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Nonbeneficial effects of glycerol on the oocyte penetrating capacity of cryopreserved and incubated human spermatozoa
CRYOBIOLOGY
22, 434-437 (1985)
Nonbeneficial Effects of Glycerol on the Oocyte Penetrating Capacity of Cryopreserved and Incubated Human Spermatozoa R. S. JEYENDRAN,*+’ *Institute
H. H. VAN DER VEN,t,2
M. PEREZ-PELAEZ,*
AND L. J. D. ZANEVELD? of Reproductive Medicine, Chicago, Illinois and tDepariment of Ob.strtrics Rush-Presbyterian St. Luke’s Medical Center, Chicago, Illinois
& Gynecology,
The effect of cryopreservation on human spermatozoa in the presence or absence of glycerol was assessed by using sperm motility, functional integr-ity of sperm membrane, and denuded hamster oocyte penetration tests. Glycerol treated cryopreserved spermatozoa yielded a significantly higher (P ( 0.01) percentage of motile sperm and percentage of sperm with functionally intact membrane immediately after thawing than the spermatozoa not treated with glycerol hut cryopreserved. However, no significant difference was ohserved between these cryopreserved spermatozoa (either treated or untreated with glycerol) on the percentage of motile sperm and the rate of oocyte penetration when the sperm were washed and incubated for 2 hr in a medium containing no glycerol. Thus, it appears glycerol may not be beneficial, since cryopreservation of spermatozoa either treated or untreated with glycerol essentially yields similar oocyte-penetrating capacity of sperm. C 1985 Academic Press. Inc.
Glycerol is the most common preservative used in the cryopreservation of human sperm (18). However, spermatozoa cryopreserved in the presence of glycerol yield significantly lower post-thaw sperm motility and penetration of denuded hamster oocytes than nonfrozen sperm (6). In addition, considerable ultrastructural and biochemical changes in the acrosome have been reported following glycerol cryopreservation (4, 5, 8, 12). We previously postulated that human spermatozoa treated with glycerol develop a dependency to glycerol, and the subsequent removal of glycerol decreases the motility of the sperm as well as their ability to penetrate denuded hamster oocytes (6). These findings may well explain the poor pregnancy rate associated with glycerol-treated cryopreserved
sperm as compared to that of fresh sperm (1,9). The present study was undertaken to further evaluate the protective cffcct of glycerol against cryoinjury of human spermatozoa. MATERIALS
434 Copynght 0 ,985 by Academic Pres. Inc. All rights of reproduction in any form reserved.
METHODS
Semen was obtained from eight healthy volunteers whose ejaculates appeared normal in all aspects (19) by masturbation after 3 to 5 days of sexual abstinence. Individual semen samples after liquefaction were either cryopreserved or used directly (unfrozen) within 3 hr of ejaculation. The pellet freezing method was used to cryopreserve the spermatozoa (13) since more uniform freezing rates are obtained by this technique than by the use of either ampules or straws (2). For cryopreservation, semen cooled to 5”C, at a rate of O.S’C/min was frozen on dry ice (-80°C) in 0.1 ml pellets. After 10 min or more on dry ice, the frozen pellets were immediately submerged into liquid nitrogen (- 196°C) and stored for not less than 30 min. This storage time was arbitrarily chosen to minimize the
Received October 15, 1984; accepted May 9, 1985. ’ To whom reprint requests should he addressed at: Institute of Reproductive Medicine, 111 N. Wabash Avenue, Suite 1016, Chicago, Ill. 60602. * Present address: Department of Obstetrics & Gynecology, University of Bonn, S-Frend-Str 25, Bonn, F.R. Germany.
001 l-2240/85 $3.00
AND
GLYCEROL
435
EFFECT ON POST-THAW HUMAN SPERM
quality deterioration of unfrozen semen control with time. Thawing was done by placing the pellets in aluminum boats that floated on water at 37°C. For the experimental protocol, semen was divided into three portions. One portion was left untreated and kept at room temperature for the same amount of time as was required to freeze and thaw the cryopreserved samples. The second portion was treated with 10% (v/v) glycerol (15) and cryopreserved as described above. The third portion was not treated with glycerol but cryopreserved. Two separate experiments were performed to compare the effects of cryopreservation on spermatozoon quality, the fresh and thawed samples of the two frozen fractions were analyzed. Experiment I. The motility of spermatozoa was assessed utilizing Makler’s counting chamber (11). The proportion of sperm with progressive motility in each of the three fractions was determined. Similarly, the functional integrity of the sperm membrane was assessed by exposing the spermatozoa to hypoosmotic stress [hypoosmotic swelling (HOS) test] and determining the proportion of sperm responding to this stress (7). Experiment 2. The ability of the spermatozoa to become capacitated and penetrate the vitelline membrane of the denuded hamster oocyte was assessed (5). Briefly, the spermatozoa in the untreated fresh and thawed samples of the two frozen fractions were washed after addition of modified Biggers, Whitten, and Whittingham’s medium containing 35 mgiml human serum albumin (“culture medium”) by centrifugation at 500g for 3 min. The sperm pellets were then resuspended in culture medium and washed two more times. The sperm concentration was adjusted to 100 x 10” per ml in the culture medium and the mixture was incubated for 2 to 3 hr at 37°C. The spermatozoa (0.02 ml, 2 x 10”) were then mixed with denuded hamster oocytes in 0.2 ml culture medium and incubated for 5 hr. The oo-
TABLE I Effect of Glycerol on the Progressive Motility and Sperm Swelling in Cryopreserved Human Spermatozoa Progressive” motility (55)
Sperm” Swelling (5?)
Unfrozen control
64 i- 2.2
61 2 7.4
Cryopreserved Glycerol treated Untreated
26 i 4.3 I? 2 2.9
40 t 7.9 25 t 5.1
Sample
’ Mean 2 SE. Mean values in each column were significantly (P < 0.05) different from each other (N = 8).
cytes were then removed, washed, fixed, stained with acetolacmoid and examined microscopically for sperm penetration. Oocytes were considered penetrated when a swollen sperm head or a male pronucleus with a corresponding sperm tail was found within the oocyte. The data were subjected to analysis of variance and the mean differences were tested for significance by Neuman-Keuls procedure (17). RESULTS AND DISCUSSION
Cryopreservation of spermatozoa yielded a significantly decreased (P < 0.01) percentage of progressively motile sperm and sperm with functionally intact membrane as compared to the unfrozen control (Table 1). Also, glycerol-treated post-thaw sperm motility and sperm with functionally intact membrane were significantly higher (P < 0.05) than the untreated but cryopreserved sperm. A significant decrease (P < 0.05) in the motility of sperm was noted during the second experiment when the cryopreserved spermatozoa treated with glycerol were washed and incubated for 2 hr at 37°C in a glycerol-free medium, as compared to sperm motility immediately after thawing (Table 2). The decrease was such that after the 2-hr incubation period, basically no differences could be observed between the spermatozoa cryopreserved in the presence
436
JEYENDRAN
ET AL.
TABLE 2 Effect of Glycerol and Incubation on the Progressive Motility and Oocyte Penetrating Ability of Cryopreserved Human Spermatozoa
Sample
Number of oocytes
Progressive motility (%)* 0 hr
2 hr
Percentage of penetrated oocytes*
Unfrozen control
95
74 + 1.8
49 2 5.1
61.4 2 6.6
Cryopreserved Glycerol treated Untreated
65
30 2 3.w.h
65
12 2 3.6”
10 2 2.9h 8 2 I.7
28.7 k 9.9 21.2 i 6.6
* Mean i SE. Mean values for the unfrozen control were significantly (P < 0.01) higher than either glycerol treated or untreated cyropreserved spermatozoa. Among the cryopreserved samples, means with a common superscript were significantly (P < 0.05) different from each other (N = 5).
or absence of glycerol. A decrease in sperm motility was noted for the unfrozen control and cryopreserved untreated spermatozoa after washing, but this decrease was not statistically significant. Cryopreservation plus incubation of spermatozoa either treated or untreated with glycerol yielded a significantly (P < 0.01) decreased percentage penetration of hamster oocytes as compared to the unfrozen control sample. Similar to the motility data obtained after washing the spermatozoa and incubating them for 2 hr, the oocyte penetration rates were essentially the same; whether the cryopreserved spermatozoa had been treated or untreated with glycerol. Thus, glycerol does not appear to aid in the ability of spermatozoa to ultimately penetrate the oocyte. We reported previously that the cryopreservation of spermatozoa treated with glycerol had a significantly lower number of ultrastructurally normal sperm, a low level of acrosin, hyaluronidase, and a decreased rate of penetration of denuded hamster oocytes as compared to spermatozoa treated with a TESTCY extender that does not contain glycerol (4, 5, 8). This decreased oocyte penetrating capacity of spermatozoa was not observed when the glycerol was maintained in the media during washing and capacitation (6). The present data confirm and extend these findings by showing that cryopreservation of untreated spermatozoa yields a
significantly decreased (P < 0.05) motility and sperm with functionally intact membrane immediately after thawing as compared to glycerol-treated cryopreserved spermatozoa, but no differences were seen in the long-term motility of spermatozoa after washing or in the rate of oocyte penetration. Therefore, it appears that glycerol does not aid in the ultimate penetrating capacity of human spermatozoa. A similar effect on sperm motility has also been reported (10). Obando and Lucena (14) reported a slight improvement in the in viva fertilizing capacity of cryopreserved spermatozoa when they were not treated with glycerol, as compared to when glycerol was present. Since cervical mucus prevents most, if not all, of the seminal fluid from entering the cervical canal (3, 16), it is likely that glycerol is also removed from the spermatozoa by this female genital tract fluid. Thus, it appears that although glycerol minimized cryoinjury during freezing and thawing, it may not be beneficial to human sperm since its removal by washing and subsequent incubation essentially yields results similar to cryopreserved spermatozoa but untreated with glycerol. REFERENCES 1. Amelar, R. D., and Dubin, L. Frozen semen-a poor form of fertility insurance. Urology 14, 53-54 (1979).
2. Graham, E. E Fundamentals of the preservation of spermatozoa. In “The Integrity of Frozen
GLYCEROL
3.
4.
5.
6.
7.
8.
9.
EFFECT
ON
POST-THAW
Spermatozoa.” Proceedings National Academy of Science USA, Vol. 4, p. 44 (1978). Hafez, E. S. E. Transport and survival of spermatozoa in the female reproductive tract. In “Human Semen and Fertility Regulation in Man” (E. S. E. Hafez. Ed.), pp. 107-121, C.V. Mosby, St. Louis (1976). Heath, E., Jeyendran, R. S., Perez-Pelaez, M., and Sobrero, A. J. Ultrastructural categorization of human spermatozoa cryopreserved in glycerol and in TESTCY. Int. J. Androl., in press (1985). Jeyendran, R. S., Van der Ven, H. H., Kennedy. W., Perez-Pelaez, M., and Zaneveld, L. J. D. Comparison of glycerol and a zwitter ion buffer system as a cryoprotective media for human spermatozoa: Effect on motility, penetration of zona-free hamster oocytes and acrosiniproacrosin. J. Androl. 5, l-5 (1984). Jeyendran, R. S., Van der Ven. H. H.. Perez-Pelaez, M., and Zaneveld, L. J. D. Effect of glycerol and cryopreservation on oocyte penetration by human spermatozoa. crndrologirr 17, 241-248 (1985). Jeyendran R. S., Van der Ven. H. H., Perez-Pelaez, M., Crabo, 6. G., and Zaneveld. L. J. D. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod. Frrfil. 70, 219-228 (1984). Joyce, C., Jeyendran. R. S., and Zaneveld, L. J. D. Release, extraction and stability of hyaluronidase associated with human spermatozoa. Comparison with the rabbit. J. Androl. 6, 152-161 (1985). Leeton, J., Selwood, T., Trounson, A., and Wood. C. Artificial donor insemination: Frozen versus fresh semen. Ausr. N.Z.J. Ohstrt. Gynrc~ol. 20, 205-207 (1980).
HUMAN
SPERM
437
10. Lucena, E., and Obando, H. Comparative study of glycerol levels as cryoprotective agents of the human sperm. Xth International Congr. Animal Reprod. and A.I. II, 193-195 (1984). 11. Makler, A. A new chamber for rapid sperm count and motility estimation. Ferril. Sferil. 30, 313318 (1978). 12. Mahadevan, M. M., and Trounson, A. 0. Relationship of fine structure of sperm head to fertility of frozen human semen. Fertil. Steril. 41, 287-293 (1984). 13. Nagase, H., and Niwa, T. Deep freezing bull semen in concentrated pellet form. Proc. Vth. Int. Congr. Animal Reprod.. Trento, Italy 4, 410-415 (1964). 14. Obando, H.. and Lucena, E. Comparative study of two extenders: Glycine vs. Test-Tris in cryopreserving human semen. Xth International Congr. Animal Reprod. and A.I. III, 203-204 (1984). 15. Sherman, J. K. Freezing and freeze-drying human spermatozoa. Fertil. Steril. 5, 357-371 (1984). 16. Sobrero, A. J. Sperm migration in the female genital tract. In “Mechanisms Concerned with Conception” (C. G. Hartman, Ed.). p. 173. Pergamon Press, Oxford (1963). 17. Snedecor. G. W., and Cochran. W. G. “Statistical Methods.” Iowa State Univ. Press. Ames. Iowa (1980). 18. Watson. P. F. The preservation of semen in mammals. In “Oxford Review of Reproductive Biology” (C. A. Finn, Ed.). Vol. I. pp. 283-305. Oxford Press, Oxford (1979). 19. Zaneveld. L. J. D.. and Polakoski, K. L. Collection and physical examination of the ejaculate. of Human Andrology” In “Techniques (E. S. E. Hafez, Ed.), pp. 147-172. Elsevieri North Holland. (1977).
22, 434-437 (1985)
Nonbeneficial Effects of Glycerol on the Oocyte Penetrating Capacity of Cryopreserved and Incubated Human Spermatozoa R. S. JEYENDRAN,*+’ *Institute
H. H. VAN DER VEN,t,2
M. PEREZ-PELAEZ,*
AND L. J. D. ZANEVELD? of Reproductive Medicine, Chicago, Illinois and tDepariment of Ob.strtrics Rush-Presbyterian St. Luke’s Medical Center, Chicago, Illinois
& Gynecology,
The effect of cryopreservation on human spermatozoa in the presence or absence of glycerol was assessed by using sperm motility, functional integr-ity of sperm membrane, and denuded hamster oocyte penetration tests. Glycerol treated cryopreserved spermatozoa yielded a significantly higher (P ( 0.01) percentage of motile sperm and percentage of sperm with functionally intact membrane immediately after thawing than the spermatozoa not treated with glycerol hut cryopreserved. However, no significant difference was ohserved between these cryopreserved spermatozoa (either treated or untreated with glycerol) on the percentage of motile sperm and the rate of oocyte penetration when the sperm were washed and incubated for 2 hr in a medium containing no glycerol. Thus, it appears glycerol may not be beneficial, since cryopreservation of spermatozoa either treated or untreated with glycerol essentially yields similar oocyte-penetrating capacity of sperm. C 1985 Academic Press. Inc.
Glycerol is the most common preservative used in the cryopreservation of human sperm (18). However, spermatozoa cryopreserved in the presence of glycerol yield significantly lower post-thaw sperm motility and penetration of denuded hamster oocytes than nonfrozen sperm (6). In addition, considerable ultrastructural and biochemical changes in the acrosome have been reported following glycerol cryopreservation (4, 5, 8, 12). We previously postulated that human spermatozoa treated with glycerol develop a dependency to glycerol, and the subsequent removal of glycerol decreases the motility of the sperm as well as their ability to penetrate denuded hamster oocytes (6). These findings may well explain the poor pregnancy rate associated with glycerol-treated cryopreserved
sperm as compared to that of fresh sperm (1,9). The present study was undertaken to further evaluate the protective cffcct of glycerol against cryoinjury of human spermatozoa. MATERIALS
434 Copynght 0 ,985 by Academic Pres. Inc. All rights of reproduction in any form reserved.
METHODS
Semen was obtained from eight healthy volunteers whose ejaculates appeared normal in all aspects (19) by masturbation after 3 to 5 days of sexual abstinence. Individual semen samples after liquefaction were either cryopreserved or used directly (unfrozen) within 3 hr of ejaculation. The pellet freezing method was used to cryopreserve the spermatozoa (13) since more uniform freezing rates are obtained by this technique than by the use of either ampules or straws (2). For cryopreservation, semen cooled to 5”C, at a rate of O.S’C/min was frozen on dry ice (-80°C) in 0.1 ml pellets. After 10 min or more on dry ice, the frozen pellets were immediately submerged into liquid nitrogen (- 196°C) and stored for not less than 30 min. This storage time was arbitrarily chosen to minimize the
Received October 15, 1984; accepted May 9, 1985. ’ To whom reprint requests should he addressed at: Institute of Reproductive Medicine, 111 N. Wabash Avenue, Suite 1016, Chicago, Ill. 60602. * Present address: Department of Obstetrics & Gynecology, University of Bonn, S-Frend-Str 25, Bonn, F.R. Germany.
001 l-2240/85 $3.00
AND
GLYCEROL
435
EFFECT ON POST-THAW HUMAN SPERM
quality deterioration of unfrozen semen control with time. Thawing was done by placing the pellets in aluminum boats that floated on water at 37°C. For the experimental protocol, semen was divided into three portions. One portion was left untreated and kept at room temperature for the same amount of time as was required to freeze and thaw the cryopreserved samples. The second portion was treated with 10% (v/v) glycerol (15) and cryopreserved as described above. The third portion was not treated with glycerol but cryopreserved. Two separate experiments were performed to compare the effects of cryopreservation on spermatozoon quality, the fresh and thawed samples of the two frozen fractions were analyzed. Experiment I. The motility of spermatozoa was assessed utilizing Makler’s counting chamber (11). The proportion of sperm with progressive motility in each of the three fractions was determined. Similarly, the functional integrity of the sperm membrane was assessed by exposing the spermatozoa to hypoosmotic stress [hypoosmotic swelling (HOS) test] and determining the proportion of sperm responding to this stress (7). Experiment 2. The ability of the spermatozoa to become capacitated and penetrate the vitelline membrane of the denuded hamster oocyte was assessed (5). Briefly, the spermatozoa in the untreated fresh and thawed samples of the two frozen fractions were washed after addition of modified Biggers, Whitten, and Whittingham’s medium containing 35 mgiml human serum albumin (“culture medium”) by centrifugation at 500g for 3 min. The sperm pellets were then resuspended in culture medium and washed two more times. The sperm concentration was adjusted to 100 x 10” per ml in the culture medium and the mixture was incubated for 2 to 3 hr at 37°C. The spermatozoa (0.02 ml, 2 x 10”) were then mixed with denuded hamster oocytes in 0.2 ml culture medium and incubated for 5 hr. The oo-
TABLE I Effect of Glycerol on the Progressive Motility and Sperm Swelling in Cryopreserved Human Spermatozoa Progressive” motility (55)
Sperm” Swelling (5?)
Unfrozen control
64 i- 2.2
61 2 7.4
Cryopreserved Glycerol treated Untreated
26 i 4.3 I? 2 2.9
40 t 7.9 25 t 5.1
Sample
’ Mean 2 SE. Mean values in each column were significantly (P < 0.05) different from each other (N = 8).
cytes were then removed, washed, fixed, stained with acetolacmoid and examined microscopically for sperm penetration. Oocytes were considered penetrated when a swollen sperm head or a male pronucleus with a corresponding sperm tail was found within the oocyte. The data were subjected to analysis of variance and the mean differences were tested for significance by Neuman-Keuls procedure (17). RESULTS AND DISCUSSION
Cryopreservation of spermatozoa yielded a significantly decreased (P < 0.01) percentage of progressively motile sperm and sperm with functionally intact membrane as compared to the unfrozen control (Table 1). Also, glycerol-treated post-thaw sperm motility and sperm with functionally intact membrane were significantly higher (P < 0.05) than the untreated but cryopreserved sperm. A significant decrease (P < 0.05) in the motility of sperm was noted during the second experiment when the cryopreserved spermatozoa treated with glycerol were washed and incubated for 2 hr at 37°C in a glycerol-free medium, as compared to sperm motility immediately after thawing (Table 2). The decrease was such that after the 2-hr incubation period, basically no differences could be observed between the spermatozoa cryopreserved in the presence
436
JEYENDRAN
ET AL.
TABLE 2 Effect of Glycerol and Incubation on the Progressive Motility and Oocyte Penetrating Ability of Cryopreserved Human Spermatozoa
Sample
Number of oocytes
Progressive motility (%)* 0 hr
2 hr
Percentage of penetrated oocytes*
Unfrozen control
95
74 + 1.8
49 2 5.1
61.4 2 6.6
Cryopreserved Glycerol treated Untreated
65
30 2 3.w.h
65
12 2 3.6”
10 2 2.9h 8 2 I.7
28.7 k 9.9 21.2 i 6.6
* Mean i SE. Mean values for the unfrozen control were significantly (P < 0.01) higher than either glycerol treated or untreated cyropreserved spermatozoa. Among the cryopreserved samples, means with a common superscript were significantly (P < 0.05) different from each other (N = 5).
or absence of glycerol. A decrease in sperm motility was noted for the unfrozen control and cryopreserved untreated spermatozoa after washing, but this decrease was not statistically significant. Cryopreservation plus incubation of spermatozoa either treated or untreated with glycerol yielded a significantly (P < 0.01) decreased percentage penetration of hamster oocytes as compared to the unfrozen control sample. Similar to the motility data obtained after washing the spermatozoa and incubating them for 2 hr, the oocyte penetration rates were essentially the same; whether the cryopreserved spermatozoa had been treated or untreated with glycerol. Thus, glycerol does not appear to aid in the ability of spermatozoa to ultimately penetrate the oocyte. We reported previously that the cryopreservation of spermatozoa treated with glycerol had a significantly lower number of ultrastructurally normal sperm, a low level of acrosin, hyaluronidase, and a decreased rate of penetration of denuded hamster oocytes as compared to spermatozoa treated with a TESTCY extender that does not contain glycerol (4, 5, 8). This decreased oocyte penetrating capacity of spermatozoa was not observed when the glycerol was maintained in the media during washing and capacitation (6). The present data confirm and extend these findings by showing that cryopreservation of untreated spermatozoa yields a
significantly decreased (P < 0.05) motility and sperm with functionally intact membrane immediately after thawing as compared to glycerol-treated cryopreserved spermatozoa, but no differences were seen in the long-term motility of spermatozoa after washing or in the rate of oocyte penetration. Therefore, it appears that glycerol does not aid in the ultimate penetrating capacity of human spermatozoa. A similar effect on sperm motility has also been reported (10). Obando and Lucena (14) reported a slight improvement in the in viva fertilizing capacity of cryopreserved spermatozoa when they were not treated with glycerol, as compared to when glycerol was present. Since cervical mucus prevents most, if not all, of the seminal fluid from entering the cervical canal (3, 16), it is likely that glycerol is also removed from the spermatozoa by this female genital tract fluid. Thus, it appears that although glycerol minimized cryoinjury during freezing and thawing, it may not be beneficial to human sperm since its removal by washing and subsequent incubation essentially yields results similar to cryopreserved spermatozoa but untreated with glycerol. REFERENCES 1. Amelar, R. D., and Dubin, L. Frozen semen-a poor form of fertility insurance. Urology 14, 53-54 (1979).
2. Graham, E. E Fundamentals of the preservation of spermatozoa. In “The Integrity of Frozen
GLYCEROL
3.
4.
5.
6.
7.
8.
9.
EFFECT
ON
POST-THAW
Spermatozoa.” Proceedings National Academy of Science USA, Vol. 4, p. 44 (1978). Hafez, E. S. E. Transport and survival of spermatozoa in the female reproductive tract. In “Human Semen and Fertility Regulation in Man” (E. S. E. Hafez. Ed.), pp. 107-121, C.V. Mosby, St. Louis (1976). Heath, E., Jeyendran, R. S., Perez-Pelaez, M., and Sobrero, A. J. Ultrastructural categorization of human spermatozoa cryopreserved in glycerol and in TESTCY. Int. J. Androl., in press (1985). Jeyendran, R. S., Van der Ven, H. H., Kennedy. W., Perez-Pelaez, M., and Zaneveld, L. J. D. Comparison of glycerol and a zwitter ion buffer system as a cryoprotective media for human spermatozoa: Effect on motility, penetration of zona-free hamster oocytes and acrosiniproacrosin. J. Androl. 5, l-5 (1984). Jeyendran, R. S., Van der Ven. H. H.. Perez-Pelaez, M., and Zaneveld, L. J. D. Effect of glycerol and cryopreservation on oocyte penetration by human spermatozoa. crndrologirr 17, 241-248 (1985). Jeyendran R. S., Van der Ven. H. H., Perez-Pelaez, M., Crabo, 6. G., and Zaneveld. L. J. D. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod. Frrfil. 70, 219-228 (1984). Joyce, C., Jeyendran. R. S., and Zaneveld, L. J. D. Release, extraction and stability of hyaluronidase associated with human spermatozoa. Comparison with the rabbit. J. Androl. 6, 152-161 (1985). Leeton, J., Selwood, T., Trounson, A., and Wood. C. Artificial donor insemination: Frozen versus fresh semen. Ausr. N.Z.J. Ohstrt. Gynrc~ol. 20, 205-207 (1980).
HUMAN
SPERM
437
10. Lucena, E., and Obando, H. Comparative study of glycerol levels as cryoprotective agents of the human sperm. Xth International Congr. Animal Reprod. and A.I. II, 193-195 (1984). 11. Makler, A. A new chamber for rapid sperm count and motility estimation. Ferril. Sferil. 30, 313318 (1978). 12. Mahadevan, M. M., and Trounson, A. 0. Relationship of fine structure of sperm head to fertility of frozen human semen. Fertil. Steril. 41, 287-293 (1984). 13. Nagase, H., and Niwa, T. Deep freezing bull semen in concentrated pellet form. Proc. Vth. Int. Congr. Animal Reprod.. Trento, Italy 4, 410-415 (1964). 14. Obando, H.. and Lucena, E. Comparative study of two extenders: Glycine vs. Test-Tris in cryopreserving human semen. Xth International Congr. Animal Reprod. and A.I. III, 203-204 (1984). 15. Sherman, J. K. Freezing and freeze-drying human spermatozoa. Fertil. Steril. 5, 357-371 (1984). 16. Sobrero, A. J. Sperm migration in the female genital tract. In “Mechanisms Concerned with Conception” (C. G. Hartman, Ed.). p. 173. Pergamon Press, Oxford (1963). 17. Snedecor. G. W., and Cochran. W. G. “Statistical Methods.” Iowa State Univ. Press. Ames. Iowa (1980). 18. Watson. P. F. The preservation of semen in mammals. In “Oxford Review of Reproductive Biology” (C. A. Finn, Ed.). Vol. I. pp. 283-305. Oxford Press, Oxford (1979). 19. Zaneveld. L. J. D.. and Polakoski, K. L. Collection and physical examination of the ejaculate. of Human Andrology” In “Techniques (E. S. E. Hafez, Ed.), pp. 147-172. Elsevieri North Holland. (1977).