1 Department of Veterinary Science, National Institute of Infectious Diseases, Toyama, Shinjuku, Tokyo 162-8640 and 2 Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan
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Abstract |
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Key words: fertilization/mouse/premature chromosome condensation/pronucleus/round spermatids
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Introduction |
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Recently, it has been demonstrated that G1-phase chromosomes that undergo premature condensation in mature metaphase II (MII) oocytes moved to either of the poles after oocyte activation (Wakayama et al., 1998). These two groups of the donor chromosomes intermingled at the first mitosis. If it is also true for spermatid G1 chromosomes, they may participate in embryo development following premature condensation and oocyte activation. In this study, mouse spermatids were injected into mature oocytes, which were kept arrested at MII to induce premature condensation of the spermatid chromosomes. The oocytes were activated while extrusion of the polar bodies was prevented, and one of the female pronuclei was removed in order to restore the diploid state. The developmental ability of embryos thus obtained was examined in vivo and in vitro.
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Materials and methods |
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Collection of spermatogenic cells
Spermatogenic cells were mechanically isolated from the seminiferous tubules of C57BL/6J males (SLC Co., Shizuoka, Japan) as described previously (Ogura and Yanagimachi, 1993). The cell suspension was washed by centrifugation twice and stored in Dulbecco's phosphate-buffered saline (DPBS) containing 0.5% bovine serum albumin (BSA, fraction V, Calbiochem, CA, USA) at 4°C for up to 4 h.
Microinsemination with round spermatids
The cover of a plastic dish (Falcon no. 1006; Becton Dickinson) was used as a microinjection chamber. A row of four small drops (12 µl each), of which two were HEPES-buffered CZB containing 0.1 g/ml polyvinyl alcohol (for oocytes) and two were 12% polyvinylpyrrolidone in HEPES-buffered CZB, was placed on the bottom of the dish and covered with silicone oil. One of the PVP drops contained a spermatogenic cell suspension. The dish was placed on the stage of an inverted microscope (Nikon TE300) equipped with Nomarski differential interference optics. Mouse round spermatids can be easily identified by their small size (about 12 µm in diameter) and round nucleus with a centrally located nucleolar structure (Ogura and Yanagimachi, 1993). An oocyte was held to the holding pipette with the metaphase II spindle at either the 2 or the 4 o'clock position. After drawing an individual spermatid into an injection pipette (45 µm inner diameter), the pipette was advanced mechanically until its tip almost reached the opposite side of the oocyte (the 9 o'clock position). The spermatid nucleus, together with a small volume of the cytoplasm, was expelled into the ooplasm. The zona pellucida and the plasma membrane of the oocyte were penetrated or broken separately by applying a few Piezo pulses as described previously (Kimura and Yanagimachi, 1995b
). All the procedures were performed at room temperature (23°C). Injected oocytes were then kept at room temperature for about 10 min before they were incubated at 37°C. Approximately 2 h later, the injected oocytes were activated by treating them in Ca2+-free CZB containing 5 mM SrCl2. The activation medium also contained 15 µM cytochalasin B to prevent extrusion of the polar bodies from the oocyte and spermatid chromosomes. Four or 8 h later, the activated oocytes were placed into a small drop of the same activation medium on a micromanipulation chamber and one of the two female pronuclei was removed with an enucleation pipette (about 15 µm inner diameter). The oocytes which had been cultured in the activation medium for 8 h were washed in fresh CZB medium and cultured for a further 24 or 72 h at 37°C. The experimental protocol, together with the spermatid injection protocol usually used, is shown diagrammatically in Figure 1
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Embryo transfer
After 24 or 72 h in culture, injected oocytes that developed to the 2-cell stage or the morula/blastocyst stage, respectively, were transferred into pseudopregnant ICR females (Clea Japan, Tokyo, Japan). Two-cell embryos were transferred into the oviducts of day 1 (the day on which vaginal plug was found) recipients and morulae and blastocysts were transferred into the uteri of day 3 recipients. On day 20, recipient females were killed and the uteri were removed. The uteri were examined for live or dead fetuses. Lactating foster mothers raised some of the live fetuses.
Statistical analyses
The data were analysed with Fisher's exact probability test.
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Results |
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Discussion |
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According to Kimura and Yanagimachi (1995b), the best protocol for spermatid conception in the mouse is to activate oocytes first and inject them with a spermatid nucleus 1 h later. The majority of oocytes (77%) were normally fertilized (two swollen pronuclei and one second polar body), while the remaining oocytes had a small spermatid-derived pronucleus. The abnormal pronuclear formation in the latter was probably due to the persistence of the spermatid nuclear membrane. This was observed more frequently when spermatid nuclei were injected into oocytes at more advanced stages in which the cytoplasmic MPF level had decreased. These small male pronuclei supported embryo development less efficiently than the regular sized male pronuclei (Kimura and Yanagimachi, 1995b). Therefore, the timing of spermatid injection and oocyte activation is critical for normal fertilization and embryo development when the usual microfertilization method is employed. With the protocol developed in this study, all the spermatids injected were transformed into swollen (pseudo)pronuclei after oocyte activation, so breakdown of the spermatid nuclear membrane must have occurred. The formation of four pronuclei, which was presumably the result of synchronous segregation of spermatid and oocyte chromosomes, was found in 73% of oocytes. This rate is comparable to that of normal fertilization with the standard spermatid injection protocol (77%, Kimura and Yanagimachi, 1995b
). In our new protocol, one of two female pronuclei is removed to restore the diploid state. As far as we could tell, the time of enucleation did not have any effect on the subsequent embryo development. The rates of normal birth after embryo transfer (24 and 14%) were within the range of our previous experiments using pre-activated oocytes (about 1035%, Ogura et al., 1996a
,b
; Tanemura et al., 1997
).
In some oocytes, two or three pronuclei were found, instead of four, and we could not perform enucleation. After spermatid injection, the oocytes were kept arrested at MII for about 2 h to induce full condensation and spindle formation. The spermatid chromosomes might have mingled with female chromosomes during this period because of the attractive forces of newly polymerized spindle microtubules, or during early pronuclear development as observed in human zygotes after spermatid injection (Tesarik and Mendoza, 1996; Barak et al., 1998
).
Our findings have implications for human clinical practice. Sperm-borne oocyte activating factor is present in human round spermatids, but it is absent or deficient in round spermatids from patients with spermatogenic failure (Tesarik et al., 1998a), and, in fact, the fertilization rate after round spermatid injection is significantly lower than that after elongated spermatid injection (25.6 versus 71%) (Kahraman et al., 1998
). Therefore, it is very probable that spermatids from these patients would undergo PCC after incorporation into oocytes, which have high MPF activity. These oocytes are classified as `unfertilized' and are presently discarded. The results of this study indicate that these oocytes may be rescued by applying external activation stimulus, followed by removal of a female pronucleus. These rescued oocytes may develop to term if spermatids have no DNA damage, which is often associated with spermiogenesis failure (Tesarik et al., 1998b
). Perhaps this can also be applied to unfertilized human oocytes after intracytoplasmic sperm injection (ICSI) if spermatozoa undergo PCC due to oocyte activation failure (Flaherty et al., 1995
). The technique of removing supernumerary pronuclei from human oocytes has already been established for rescuing polyspermic zygotes (Gordon et al., 1989
).
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Acknowledgments |
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Notes |
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References |
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Chatot, C.L., Ziomek, C.A., Bavister, B.D. et al. (1989) An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J. Reprod. Fertil., 86, 679688.[Abstract]
Flaherty, S.P., Payne, D., Swann, N.J. et al. (1995) Assessment of fertilization failure and abnormal fertilization after intracytoplasmic sperm injection (ICSI). Reprod. Fertil. Dev., 7, 197210.[ISI][Medline]
Gordon, J.W., Grunfeld, L., Garrisi, G.J. et al. (1989) Successful microsurgical removal of a pronucleus from tripronuclear human zygotes. Fertil. Steril., 52, 367372.[ISI][Medline]
Kahraman, S., Polat, G., Samli, M. et al. (1998) Multiple pregnancies obtained by testicular spermatid injection in combination with intracytoplasmic sperm injection. Hum. Reprod., 13, 104110.[Abstract]
Kimura, Y. and Yanagimachi, R. (1995a) Development of normal mice from oocytes injected with secondary spermatocyte nuclei. Biol. Reprod., 53, 855862.[Abstract]
Kimura, Y. and Yanagimachi, R. (1995b) Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring. Development, 121, 23972405.
Kimura, Y., Tateno, H., Handel, M.A. et al. (1998) Factors affecting meiotic and developmental competence of primary spermatocyte nuclei injected into mouse oocytes. Biol. Reprod., 59, 871877.
Ogura, A., Yamamoto, Y., Suzuki, O. et al. (1996a) In vitro fertilization and microinsemination with round spermatids for propagation of nephrotic genes in mice. Theriogenology, 45, 11411149.[ISI]
Ogura, A., Matsuda, J., Asano, T. et al. (1996b) Mouse oocytes injected with cryopreserved round spermatids can develop into normal offspring. J. Assist. Reprod. Genet., 13, 431434.[ISI][Medline]
Ogura, A., Suzuki, O., Tanemura, K. et al. (1998) Development of normal mice from metaphase I oocytes fertilized with primary spermatocytes. Proc. Natl Acad. Sci. USA, 95, 56115615.
Ogura, A. and Yanagimachi, R. (1993) Round spermatid nuclei injected into hamster oocytes form pronuclei and participate in syngamy. Biol. Reprod., 48, 219225.[Abstract]
Tanemura, K., Wakayama, T., Kuromoto, K. et al. (1997) Birth of normal young by microinsemination with frozen-thawed round spermatids collected from aged azoospermic mice. Lab. Anim. Sci., 47, 203204.[Medline]
Tesarik, J. and Mendoza, C. (1996) Spermatid injection into human oocytes. I. Laboratory techniques and special features of zygote development. Hum. Reprod., 11, 772779.[Abstract]
Tesarik, J., Sousa, M., Greco, E. et al. (1998a) Spermatids as gametes: indications and limitations. Hum. Reprod., 13, 89107.[Medline]
Tesarik, J., Greco, E., Cohen-Bacrie, P. et al. (1998b) Germ cell apoptosis in men with complete and incomplete spermiogenesis failure. Mol. Hum. Reprod., 4, 757762.[Abstract]
Wakayama, T., Perry, A.C.F., Zuccotti, M. et al. (1998) Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature, 394, 369374.[ISI][Medline]
Submitted on October 27, 1998; accepted on January 8, 1999.