Molecular Human Reproduction, Vol. 5, No. 3, 227-233,
March 1999
© 1999 European Society of Human Reproduction and Embryology
Effect of post-ovulatory age and calcium in the injection medium on the male pronucleus formation and metaphase entry following injection of human spermatozoa into golden hamster oocytes
Infertility Centre, Department of Obstetrics and Gynaecology, University Hospital, De Pintelaan 185, Ghent, B9000 Belgium
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Abstract |
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The occurrence of parthenogenetic activation is a major hurdle in obtaining sperm chromosome metaphases after heterospecific intracytoplasmic sperm injection (ICSI) of golden hamster oocytes with human spermatozoa. We addressed two potential contributors to parthenogenetic activation namely, post-ovulatory age of the oocyte and Ca2+ content of the injection medium. In serial experiments, hamster oocytes were retrieved at 11.5, 13, 16 and 21 h after the ovulatory dose of human chorionic gonadotrophin (HCG) and microinjected with human spermatozoa suspended alternately in a regular (1.9 mM Ca2+) or a Ca2+-free medium. A progressive decrease in the rates of male pronucleus (MPN) formation and metaphase entry and increase in the rates of parthenogenetic activation without male pronucleus occurred with increasing post-ovulatory age. The favourable influence of Ca2+-free injection medium on the mean rates of MPN and metaphase entry was restricted to the relatively older oocytes (MPN 16 h: 49.5 versus 32.3%, P < 0.008; 21 h: 22.2 versus 11.1%, P < 0.001; metaphase entry 16 h: 36.8 versus 25.1%, P < 0.02; 21 h: 13.3 versus 5.2%, P < 0.01 in the Ca2+-free and regular groups respectively). Our data confirm the increased activation sensitivity with post-ovulatory ageing and its adverse influence on the MPN formation and metaphase entry after heterospecific ICSI of hamster oocytes.
heterospecific fertilization/ICSI/male pronucleus formation/parthenogenetic activation/post-ovulatory age
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Introduction |
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Zona-free Syrian golden hamster oocytes have a unique property to undergo heterospecific fertilization when inseminated with spermatozoa from a variety of mammalian species including human. This property has been utilized for assessing human sperm function, obtaining sperm chromosome metaphases and to study heterospecific gamete interactions (Rudak et al., 1978
; Rogers et al., 1979; Martin, 1983; Yanagimachi, 1984; Maleszewski et al., 1995). Furthermore, hamster oocytes also undergo heterologous fertilization after sperm microinjection, similar to the inseminated zona-free oocytes (Uehara and Yanagimachi, 1976; Perrault et al., 1988; Yanagida et al., 1991). However, a significant fraction of the hamster oocytes microinjected with spermatozoa undergo oocyte activation without forming the male pronucleus (MPN) and finally not many of the injected oocytes reach the first mitotic metaphase (Goud et al., 1998a; Martin et al., 1988). Nevertheless, microinjection of the hamster oocytes still remains a practical alternative to karyotyping human spermatozoa due to the ease of injection without oocyte damage and the possibility of applying the recently improved culture techniques for hamster oocytes and embryos (Barnett and Bavister, 1992). Besides, the technique of human sperm karyotyping using zona-free hamster oocytes is well established and provides a suitable background data on structural chromosome analysis of human spermatozoa for reference or comparison (Martin, 1995). On the other hand, application of the zona-free hamster fertilization technique is not suitable for sperm samples with low concentration and motility (Brandriff et al., 1985) and injection of oocytes from other species, e.g mice, is technically difficult although possible with equipment modifications and a reasonable skill (Markert, 1983; Kimura and Yanagimachi, 1995; Rybouchkin et al., 1995). Therefore, further research into the possible means to improve the results of heterospecific hamster intracytoplasmic sperm injection (ICSI) is warranted.
In order to obtain analysable sperm chromosome spreads, the development of the heterospecific zygotes to the first mitotic metaphase is critical. A major hurdle for all the sperm injected hamster oocytes to develop to the first metaphase is the occurrence of oocyte activation without MPN formation in a fraction of these oocytes. This is presumably due to spontaneous and/or injection related parthenogenetic activation. One of the factors leading to this phenomenon is the presence of Ca2+ in the injection medium, because omission of Ca2+ from the medium during injection results in a significant improvement in the development of the sperm injected hamster oocytes to the first metaphase (Goud et al., 1998a). Another factor contributing to this phenomenon might be the post-ovulatory age of the oocytes. Activation sensitivity of mammalian oocytes is known to increase with post-ovulatory age (Fulton and Whittingham, 1978; Nagai, 1987; Collas et al., 1989; Kubiak, 1989; Fissore and Robl, 1992). Moreover, hamster oocytes are known to activate even spontaneously with post-ovulatory ageing (Austin, 1956; Yanagimachi and Chang, 1961; Longo, 1974). Therefore, injection of post-ovulatory young oocytes could possibly reduce the incidence of parthenogenetic activation without MPN formation and consequently enhance the rates of MPN formation and metaphase entry (Goud et al., 1998a). Therefore, we investigated the developmental progression of human sperm nuclei through the stages of pronucleus formation and metaphase entry following heterologous ICSI into hamster oocytes retrieved at different post-ovulatory ages and injected with spermatozoa in regular or Ca2+-free medium. A post-ovulatory age-dependent increase in the oocyte activation sensitivity and its adverse effect on the MPN formation and metaphase entry was clearly evident.
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Materials and methods |
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Experimental design
Hamster oocytes were obtained from the oviducts of superovulated animals at 13 h (seven experiments), 16 h (eight experiments) and 21 h (six experiments) after human chorionic gonadotrophin (HCG) and from follicles at 11.5 h after HCG (six experiments). In each experiment, sibling oocytes were alternately injected with spermatozoa suspended in either a regular (1.9 mM Ca2+) or a Ca2+-free medium. The injected oocytes were cultured and evaluated for the development and progression of the injected sperm nucleus through interphase and the following first mitotic metaphase after treatment with a microtubule inhibitor. In one experiment, heterospecific zygotes resulting from human sperm microinjected hamster post-ovulatory young oocytes were allowed to cleave in culture.
Chemicals, reagents and culture media
All chemicals were obtained from Sigma Chemical Co (St Louis, MO, USA) unless otherwise specified. The media used for handling and culture were hamster embryo culture medium-3 (HECM-3) with or without HEPES respectively, containing 1.9 mM Ca2+ (McKiernan et al., 1991; Barnett and Bavister, 1992). Calcium-free injection medium was prepared by omitting Ca2+ during media preparation and including 100 µM Ethylene glycol-bis-N,N,N'N'-tetraacetic acid (EGTA) in the handling medium. This medium was used for sperm washing in all the experiments and for sperm retrieval and injection in the Ca2+-free groups. For culture of the heterospecific zygotes, HECM-3 culture medium was supplemented with 0.4 µg/ml colcemid (Demecolchicine) except in one experiment where the heterospecific zygotes were allowed to cleave.
Hamster care, superovulation and oocyte retrieval
Female golden hamsters (6–8 weeks old; 100–150 g) were obtained from Charles River Germany (IFFA Credo, Brussels, Belgium) and were allowed to adjust to our light conditions (14 h light and 10 h dark). Superovulation, oviductal oocyte retrieval and oocyte preparation for ICSI were performed as before (Goud et al., 1998a). Follicular oocytes were retrieved at 11.5 h post-HCG by puncturing prominent antral follicles from the excised ovaries in handling medium under mineral oil. The oocyte–cumulus complexes were cultured for 1–2 h in HECM-3 culture medium supplemented with 2.5% hamster serum and metaphase II (MII) stage oocytes were subjected to ICSI.
Sperm preparation and intracytoplasmic sperm injection (ICSI)
The spermatozoa were obtained from sperm donors and male partners of patients attending our ART centre. In both cases, semen analyses were normal and a prior consent was obtained. The sperm samples were washed with Ca2+-free medium and retrieved by side migration (Dozortsev et al., 1996) in a dish containing either the regular or Ca2+-free HECM-3 Hepes medium according to the protocol. Care was taken to keep the duration of this final pre-ICSI step as short as possible to avoid a confounding influence of Ca2+-free medium on spermatozoa. The procedure of ICSI was as described before (Goud et al., 1998a). Special care was taken to inject sibling oocytes alternately with spermatozoa either in the regular (1.9 mM Ca2+) or in the Ca2+-free injection medium. The oocyte holding media in all cases contained Ca2+. Therefore, excessive aspiration of the cytoplasm during ICSI was avoided to prevent entry of the oocyte holding medium into the oocyte alongside the injection pipette (Goud et al., 1997).
Pronuclear check, fixation procedure and staining
The oocytes were cultured in HECM-3 and checked for pronucleus (PN) formation at 12 h. The oocytes that formed male pronuclei were cultured further in the presence of 0.4 µg/ml colcemid and fixed after PN disappearance (Dyban et al., 1983; Goud et al., 1998a). The sperm chromosome metaphases were subjected to G-banding (Benet et al., 1986).
The pilot experiment
The pilot experiment was designed to note the occurrence of first cleavage after heterospecific ICSI of post-ovulatory young hamster oocytes. Oocytes were retrieved from enlarged antral follicles of superovulated hamsters at 11.5 h post-HCG, checked after 1 h culture and MII stage oocytes were injected with spermatozoa in regular (1.9 mM Ca2+) medium. The PN check was performed at 12 h post-ICSI and the resultant hamster–human heterospecific zygotes were further cultured for a period of 24 h to note the occurrence of PN disappearance and cleavage. The PN stage arrested heterospecific zygotes were fixed and processed for fluoresecent in-situ hybridization (FISH) using probes specific for human chromosomes X, Y and 1 (Harper et al., 1994; Laverge et al., 1997).
Statistical analysis
The results were analysed by the Wilcoxon paired rank-sum test (Siegel, 1956) for the comparison of the sibling oocytes (Prism Graphpad software, San Diego, CA, USA) and the Fisher's exact test for 2x2 contingency table for the non-sibling oocytes.
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Results |
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Follicular oocytes
In total, 201 oocytes were collected from the follicles at 11.5 h post-HCG. After culture for 1–2 h and removing the cumulus cells, 193 oocytes were found to be suitable for microinjection (MII stage with normal looking ooplasm). The rates of survival, MPN formation, metaphase entry and oocyte activation without MPN formation were similar in each oocyte subgroup injected with spermatozoa suspended in the regular (1.9 mM Ca2+) or the Ca2+-free injection medium. However, in comparison to the corresponding subgroups among the oviductal oocytes retrieved at 16 and 21 h, the rate of MPN formation in the regular subgroup and that of metaphase entry in both the subgroups among the follicular oocytes were significantly higher (P < 0.005). The rates of oocyte activation without MPN formation were higher in both the subgroups than their corresponding subgroups of oviductal oocytes obtained at 16 and 21 h (Table I
).
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Oviductal oocytes
Among the oocytes retrieved from the oviducts, 166, 243 and 220 oocytes were injected with spermatozoa suspended either in the regular (1.9 mM Ca2+) or the Ca2+-free injection medium in the 13, 16 and 21 h (post-HCG) groups respectively.
In the 13 h group, the rates of survival, MPN formation and metaphase entry in all the oocyte subgroups were similar to those in the follicular oocyte subgroups. Also, these rates were similar in the sibling oocyte subgroups injected with spermatozoa in the regular or the Ca2+-free injection medium. However, the rates of MPN formation and metaphase entry in each oocyte subgroup within the 13 h group was higher than the corresponding (regular or Ca2+-free) subgroups in the 16 and 21 h groups and the rates of oocyte activation without male PN formation in the regular subgroup were lower than those in the corresponding subgroups in the 16 h and 21 h groups (Table I
).
In the 16 h group, the rates of MPN formation and metaphase entry in both the subgroups were lower than the follicular as well as oviductal post-ovulatory young oocytes. The rates of oocyte activation without MPN formation were higher in both the subgroups than their corresponding subgroups in the follicular group and the regular subgroup in the 13 h oviductal group (Table I). However, between the sibling oocyte subgroups within the 16 h group, more oocytes injected with spermatozoa in Ca2+-free medium developed MPN and entered metaphase, when compared with the regular injection medium (Table I). The survival rates in the Ca2+-free subgroup were lower than those in the regular subgroup.
Among the oocytes retrieved at 21 h post-HCG and injected with spermatozoa, the rates of MPN formation and metaphase entry were lower than those in the corresponding subgroups in the 16 h group (Table I). However, among the sibling oocyte subgroups, significantly higher rates of MPN formation and metaphase entry were noted in the Ca2+-free compared to the regular subgroup. On the other hand, the rates of oocyte activation without MPN formation were similar in the subgroups injected with spermatozoa with the regular or the Ca2+-free medium. The oocyte survival rate was lower in the oocytes injected with spermatozoa in the Ca2+-free injection medium compared with the regular medium.
Thus, the mean rates of MPN formation and metaphase entry were the highest in post-ovulatory young oocytes from follicles and oviducts irrespective of the Ca2+ content in the injection medium. The rates of MPN formation and metaphase entry fell progressively with post-ovulatory ageing in the 16 and 21 h groups. On the other hand, the rate of oocytes undergoing activation without formation of the MPN increased with post-ovulatory age at 16 and 21 h groups (Table I). A difference in the rates of MPN formation and metaphase entry between the oocyte subgroups injected with the Ca2+-free and the regular injection medium was noted only in the 16 and the 21 h but not the post-ovulatory young follicular or oviductal oocytes. The rates of oocyte activation without MPN formation were higher in the oocyte subgroups injected with spermatozoa in the regular versus the Ca2+-free injection medium in the 16 h group and not in the 21 h group.
The number of sperm chromosome spreads followed the same pattern as the rates of metaphase entry with the highest rates among post-ovulatory young follicular and oviductal oocytes retrieved at 11.5 or 13 h after HCG. The yield of chromosome spreads fell progressively with post-ovulatory ageing and very few sperm chromosome spreads were obtained in the 21 h group (Figure 1). A chromosome metaphase spread obtained after heterospecific ICSI is depicted in Figure 2.
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In the pilot experiment, 40 MII stage oocytes retrieved from the follicles at 11.5 h were microinjected with spermatozoa. Male PN formation occurred in 25 oocytes. On further culture of these heterospecific zygotes, the pronuclei disappeared in 16 and 7 underwent the first cleavage (Figure 3
). None of the heterospecific zygotes cleaved beyond the 2-cell stage. Among the remaining nine heterospecific zygotes that had arrested in the PN stage, six were successfully processed for FISH. In each these six heterospecific zygotes, signals for human chromosomes X and 1 or Y and 1 were noted in one of the two pronuclei (Figure 4).
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Discussion |
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The application of ICSI to hamster oocytes enables the study of fertilization-related phenomena, e.g. cortical granule loss and chromatin decondensation (Lanzendorf et al., 1988
; Perrault et al., 1988; Yanagida et al., 1991; Hoshi et al., 1992). Likewise, a number of phenomena related to specific features of gametes may also be studied by purposely selecting such gametes during ICSI (Ogura and Yanagimachi, 1993; Lee et al., 1996; Rybouchkin et al., 1996). Post-ovulatory age and the presence or absence of Ca2+ were the two targets of our investigation for reducing the incidence of parthenogenetic activation and the effect of both these factors was evident from our study. Our results indicate that the rates of oocyte activation with MPN and without MPN have an opposite pattern of change related to post-ovulatory age. This confirms their inverse correlation to each other as shown in our previous study (Goud et al., 1998a).
Sensitivity of the oocytes to activation is known to increase with increasing post-ovulatory age (Fulton and Whittingham, 1978; Collas et al., 1989; Fissore and Robl, 1992; McConnel et al., 1995, Xu et al., 1997). Therefore, it is possible that the threshold for oocyte activation is relatively high in post-ovulatory young oocytes allowing oocyte activation only when a stimulus of sufficient strength, e.g. the fertilizing spermatozoon itself is present. On the other hand, these oocytes would be relatively resistant to activation in response to 1.9 mM Ca2+ in the injection medium. This hypothesis is strengthened by the fact that the rates of oocyte activation without MPN formation were similar and low and those of MPN formation and metaphase entry were similar and high in the subgroups of young oocytes injected with spermatozoa in the regular or the Ca2+-free medium.
At 16 h post-HCG, the threshold for oocyte activation was presumably lowered, compared with those retrieved at 11.5 h (follicular) and 13 h (oviductal). Thus, a fraction of oocytes from the 16 h group must have become sensitive to activation in response to the injection medium containing 1.9 mM Ca2+. In this fraction of the oocytes, formation of male PN could still occur when Ca2+ was omitted from the injection medium. This explains both the relatively higher rates of oocyte activation without MPN formation, and the beneficial effect of the Ca2+-free injection medium on the MPN formation in the 16 h group.
In the 21 h post-HCG group, the threshold for activation may have been reduced even further. Therefore, a larger fraction of oocytes underwent parthenogenetic activation in the 21 h group in comparison to other groups. However, the rates of oocyte activation without male PN formation were similar in both subgroups within the 21 h group. This may be due to the occurrence of spontaneous activation because of further post-ovulatory ageing of already old oocytes by the time of the PN check. However, although very low, the rate of MPN formation was still higher in the Ca2+-free subgroup compared with its counterpart, indicating that oocyte activation in response to the injection medium was prevented at least in a small fraction of oocytes allowing the male PN formation after ICSI.
Thus overall, our results are indicative of a gradual reduction in the oocyte threshold to activation stimuli with post-ovulatory age (Xu et al., 1997). The best rates of MPN formation and metaphase entry following ICSI occurred in the post-ovulatory young oocytes and dropped significantly even at 16 h post-HCG. Thus, hamster oocytes seem to have a rather narrow time window for optimum fertilization following ICSI (Ducibella, 1996). Therefore, for optimum results, ICSI should be performed only within this narrow time frame.
A decrease in the oocyte threshold for activation with increasing post-ovulatory age may be related either to an increase in the sensitivity of the Ca2+-release machinery or due to the decreased threshold of the cell cycle activation mechanisms in response to the Ca2+ release. Mammalian oocytes acquire the capability to activate during meiotic maturation (Kubiak, 1989; Fujiwara et al., 1993). This is related to the ultrastructural alterations in the endoplasmic reticulum and the redistribution as well as up-regulation of the inositol 1,4,5 trisphosphate receptor (IP3R) operated Ca2+ channels in the oocyte (Mehlmann et al., 1995, 1996; Shiraishi et al., 1995). Although this mechanism seems to explain how oocytes acquire activation capability during meiotic maturation, it is insufficient to explain why the oocyte activation threshold falls during post-ovulatory ageing. In fact, expression of IP3R may even decrease with post-ovulatory ageing as seen in the bovine oocytes (He et al., 1997). Thus, alteration in the IP3R expression seems to be an unlikely cause of increased oocyte activation sensitivity of post-ovulatory old oocytes. However, post-ovulatory ageing may lead to sensitization of the Ca2+-release mechanisms due to a dysfunctional Ca2+ regulation by the oocyte. The latter may be secondary to failure of the Ca2+ pump in the endoplasmic reticulum and may result in a defective membrane transport and accumulation of Ca2+ within the oocyte. Therefore, there may be a delay in return of the cytosolic Ca2+ levels to the baseline (Igarashi et al., 1997). These factors may result in sensitization of the IP3R which may at least partly contribute to the time-dependent change in oocyte sensitivity to activation stimuli.
One other factor may be related to the increase in the sensitivity of the cell cycle mechanisms to rise in cytosolic Ca2+ within the oocytes. In most mammalian species, the final stages of oocyte maturation are initiated in response to the luteinizing hormone (LH) surge and the oocyte undergoes germinal vesicle breakdown and polar body extrusion to rearrest at the MII stage awaiting fertilization. This stage is characterized by increased activity of M-phase promoting factor (MPF), which is a complex of p34cdc2 kinase and cyclin B. After fertilization, the oocyte exits from MII and enters into interphase. This metaphase–interphase transition is accompanied by a decrease in the MPF activity and also a subsequent decrease in mitogen-activated protein (MAP) kinase activity (Verlhac et al., 1994; Moos et al., 1995). However, prolonged delay prior to fertilization and residence in the oviducts results in changes of post-ovulatory ageing in these oocytes. These changes mainly consist of a time dependent progression of such oocytes into an interphase like state characterized by a decrease in the MPF as well as MAP kinase activities (Xu et al., 1997). Oocytes in such a partially activated state would be prone to activation with the slightest stimulus e.g. 1.9 mM Ca2+ in the injection medium. This altered response of the oocyte to ICSI with regular injection medium was augmented with post-ovulatory age in the16 and 21 h groups.
The timing and the duration of the chromatin decondensation activity in the oocyte is cell cycle-dependent (Usui and Yanagimachi, 1976). Thus, in an oocyte that progresses rapidly to interphase, the time for decondensation of the sperm chromatin may be too inadequate to allow complete remodelling of the sperm nucleus and its transformation into a pronucleus (Komar, 1982; Jedliki et al., 1986). This is evident from the slightly swollen and partially decondensed sperm nuclei seen in the oocytes that undergo oocyte activation with or without only female PN formation after ICSI (Goud et al., 1998a). The phenomenon of increased oocyte sensitivity to undergo activation without MPN formation was recently noted to occur also in human oocytes when ICSI was delayed after in-vitro maturation (Goud et al., 1998b).
Thus, the temporal window for optimum sperm chromatin decondensation and development to the first metaphase of hamster oocytes is limited to a short time following ovulation or maturation. Therefore, to obtain optimum results, one needs to inject the hamster oocytes within this period. Application of this technique will result in an optimal number of sperm karyotypes after heterospecific hamster oocyte ICSI. This reasoning may also be extended to other species including humans where performing insemination or ICSI within the narrow temporal window could be crucial for optimum fertilization and development (Ducibella, 1996; Goud et al., 1998b).
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Acknowledgments |
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The authors wish to sincerely thank Mrs Vera David for taking care of the animals and Mr Georges Van Maele for his advice regarding the statistics. The current study was supported by a grant awarded by the University of Ghent.
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1 To whom correspondence should be addressed
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References |
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Submitted on April 23, 1998; accepted on November 10, 1998.
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