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Isolation and Characterization of Dynein ATPase from Human Sperm
Accepted 241
242
PREVENTION OF ANTISPERM ANTIBODIES AND INFERTILITY AFTER UNILATERAL VASECTOMY BY PRE-OPERATIVE IMMUNOSUPPRESSJON. Daniel L. Kessler,* Gabriel J. Michael,* Patricia Darnen* Cincinnati, OH (Presentation to be made by Dr. Kessler) Certain unilateral conditions such as testicular torsion, tumor, or varicocele have been associated with We suspected that a unilateral decreased fertility. vasectomy might also be associated with decreased fertility as a result of the production of antisperm antibodies. We have studied this in a mouse model. In a prior study, male DBA/lJ mice were found to have a significantly increased antisperm antibody titers and decreased fertility 3 months after unilateral vasectomy. We now report on a study using the same model to see if fertility can be maintained by pre-treating the mice with an immunosuppressive regimen. Thirty-six male DBA/lJ mice were given either cortisone acetate, cyclosporine A or gavage fed sperm for one week prior to unilateral vasectomy. Eighteen untreated mice also underwent unilateral vasectomy. An additional 18 mice had a vasectomy and unilateral orchiectomy, orchiectomy alone or sham surgery and served Post-operative fertility was determined by as controls. matings and antisperm antibody titers were measured. After 3 months, fertility in the ligation group was 67%, while it was 100% in the control groups. Fertility in the group pre-treated with cortisone acetate was 69%, while in the cyclosporine A and antigen fed groups, it was 83% and 90% respectively. At 3 months antisperm antibodies were detectable in 58% of the mice in the cyclosporine A group, while they were only detectable in 23% of the cortisone acetate and 27% of the antigen fed groups. We conclude that pre-treating mice with cyclosporine A or sperm antigens may help maintain fertility after a unilateral vasectomy in DBA/lJ mice. However, this protective effect of immunosuppression did not seem to correlate well with suppression of the post-vasectomy antibody response as measured by our assay.
ISOLATION AND CHARACTERIZATION OF DYNEIN ATPase FROM HUMAN SPERM. John D. McConnell, Kelley Black* and Larry Johnson~ Dallas, TX ( Presentation to be made by Dr. McConnell) Dynein is the adenosine triphosphatase (ATPase) containing complex of polypeptides that constitute the axonemal arms of the sperm tail. Energy transducing interactions between the dynein arms and the microtubular system in the sperm tail provide the driving force for sperm motility. Dynein arm defects, which produce immotile sperm in man, have been characterized at the ultrastructural level. In an effort to explore these defects of sperm motility at the biochemical level, we have developed techniques to isolate outer arm dynein from human sperm. Sperm from donors with normal sperm motility were washed three times with buffered saline and then demembranated with O. 5% Nonidet P-40 ( NP-40). Demembranated sperm were washed with buffer and resuspended in O. 6M NaCl buffer for 10 min at 4°C. The supernatant obtained after centrifugation at 100, OOOg for 30 min was designated crude dynein extract. Electron microscopy demonstrates that exposure to O. 5% NP-40 does not alter axonemal structure, whereas, 0. 6M NaCl leads to fairly selective extraction of the outer dynein arms. ATPase activity of the crude dynein extract is inhibited (;>50%) by 5uM vanadate and O. 5mM erythro-9[ 3-( 2-hydroxynonyl) J adenine, known inhibitors of dyne in in other species. Gel electrophoresis of the human sperm extract demonstrates the presence of several high molecular subunits (}300 ,000 kd) characteristic of dynein heavy chains. These data suggest that outer arm dynein can be extracted from human sperm by high-salt buffer. It is hoped that further characterization of this enzyme complex will provide insight into disorders of sperm motility.
243
244
A COMPARISON OF THE EFFECT ON FERTILITY OF VARICOCELES EXPERIMENTALLY INDUCED IN EITHER JUVENILE OR ADULT RABBITS. *Thomas E. Shook, Charleston, SC; Leroy M. Nyberg, Jr., Farmington, CT (Presentation to be made by Dr. Shook) The presence of a varicocele in the adult male has been correlated with infertility. Neither the size nor the time of occurance of the varicocele has been shown to correlate with the degree of fertility. In this study we evaluated the effect of a unilateral varicocele, experimentally induced in either juvenile (prepubertal) or adult rabbits, on subsequent adult testis function. The juvenile rabbits were 21 days old and the adults were 51 days. Ten rabbits were in each surgical group. Varicoceles were induced by partial occlusion of the spermatic vein. Animals were sacrificed one month after surgery. There were ten sham-operated and five control rabbits in each age group. Intrascrotal temperatures were recorded, cytotoxic sperm antibodies were determined, and the testes were histologically evaluated for spermatogenesis. Intrascrotal temperatures were elevated in both age groups of rabbits with varicoceles. Histologically the ipsilateral testes of both age groups demonstrated a decrease in the number of functioning seminiferous tubules and germ cells. There was a significantly greater decrease seen in the juvenile rats. No changes were seen in the contralateral testes. Cytotoxic sperm antibody titers were present in all animals with varicoceles. None were in the controls. The juveniles had a significantly greater decrease in the dilution titer than the adults. Thus, the induction of a unilateral varicocele in juvenile rabbits produced a markedly greater effect on spermatogenesis and testis function in adulthood than in the animals with adult onset of varicocele.
THE INFLUENCE OF URETHRAL AND PARAURETHRAL LEASONS IN THE FORMATION OF POST TRAUMATIC STRICTURES:EXPERIMENTAL STUDY. Roman A. Zink and Max Weber*, Wuerzburg; Max Weiss*, Munich,FRG (Presentation to be made by Dr.Zink) It should be investigated to which extent urethral strictures occure after leasons which involve the urethra alone or the urethra plus the periurethral tissue. The experiments were performed in 56 rabbits, which had previously received a vesicocutaneostomy. The proximal urethra was exposed and mobilized from a small perineal incision in 5 sham operated animals. In 16 animals a cylindrical segment of 4 mm length was additionally excised from the urethra. In 35 other animals a gross urethral and periurethral leason was obtained by tearing a 4 mm segment together with the periurethral tissue out of the skin incision. Urethrogramms were performed after the operation and in time intervalls up to 3 days, 3 weeks and 3 months, when the animals were terminated. The bladder and urethra including the periurethral tissue was then dissected, macro- and microscopically investigated. Neither urethral strictures nor severe inflammatory reactions could be detected in all sham operated animals and in the other groups during the first 3 days. Up to 3 weeks after mere urethral trauma (UT) severe inflammation and stricture formation was seen in 8 %, wheras 24 % of the animals developed severe inflammatory changes and 41 % strictures after urethral plus periurethral trauma (UPT). Mild inflammation was seen in approximatly 1/3 of the UT and UPT group. After 3 months 29 % of the UPT animals developed severe inflammation and 53 % strictures. According to these findings the formation of urethral strictures correlates with the extent of the inflammatory reaction and rather depends upon the extent of the periurethral lesion than from the urethral lesion itself. This should be considered in urethral surgery and emphasizes the importance of avoiding unduely catheterisation attempts in patients with urethral trauma in order to avoid additional periurethral lesions.
164A
242
PREVENTION OF ANTISPERM ANTIBODIES AND INFERTILITY AFTER UNILATERAL VASECTOMY BY PRE-OPERATIVE IMMUNOSUPPRESSJON. Daniel L. Kessler,* Gabriel J. Michael,* Patricia Darnen* Cincinnati, OH (Presentation to be made by Dr. Kessler) Certain unilateral conditions such as testicular torsion, tumor, or varicocele have been associated with We suspected that a unilateral decreased fertility. vasectomy might also be associated with decreased fertility as a result of the production of antisperm antibodies. We have studied this in a mouse model. In a prior study, male DBA/lJ mice were found to have a significantly increased antisperm antibody titers and decreased fertility 3 months after unilateral vasectomy. We now report on a study using the same model to see if fertility can be maintained by pre-treating the mice with an immunosuppressive regimen. Thirty-six male DBA/lJ mice were given either cortisone acetate, cyclosporine A or gavage fed sperm for one week prior to unilateral vasectomy. Eighteen untreated mice also underwent unilateral vasectomy. An additional 18 mice had a vasectomy and unilateral orchiectomy, orchiectomy alone or sham surgery and served Post-operative fertility was determined by as controls. matings and antisperm antibody titers were measured. After 3 months, fertility in the ligation group was 67%, while it was 100% in the control groups. Fertility in the group pre-treated with cortisone acetate was 69%, while in the cyclosporine A and antigen fed groups, it was 83% and 90% respectively. At 3 months antisperm antibodies were detectable in 58% of the mice in the cyclosporine A group, while they were only detectable in 23% of the cortisone acetate and 27% of the antigen fed groups. We conclude that pre-treating mice with cyclosporine A or sperm antigens may help maintain fertility after a unilateral vasectomy in DBA/lJ mice. However, this protective effect of immunosuppression did not seem to correlate well with suppression of the post-vasectomy antibody response as measured by our assay.
ISOLATION AND CHARACTERIZATION OF DYNEIN ATPase FROM HUMAN SPERM. John D. McConnell, Kelley Black* and Larry Johnson~ Dallas, TX ( Presentation to be made by Dr. McConnell) Dynein is the adenosine triphosphatase (ATPase) containing complex of polypeptides that constitute the axonemal arms of the sperm tail. Energy transducing interactions between the dynein arms and the microtubular system in the sperm tail provide the driving force for sperm motility. Dynein arm defects, which produce immotile sperm in man, have been characterized at the ultrastructural level. In an effort to explore these defects of sperm motility at the biochemical level, we have developed techniques to isolate outer arm dynein from human sperm. Sperm from donors with normal sperm motility were washed three times with buffered saline and then demembranated with O. 5% Nonidet P-40 ( NP-40). Demembranated sperm were washed with buffer and resuspended in O. 6M NaCl buffer for 10 min at 4°C. The supernatant obtained after centrifugation at 100, OOOg for 30 min was designated crude dynein extract. Electron microscopy demonstrates that exposure to O. 5% NP-40 does not alter axonemal structure, whereas, 0. 6M NaCl leads to fairly selective extraction of the outer dynein arms. ATPase activity of the crude dynein extract is inhibited (;>50%) by 5uM vanadate and O. 5mM erythro-9[ 3-( 2-hydroxynonyl) J adenine, known inhibitors of dyne in in other species. Gel electrophoresis of the human sperm extract demonstrates the presence of several high molecular subunits (}300 ,000 kd) characteristic of dynein heavy chains. These data suggest that outer arm dynein can be extracted from human sperm by high-salt buffer. It is hoped that further characterization of this enzyme complex will provide insight into disorders of sperm motility.
243
244
A COMPARISON OF THE EFFECT ON FERTILITY OF VARICOCELES EXPERIMENTALLY INDUCED IN EITHER JUVENILE OR ADULT RABBITS. *Thomas E. Shook, Charleston, SC; Leroy M. Nyberg, Jr., Farmington, CT (Presentation to be made by Dr. Shook) The presence of a varicocele in the adult male has been correlated with infertility. Neither the size nor the time of occurance of the varicocele has been shown to correlate with the degree of fertility. In this study we evaluated the effect of a unilateral varicocele, experimentally induced in either juvenile (prepubertal) or adult rabbits, on subsequent adult testis function. The juvenile rabbits were 21 days old and the adults were 51 days. Ten rabbits were in each surgical group. Varicoceles were induced by partial occlusion of the spermatic vein. Animals were sacrificed one month after surgery. There were ten sham-operated and five control rabbits in each age group. Intrascrotal temperatures were recorded, cytotoxic sperm antibodies were determined, and the testes were histologically evaluated for spermatogenesis. Intrascrotal temperatures were elevated in both age groups of rabbits with varicoceles. Histologically the ipsilateral testes of both age groups demonstrated a decrease in the number of functioning seminiferous tubules and germ cells. There was a significantly greater decrease seen in the juvenile rats. No changes were seen in the contralateral testes. Cytotoxic sperm antibody titers were present in all animals with varicoceles. None were in the controls. The juveniles had a significantly greater decrease in the dilution titer than the adults. Thus, the induction of a unilateral varicocele in juvenile rabbits produced a markedly greater effect on spermatogenesis and testis function in adulthood than in the animals with adult onset of varicocele.
THE INFLUENCE OF URETHRAL AND PARAURETHRAL LEASONS IN THE FORMATION OF POST TRAUMATIC STRICTURES:EXPERIMENTAL STUDY. Roman A. Zink and Max Weber*, Wuerzburg; Max Weiss*, Munich,FRG (Presentation to be made by Dr.Zink) It should be investigated to which extent urethral strictures occure after leasons which involve the urethra alone or the urethra plus the periurethral tissue. The experiments were performed in 56 rabbits, which had previously received a vesicocutaneostomy. The proximal urethra was exposed and mobilized from a small perineal incision in 5 sham operated animals. In 16 animals a cylindrical segment of 4 mm length was additionally excised from the urethra. In 35 other animals a gross urethral and periurethral leason was obtained by tearing a 4 mm segment together with the periurethral tissue out of the skin incision. Urethrogramms were performed after the operation and in time intervalls up to 3 days, 3 weeks and 3 months, when the animals were terminated. The bladder and urethra including the periurethral tissue was then dissected, macro- and microscopically investigated. Neither urethral strictures nor severe inflammatory reactions could be detected in all sham operated animals and in the other groups during the first 3 days. Up to 3 weeks after mere urethral trauma (UT) severe inflammation and stricture formation was seen in 8 %, wheras 24 % of the animals developed severe inflammatory changes and 41 % strictures after urethral plus periurethral trauma (UPT). Mild inflammation was seen in approximatly 1/3 of the UT and UPT group. After 3 months 29 % of the UPT animals developed severe inflammation and 53 % strictures. According to these findings the formation of urethral strictures correlates with the extent of the inflammatory reaction and rather depends upon the extent of the periurethral lesion than from the urethral lesion itself. This should be considered in urethral surgery and emphasizes the importance of avoiding unduely catheterisation attempts in patients with urethral trauma in order to avoid additional periurethral lesions.
164A