gynecol 64:628, 1952 7. Quesada EM, Dukes CD, Deen GH, Franklin RR: Genital infection and sperm agglutinating antibodies in infertile men. J Urol 99:106, 1968 8. Wu DH: Personal communication 9. Dahlberg B: Asymptomatic bacteriospermia and male infertility. Presented at the International Conference of Andrology, Wayne State University, Detroit, Mich, April 26, 1975 10. Keller DW, Polakoski KL: L-Arginine stimulation of human sperm motility in vitro. BioI Reprod 13:154, 1975 11. Paulson JD, Polakoski KL: Removal of extraneous material from the ejaculate. Presented at the First International Congress of Andrology, Barcelona, Spain, July 1976 12. Paulson JD, Polakoski KL: Preparation of human semen by glass wool columns. In Techniques in Human Andrology, Edited by ESE Hafez. New York, Elsevier Publishing Co. In press 13. Eliasson R, Treichl L: Supravital staining of human spermatozoa. Fertil Steril 22:134, 1972