1 Kapiolani Medical Center, Department of Obstetrics and Gynecology, University of Hawaii School of Medicine, Honolulu, HI 96826, USA and 2 Department of Obstetrics and Gynecology, Yamanashi Medical University, Shimokato 1110, Tamaho, Nakakoma, Yamanashi, 409-3898, Japan
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: embryo development/fertilization/ICSI/PVP/sperm immobilization
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Clinically, problems relating to the use of immotile sperm remain unclear since their membranes may have degraded prior to injection. This situation can arise with severely asthenospermic specimens, with testicular biopsies from non-obstructive azoospermic patients, with frozenthawed sperm, or in cases where sperm are held in polyvinylpyrrolidone (PVP) for logistical reasons before use. It was recently reported that mouse and human sperm immobilized by sonication, and kept for up to 2 h in culture, showed an increase in structural chromosome aberrations (Tateno et al., 2000). They concluded that prolonged exposure of sperm nuclei to culture medium, rather than sonication, caused the aberrations. They further showed that use of a K+-rich nuclear isolation medium (NIM) partially protected against chromosome damage (Suzuki et al., 1998
; Tateno et al., 2000
). Sperm disruption in isotonic NaCl for 2 h after sonication also impaired normal embryo development (Kuretake et al., 1996
), consistent with a genetic cause for developmental damage. PVP is thought to have a sperm membrane-stabilizing effect, even at low concentrations (Dozortsev et al., 1995
). In vitro, this may help to protect the sperm from degradative damage. Therefore, the purpose of the present study was to systematically evaluate the effects of immobilizing sperm for defined intervals in PVP or NIM buffers on oocyte activation and development to the blastocyst stage.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Media
The medium for the culture of mouse oocytes was ChatotTascaZiomek (CZB) medium supplemented with 5.56 mmol/l D-glucose and 5 mg/ml bovine serum albumin (BSA) (Chatot et al., 1989, 1990
). The medium for collection of oocytes from oviducts and for micromanipulation was a modified CZB (HEPESCZB) containing 20 mmol/l HEPES, 5 mmol/l NaHCO3 and 0.1 mg/ml polyvinyl alcohol (PVA, cold water soluble) instead of BSA. The pH value of both media was 7.4 under 5% CO2 in air (37°C) or room air respectively.
The medium used for isolation of sperm heads (nuclei) was modified nucleus isolation medium (NIM) (Kuretake et al., 1996). This consisted of 123.0 mmol/l KCl, 2.6 mmol/l NaCl, 7.8 mmol/l NaH2PO4, 1.4 mmol/l KH2PO4 and 3 mmol/l EDTA (disodium salt). We used phenylmethylsulphonyl fluoride (protease inhibitor)-free NIM. Its pH value was adjusted to 7.2 by addition of a small quantity of 1 mol/l KOH.
Preparation of mouse oocytes
B6D2F1 female mice, 812 weeks old, underwent ovulation induction by i.p. injection of 5 IU pregnant mare's serum gonadotrophin followed by 5 IU hCG 48 h later. Oocytes were collected from oviducts ~14 h after hCG injection. The oocytes were freed from cumulus cells by exposure to 0.1% hyaluronidase in HEPESCZB for a few minutes. They were rinsed and incubated in CZB for up to 4 h at 37°C under 5% CO2 in air before sperm injection.
Preparation of human and mouse sperm
Human semen samples were obtained from fertile volunteers. The semen parameters were normal by published standards (World Health Organization, 1992). Semen samples were allowed to liquefy for 1h, mixed with 10 ml of CZB, and centrifuged at 500 g for 5 min. The sperm pellet was resuspended in 0.5 ml of CZB, then layered beneath 1 ml of CZB for 1 h at 37°C. Motile swim-up sperm were retrieved and transferred to another tube.
The cauda epididymides of a B6D2F1 male mouse were cut with a pair of scissors, and a dense sperm mass squeezed out into a 5 ml tube containing 1.5 ml of CZB. After incubation at 37°C under 5% CO2 in air for ~1 h, the upper portion of the medium was collected and examined. Over 80% of sperm displayed high motility.
Immobilization of sperm and ICSI
A small amount of human sperm suspension was suspended in 5 µl HEPESCZB containing a 10% PVP (mol. wt 360 000) (CZBPVP). Human sperm were immobilized by drawing a single motile spermatozoon, tail first, midway into the injection pipette. At that point, several piezo-pulses were applied to the tail until the spermatozoon became completely immotile (Kimura and Yanagimachi, 1995). For the mouse, a small amount of epididymal sperm suspension was first suspended in drops of CZBPVP and a single spermatozoon was drawn, tail first, into the injection pipette just to the headmidpiece junction. Application of a few piezo-pulses separated the head from the tail (Kuretake et al., 1996
).
Control human immobilized sperm were injected immediately into mouse oocytes as previously described (Kimura and Yanagimachi, 1995). Immobilized human sperm in experimental groups were transferred into another drop of CZBPVP and kept for 124 h at room temperature before injection. Similarly, isolated mouse heads in experimental groups were transferred into either CZBPVP or PVP-free NIM buffer and maintained there for 124 h at room temperature before injection. Sperm incubation intervals in all cases were staggered so that all retrieved fresh mouse oocytes were injected within a 24 h time interval.
Examination of oocytes and embryos
After injection with human sperm or mouse sperm heads, mouse oocytes were incubated in CZB for 68 h and examined with an Olympus IM-6 microscope with Hoffman differential interference optics for evidence of activation. Two types of activation were scored. First, a `normal' group exhibited two pronuclei and a second polar body (2PN + PB2). Second, a `total' number of activated oocytes was tallied by including oocytes exhibiting either 1PN + PB2 or 3PN without PB2.
Oocytes fertilized with mouse sperm heads were cultured in CZB medium. Embryos were examined at 24 h intervals for up to 120 h to evaluate development in vitro to the blastocyst stage.
Statistical analysis
Results were compared using the 2-test with the Yate's correction. Differences at P < 0.05 were considered significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The effects of maintaining mouse sperm in CZBPVP buffer or NIM on developmental arrest are shown in Figures 1 and 2 respectively. In CZBPVP there was a time-dependent decline in blastocyst development beginning with the 1 h time interval, where the majority of embryos arrested at the morula stage. This pattern of arrest was similar during 4 h. However, developmental arrest occurred even earlier than the 4-cell stage when utilizing sperm maintained for 6 or 24 h prior to injection. Figure 2
shows that NIM partially protected embryos from arrest only for 12 h. By 3 h, arrest at the morula stage was similar to that seen in the CZBPVP group.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Results indicate that fertilization rates are relatively unaffected with increasing time intervals from immobilization to injection using either entire human or detached mouse sperm heads. There were no significant differences in fertilization rates using human sperm over any interval tested up to 24 h after immobilization in CZBPVP medium (Table I). SOAF are not very species-specific, which allows mouse oocytes to be activated by human sperm (Rybouchkin et al., 1995
). SOAF appears to be localized within the perinuclear theca of the sperm head (Kimura et al., 1998
; Perry et al., 1999
). While there was a reduction in oocyte activation using isolated mouse sperm heads after a 6 h maintenance period, the decline over controls never fell below 82% (Table II
) even after 24 h. This could be explained by a more rapid degradative process compared with the human sperm given that the mouse sperm head is completely dissociated from its tail. (Human sperm heads cannot be easily dissociated from tails using piezo, making a direct comparison difficult in these experiments). Since human sperm membranes may be more stable than those of mouse sperm (Kasai et al., 1999
), one cannot conclude that there are obvious species differences in this regard. Immotile ejaculated human sperm can fertilize oocytes but often at a lower rate (Nagy et al., 1995
). This could be explained by more extensive degradative damage with sperm immotile for >24 h. Finally, the results do not address whether qualitative aspects of oocyte activation (e.g. the frequency of calcium transients) might also be different over the different time intervals tested.
Embryo development was a more sensitive indicator that time-dependent damage occurs following sperm immobilization (Table II). Using CZBPVP buffer as the holding medium, blastocyst development was impaired at the earliest interval assayed (1 h) for mouse sperm. This showed a further time-dependent effect such that virtually no blastocyst development occurred by 46 h. Additional experiments would be needed to rule out that developmental effects might occur within several minutes of sperm head separation. Holding the sperm heads in the K+-rich NIM buffer protects from this developmental effect only for ~2 h. Thereafter, the time-dependent decline in development occurred as seen in the CZBPVP buffer.
Using either CZBPVP or NIM buffer, the clearest block in development occurred at the morula stage (Table II). However, with increasing time intervals after immobilization, earlier cleavage stages were arrested as well. The mouse embryonic genome becomes increasingly active by the 2-cell stage, including paternal markers (Wudl and Chapman, 1976
; Sawicki et al., 1981
; Latham, 1999
). Thus, the developmental data are consistent with some effect on appropriate genome activation or expression. This is further supported by the protective effect in NIM buffer (Table II
). Using cytogenetic analysis, NIM has been shown to protect the mouse sperm from genetic damage from the extracellular environment after membrane disruption (Tateno et al., 2000
). The Na+ and K+ concentrations in bovine sperm have been estimated at 14 ± 2 and 120 ± 5 mmol/l respectively (Babcock, 1983
), and NIM is formulated to maintain a K+-rich ionic environment around the disrupted sperm. Sperm chromatin may be susceptible to ion-dependent genetic damage. This protection, however, is limited and the data do not rule out additional epigenetic developmental mechanisms that have been described through nuclear transfer experiments and involving cytoplasmic interactions (Reik et al., 1993
; Roemer et al., 1997
).
The mouse data described cannot yet be extrapolated to the human since isolated human sperm heads were not evaluated; however, the data support the hypothesis that prolonged exposure to the extracellular environment could pose a developmental risk for the membrane-compromised human sperm heads. First, structural damage to human chromosomes after injection into mouse oocytes has been described, although mouse chromosomes may be affected more rapidly (Tateno et al., 2000). Second, it has been reported that injection of isolated sperm heads may lead to chromosomal errors in the resulting embryos (Colombero et al., 1999
). However, it remains to be ruled out that this is secondary to damage to the sperm centrosome, which is of paternal origin in the human. In the mouse, the sperm centriole is lost during testicular and epididymal transit and is not required for normal fertilization and development (Manandhar et al., 1998
). Third, there is evidence that human sperm chromatin is sensitive to some stresses such as sonication (Kuretake et al., 1996
). Human sperm have less disulphide bonding within protamines (Perreault et al., 1988
), and ~15% of human sperm DNA remains bound to nucleohistones (Gatewood et al., 1987
; Choudhary et al., 1995
).
On the other hand, human sperm also have some measure of protection from the types of conditions reported here using isolated mouse epididymal sperm heads. Ejaculated human sperm chromatin takes up considerable amounts of zinc from the seminal fluid which acts to stabilize nuclear proteins (Kvist et al., 1985). In addition, human sperm heads are rarely used for ICSI as described here for the mouse. The mouse sperm tail is routinely removed prior to ICSI to improve oocyte survival, and this does not appear to pose a developmental risk as long as the heads are injected in a timely manner (Ron-El et al., 1995
; Kuretake et al., 1996
). Nevertheless, the data are consistent with the hypothesis that sperm-immobilizing conditions could pose developmental risks which would not be apparent from fertilization rate or normal activation rate (2PN) endpoints alone.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahmadi, A. and Ng, S.C. (1999) Influence of sperm plasma membrane destruction on human sperm head decondensation and pronuclear formation. Arch. Androl., 42, 17.[ISI][Medline]
Babcock, D.F. (1983) Examination of the intracellular ionic environment and of ionophore action by null point measurements employing the fluorescein chromophore. J. Biol. Chem., 258, 63806389.
Bergh, M.V., Bertrand, E., Biramane, J. and Englert, Y. (1995) Importance of breaking a spermatozoon's tail before intracytoplasmic injection: a prospective randomized trial. Hum. Reprod., 10, 28192820.[ISI][Medline]
Catt, J. and O'Neill, C. (1995) Manipulation of sperm before intracytoplasmic sperm injection improves fertilization rates. Fertil. Steril., 64, 12101212.[ISI][Medline]
Chatot, C.L., Ziomek, C.A., Bavister, B.D., Lewis, J.L. and Torres, I. (1989) An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J. Reprod. Fertil., 86, 679688.[Abstract]
Chatot, C.L., Lewis, J.L., Torres, I. and Ziomek, C.A. (1990) Development of 1-cell embryos from different strains of mice in CZB medium. Biol. Reprod., 42, 432440.[Abstract]
Choudhary, S.K., Wykes, S.M., Kramer, J.A., Mohamed, A.N., Koppitch, F., Nelson, J.E. and Krawetz, S.A. (1995) A haploid expressed gene cluster exists as a single chromatin domain in human sperm. J. Biol. Chem., 270, 87558762.
Colombero, L.T., Moomjy, M., Sills, E.S. Rosenwaks, Z. and Palermo, G.D. (1999) The role of structural integrity of the fertilising spermatozoon in early human embryogenesis. Zygote, 7, 157163.[ISI][Medline]
Dozortsev, D., Rybouchkin, A., Sutter, P.D. and Dhont, M. (1995) Sperm plasma membrane damage prior to intracytoplasmic sperm injection: a necessary condition for sperm nucleus decondensation. Hum. Reprod., 10, 29602964.[Abstract]
Dozortsev, D., Qian, C., Ermilov, A., Rybouchkin, A., Sutter, P.D. and Dhont, M. (1997) Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 minutes after injection as a result of the spermoocyte interaction. Hum. Reprod., 12, 27922796.[Abstract]
Fishel, S., Lisi, F., Rinaldi, L., Green, S., Hunter, A., Dowell, K. and Thornton, S. (1995) Systematic examination of immobilizing spermatozoa before intracytoplasmic sperm injection in the human. Hum. Reprod., 10, 497500.[ISI][Medline]
Gatewood, J.M., Cook, G.R., Balhorn, R., Bradbury, E.M. and Schmid, C.W. (1987) Sequence-specific packaging of DNA in human sperm chromatin. Science, 236, 962964.[ISI][Medline]
Gerris, J., Mangelschots, K., Royen, E.V., Joostens, M., Eestermans, W. and Ryckaert, G. (1995) ICSI and severe male-factor infertility: breaking the sperm tail prior to injection. Hum. Reprod., 10, 484486.[ISI][Medline]
Goto, K. (1993) Bovine microfertilization and embryo transfer. Mol. Reprod. Dev., 36, 288290.[ISI][Medline]
Goto, K., Kinoshita, A., Takuma, Y. and Ogawa, K. (1990) Fertilization of bovine oocytes by the injection of immobilized, killed spermatozoa. Vet. Rec., 127, 517520.[ISI][Medline]
Hoshi, K., Yanagida, K., Yazawa, H., Katayose, H. and Sato, A. (1995) Intracytoplasmic sperm injection using immobilized or motile human spermatozoon. Fertil. Steril., 63, 12411245.[ISI][Medline]
Kasai, T., Hoshi, K. and Yanagimachi, R. (1999) Effect of sperm immobilisation and demembranation on the oocyte activation rate in the mouse. Zygote, 7, 187193.[ISI][Medline]
Kimura, Y. and Yanagimachi, R. (1995) Intracytoplasmic sperm injection in the mouse. Biol. Reprod., 52, 709720.[Abstract]
Kimura, Y., Yanagimachi, R., Kuretake, S., Bortkiewicz, H., Perry, A.C.F. and Yanagimachi, H. (1998) Analysis of mouse oocyte activation suggests the involvement of sperm perinuclear material. Biol. Reprod., 58, 14071415.[Abstract]
Kuretake, S., Kimura, Y., Hoshi, K. and Yanagimachi, R. (1996) Fertilization and development of mouse oocytes injected with isolated sperm heads. Biol. Reprod., 55, 789795.[Abstract]
Kvist, U., Bjorndahl, L., Roomans, G.M. and Lindholmer, C. (1985) Nuclear zinc in human epididymal and ejaculated spermatozoa. Acta Physiol. Scand., 125, 297303.[ISI][Medline]
Latham, K.E. (1999) Mechanisms and control of embryonic genome activation in mammalian embryos. Int. Rev. Cytol., 193, 71124.[ISI][Medline]
Manandhar, G., Sutovsky, P., Joshi, H.C., Stearns, T. and Schatten, G. (1998) Centrosome reduction during mouse spermiogenesis. Dev. Biol., 203, 424434.[ISI][Medline]
Martin, R.H., Ko, E. and Rademaker, A. (1988) Human sperm chromosome complements after microinjection of hamster eggs. J. Reprod. Fertil., 84, 179186.[Abstract]
Nagy, Z.P., Liu, J., Joris, H., Verheyen, G., Tournaye, H., Camus, M., Derde, M.P., Devroey, P. and Van Steirteghem, A.C. (1995) The result of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters. Hum. Reprod., 10, 11231129.[Abstract]
Palermo, G., Joris, H., Devroey, P. and Van Steirteghem, A.C. (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 340, 1718.[ISI][Medline]
Palermo, G.D., Cohen, J., Alikani, M., Adler, A. and Rosenwaks, Z. (1995) Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility. Fertil. Steril., 63, 12311240.[ISI][Medline]
Palermo, G.D., Schlegel, P.N., Colombero, L.T., Zaninovic, N., Moy, F. and Rosenwaks, Z. (1996) Aggressive sperm immobilization prior to intracytoplasmic sperm injection with immature spermatozoa improves fertilization and pregnancy rates. Hum. Reprod., 11, 10231029.[Abstract]
Perreault, S.D., Barbee, R.R., Elstein, K.H., Zucker, R.M. and Keefer, C.L. (1988) Interspecies differences in the stability of mammalian sperm nuclei assessed in vivo by sperm microinjection and in vitro by flow cytometry. Biol. Reprod., 39, 157167.[Abstract]
Perry, A.C.F., Wakayama, T. and Yanagimachi, R. (1999) A novel trans-complementation assay suggests full mammalian oocyte activation is coordinately initiated by multiple, submembrane sperm components. Biol. Reprod., 60, 747755.
Reik, W., Romer, I., Barton, S.C., Surani, M.A., Howlett, S.K. and Klose, J. (1993) Adult phenotype in the mouse can be affected by epigenetic events in the early embryo. Development, 119, 933942.
Roemer, I., Reik, W., Dean, W. and Klose, J. (1997) Epigenetic inheritance in the mouse. Curr. Biol., 7, 277280.[ISI][Medline]
Ron-El, R., Liu, J., Nagy, Z., Joris, H., Abbeel, E.V. and Van Steirteghem, A.C. (1995) Intracytoplasmic sperm injection in the mouse. Hum. Reprod., 10, 28312834.[Abstract]
Rybouchkin, A., Dozortsev, D., Sutter, P.D., Qian, C. and Dhont, M. (1995) Intracytoplasmic injection of human spermatozoa into mouse oocytes: a useful model to investigate the oocyte-activating capacity and the karyotype of human spermatozoa. Hum. Reprod., 10, 11301135.[Abstract]
Rybouchkin, A., Sutter, P.D. and Dhont, M. (1996) Unprotected freezing of human spermatozoa exerts a detrimental effect on their oocyte activating capacity and chromosome integrity. Zygote, 4, 263268.[ISI][Medline]
Sawicki, J.A., Magnuson, T. and Epstein, C.J. (1981) Evidence for expression of the paternal genome in the two-cell mouse embryo. Nature, 294, 450451.[ISI][Medline]
Suzuki, K., Yanagida, K. and Yanagimachi, R. (1998) Comparison of the media for isolation and storage of round spermatid nuclei before intracytoplasmic injection. J. Assist. Reprod. Genet., 15, 154158.[ISI][Medline]
Tateno, H., Kimura, Y. and Yanagimachi, R. (2000) Sonication per se is not as deleterious to sperm chromosomes as previously inferred. Biol. Reprod., 63, 341346.
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction, 3rd edn. Cambridge University Press, Cambridge.
Wudl, L. and Chapman, V. (1976) The expression of ß-glucuronidase during preimplantation development of mouse embryos. Dev. Biol., 48, 104109.[ISI][Medline]
Yanagida, K., Katayose, H., Yazawa, H., Kimura, Y., Konnai, K. and Sato, A. (1998) The usefulness of a piezo-micromanipulator in intracytoplasmic sperm injection in humans. Hum. Reprod., 14, 448453.
Yanagimachi, R. (1981) Mechanisms of fertilization in mammals. In Mastroianni, L. and Biggers, J.D. (eds), Fertilization and Embryonic Development In Vitro, Plenum Press, New York, pp. 133134.
Submitted on October 1, 2001; resubmitted on March 13, 2002; accepted on May 17, 2002.