1 Department of Anatomy and Developmental Biology and 2 Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK
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Abstract |
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Key words: calcium/fertilization/in vitro/mouse/oocyte
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Introduction |
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The trigger that stimulates the transformation of the oocyte into a developing embryo is a sperm-induced increase in intracellular Ca2+ at the time of fertilization. In mammals, the increase in Ca2+ originates from intracellular stores and takes the form of a series of Ca2+ oscillations that continue for 34 h (Cuthbertson and Cobbold, 1985; Miyazaki et al., 1986
; Kline and Kline, 1992
; Jones et al., 1995
). The repetitive nature of this signal is essential for complete oocyte activation. A single sperm-induced Ca2+ increase is insufficient to stimulate the completion of meiosis as indicated by the extrusion of the second polar body (Kline and Kline, 1992
; Ozil and Swann, 1995
). More recent studies indicate that multiple sperm-induced Ca2+ oscillations are necessary to ensure progression into the first embryonic cell cycle (Lawrence et al., 1998
). The requirement of repetitive Ca2+ transients at fertilization is most likely due to the need to stimulate and maintain the degradation of cyclin B and hence ensure exit from meiosis and entry into interphase of the first mitotic division (Collas et al., 1995
). Given the central role of sperm-induced Ca2+ transients in the initiation of early development, it is essential that the oocyte develops the capacity to respond to spermatozoa in such a manner.
The development of Ca2+ release mechanisms during oocyte maturation represents a major change in the physiology of the oocyte and ensures the appropriate response to the fertilizing spermatozoa. Ca2+ transients in response to spermatozoa, sperm factors, inositol trisphosphate and Ca2+ ionophores have all been shown to increase during oocyte maturation (for review see Carroll et al., 1996). A number of mechanisms have been proposed to explain these modifications, including an increase in the levels and changes in the regulation of the inositol trisphosphate receptor (InsP3 R) (Fujiwara et al., 1993; Mehlman and Kline, 1994; Jones et al., 1995
), changes in the structure of the endoplasmic reticulum (ER) (Mehlman et al., 1995), or changes in the size of the Ca2+ store itself (Carroll et al., 1994
; Mehlman and Kline, 1994; Jones et al., 1995
; Herbert et al., 1997
).
Although it is well established that Ca2+ signalling systems are modified during oocyte maturation, there is considerable discrepancy between studies as to the extent of these changes (Mehlman and Kline, 1994; Jones et al., 1995). In addition, it is not known when during oogenesis the developing oocyte becomes competent to undergo these maturation-associated changes or whether they are affected by the conditions in which the oocyte is matured. Oocytes recovered from juvenile mice provide an excellent model system to investigate these questions, as they provide a relatively homogeneous cohort of oocytes of increasing developmental capacity (Eppig and Schroeder, 1989
). Using this system, it has been established that the developmental competence of oocytes recovered from mice between the ages of 1624 days increases with the age of the oocyte donor (Eppig and Schroeder, 1989
). In order to determine if different patterns of Ca2+ signalling may account for these developmental differences, we have examined the ability of ovulated and in-vitro matured oocytes from mice of 19 and 24 days of age to generate Ca2+ transients in response to spermatozoa.
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Materials and methods |
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For the collection of ovulated oocytes mice were injected with 5 IU of human chorionic gonadotrophin (HCG) 4852 h after PMSG. In order to standardize the nomenclature for determining the age of oocyte donors, the age of the donors of mature eggs as the age at which HCG was administered was defined. As such the mice are 19 or 24 days old when the oocytes were recovered. The oviducts were removed 1416 h after HCG and the cumulus masses were released into M2 containing 0.3 mg hyaluronidase/ml. The cumulus-free oocytes were collected, washed three times in M2 and transferred to a drop of M2 under oil prior to IVF.
IVF and embryo culture
Insemination drops consisted of 100 µl drops of medium T6 containing 10 mg/ml of bovine serum albumin (BSA) (Fraction V, Sigma, Poole, Dorset, UK) under paraffin oil. Mice of proven fertility were killed by cervical dislocation and the epididimydes were removed into 1 ml of T6 medium in a Petri dish. Sterile needles were used to puncture each epididymis and release the spermatozoa. The dish was placed in the incubator for 15 min allowing time for the spermatozoa to swim into a suspension. This suspension was diluted 1:10 into a pre-equilibrated insemination drop. The diluted sperm suspension was incubated for a further 2 h before adding the in-vitro matured or ovulated oocytes. The oocytes were incubated with spermatozoa for 45 h before the oocytes were collected and washed in CZB medium (Chatot et al., 1989). Development to the 2-cell stage and beyond was performed in 30 µl drops of CZB medium covered with paraffin oil and maintained in the incubator at 37°C in 5% CO2. In order to support development beyond the morula stage, it was necessary to transfer morulae into CZB containing 5.5 mmol/l glucose on day 3 of culture (Chatot et al., 1990
). Throughout the culture period, development was assessed at 24h intervals for a total of 72h post-fertilization. At each assessment, the proportion of oocytes in the different stages of development was recorded.
Measurement of intracellular Ca2+
To monitor changes in the level of intracellular Ca2+, oocytes were loaded with the Ca2+-sensitive fluorescent dye, Fura-red (Molecular Probes, Eugene, OR, USA). For loading, oocytes were incubated in 2 µmol/l of Fura-red AM for 15 min at 37°C. To improve loading the medium also contained 0.05% pluronic F-127 (Molecular Probes) and to prevent extrusion and compartmentalization of the dye into organelles, 250 µmol/l of the anion pump inhibitor sulphinpyrazone was also included. After loading, the oocytes were removed from the Fura-red and maintained in M2 containing sulphinpyrazone. The zona pellucida was removed by a brief incubation in acidified Tyrode's medium (pH 2.5, Sigma). The oocytes were washed in M2 and transferred to a heated chamber containing 500 µl of M2 without BSA on the stage of a Nikon Diaphot microscope. A further 500 µl of M2 with BSA and sulphinpyrazone was added to the stage and the medium was covered with oil.
Fura-red was excited sequentially at 440 and 490 nm and the fluorescence was collected using a 20x0.75 NA (numerical aperture) objective. The emitted light was passed through a long pass filter (>510 nm) and the data collected using a Newcastle Photometrics Multipoint system.
Statistics
Comparisons of the proportions of oocytes developing to different stages of development were compared using a 2 test. For analysis of the Ca2+ transients the mean numbers of oscillations generated in each group were compared by Student's t-test and the proportions that continued to oscillate for 1 or 2 h by
2 test.
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Results |
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Effect of oocyte donor and in-vitro maturation on the ability to generate Ca2+ transients in response to spermatozoa
To investigate the possibility that oocytes with limited developmental capacity show an abnormal response to spermatozoa, we monitored intracellular Ca2+ in ovulated and in-vitro matured oocytes from 19- and 24-day old donors. Although oocytes from all groups were capable of generating Ca2+ oscillations in response to spermatozoa, it was clear that some conditions limited the ability to generate Ca2+ transients. Representative traces of fertilization-induced Ca2+ transients are shown for ovulated oocytes from young and old donors (Figure 2). In order to quantify different Ca2+ responses, the mean number of oscillations was measured in the first 2 h (Figure 3A
) and the proportion of oocytes that continued oscillating after 1 or 2 h (Figure 3B
). Oocytes from older donors generated significantly more oscillations in the first 2 h and continued to oscillate for longer than oocytes from younger donors (Figure 3A,B
). In oocytes from 24-day old donors, in-vitro maturation had no effect on the ability to generate Ca2+ transients in response to spermatozoa. In contrast, in-vitro maturation significantly decreased the number of Ca2+ transients generated in response to spermatozoa in oocytes from younger donors (Figure 3A,B
).
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Discussion |
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Acquisition of developmental competence in the final stages of oocyte growth
Our studies confirm previous observations that suggest a gradual acquisition of developmental competence during oocyte growth. We found that oocytes from 19-day old donors cleaved to the 2-cell stage 1015% less frequently than oocytes derived from 24-day old donors. However, the major difference was apparent in the relative abilities to develop to the blastocyst stage in vitro. Over 40% of ovulated oocytes from 24-day old females developed to the blastocyst stage compared with <10% for oocytes from 19-day old females. The idea that growing oocytes acquire the ability to progress through the developmental process is well established. Much of this work has been carried out using the juvenile mouse as a model system, where, similar to the present study, the most dramatic effect is the increased ability to develop from 2-cells to blastocysts in culture (Eppig and Schroeder, 1989; Eppig et al., 1994
). A major difference between our studies and those of Eppig and Schroeder (Eppig and Schroeder, 1989
) is that the donors in our studies were primed with PMSG prior to recovery of the immature oocytes. The finding that the developmental differences are maintained, despite priming with PMSG, suggests that hormonal stimulation of the follicular environment with exogenous gonadotrophins is not capable of improving the developmental competence of oocytes from young mice. This result would indicate that follicles falling into the smaller antral stages contain oocytes that cannot be forced into a developmentally viable state by treatment with gonadotrophins. This is supported by our findings that ovulated and in-vitro matured oocytes from the young donors show a similar low rate of development. Thus it appears that the factors responsible for improving developmental competence accumulate between 19 and 24 days gestation, equivalent to the final few days of follicular development in the mouse. Furthermore, it is apparent that these factors are not induced by hormonal priming and are therefore likely to represent oocyte-specific modifications required for early embryonic development.
Ability to generate Ca2+ transients in response to spermatozoa increases in the final stages of oocyte growth and during maturation
A number of oocyte-specific factors are likely to be required for implementing the developmental programme. One necessary contribution to the successful transition from oocyte to embryo is the ability to generate a physiological response to the fertilizing spermatozoon. Here we provide evidence that the failure of oocytes to generate Ca2+ transients in response to spermatozoa may be one of the contributing factors to the poor rates of development in embryos derived from young oocytes. This is suggested by the association of the ability to generate Ca2+ transients in response to spermatozoa and the developmental competence of the oocyte. For example, oocytes from 24-day old donors generate more Ca2+ transients and have a greater developmental capacity than oocytes from 19-day old donors. Support for the idea that Ca2+ transients at activation can influence preimplantation development has been accumulating in recent years. The most dramatic example of this is the finding that the frequency and strength of electric field pulses at the time of oocyte activation dramatically influence the rate of preimplantation development (Ozil, 1990). Other studies have shown that the ratio of inner cell mass to trophectoderm cells can be changed by different patterns of Ca2+ oscillations in the first cell cycle (Bos-Mikich et al., 1997
) and that the frequency of Ca2+ transients at activation influences the rate of implantation after embryo transfer (Swann and Ozil, 1994
). Although the mechanism of this effect of Ca2+ transients remains unclear, it has been demonstrated that Ca2+ increases can lead to quantitative and qualitative differences in mRNA species in developing embryos (Rout et al., 1997
). Further studies are required to determine a causal link between the ability to generate Ca2+ transients in growing oocytes and developmental competence.
Our studies clearly indicate that changes occur in growing oocytes that modify their capacity for releasing Ca2+. Previously it had been demonstrated that dramatic changes in Ca2+ release mechanisms occurred during oocyte maturation (Fujiwara et al., 1993; Carroll et al., 1994
; Mehlman and Kline, 1994; Jones et al., 1995
). Although all studies have described the same general phenomenon, a decrease in the capacity for Ca2+ signalling in immature oocytes, the effect of oocyte maturation on the ability to generate Ca2+ transients at fertilization was different in different laboratories. Mehlman and Kline (Mehlman and Kline, 1994) described smaller transients that persisted, while Jones et al. (Jones et al., 1995
) found a rapid damping of the Ca2+ transients such that only one to three transients were generated. Our revisit to this question confirms that Ca2+ signalling is modified during oocyte maturation and that oscillations do stop earlier in immature oocytes, although not as early as indicated in the study of Jones et al. We found that immature oocytes generated about half the number of oscillations in a 2 h time window and only 20% of immature (compared to 80% of mature) oocytes continued oscillating for >2 h. Explanations for the subtle differences experienced in different laboratories may come from the use of different Ca2+ indicators, different levels of illumination and different strains of mice. Although some quantitative differences are apparent, the present study and those described above still indicate that changes in the ability to generate Ca2+ transients in response to spermatozoa are initiated during oocyte growth and continue through oocyte maturation.
In-vitro maturation can affect the ability to generate Ca2+ transients in response to spermatozoa
Since the capacity to generate Ca2+ transients in response to spermatozoa increases during oocyte maturation, systems for in-vitro maturation must support this developmental modification. Our study illustrates that in-vitro maturation further reduces the ability of oocytes from young donors to generate Ca2+ transients in response to spermatozoa. This further decrease in the ability to release Ca2+ may explain the decreased rate of 2-cell formation compared with ovulated controls. As described in the Introduction, there is a strong relationship between Ca2+ oscillations and egg activation such that sufficient sperm-induced Ca2+ transients are necessary to ensure complete oocyte activation. The requirement for multiple transients is most likely due to the observation that cyclin B is continuously synthesized in the MII oocyte (Kubiak et al., 1993) with the result that maturation promoting factor levels remain high unless Ca2+-activated cyclin B degradation is maintained. In order to understand the limitations of in-vitro maturation, it will be necessary to elucidate the exact mechanisms responsible for the changes in Ca2+ signalling that occur during oocyte maturation. This knowledge may provide the necessary basis for developing systems for in-vitro maturation that support optimal development.
The findings in this study have implications for the treatment of human infertility. Our results indicate that during the final stages of oocyte growth and during residence in the antral follicle oocytes accumulate factors, or undergo modifications, necessary for preimplantation development. One of these modifications is the ability to respond fully to spermatozoa at the time of fertilization. There are a number of observations illustrating that a significant proportion of apparently unfertilized human oocytes have in fact been penetrated by spermatozoa but have not formed pronuclei (Schmiadi and Kentenich, 1989; Calafel et al., 1991
; Van Blerkom et al., 1994
). Our results suggest that one possible reason for this developmental arrest is the lack of maternal modifications necessary to generate sufficient Ca2+ oscillations in response to spermatozoa. It is also reasonable to conclude from our data that the use of in-vitro maturation systems in IVF programmes, where human oocytes also undergo maturation-associated changes in Ca2+ signalling (Herbert et al., 1997
), may limit the ability of oocytes to generate Ca2+ transients in response to spermatozoa.
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Acknowledgments |
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Notes |
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References |
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Submitted on November 18, 1999; accepted on February 25, 2000.