Non-invasive rapid time-lapse imaging combined with PIV analysis of cytoplasmic events after fertilization described here shows that sperm entry into the mouse egg triggers a series of unexpected rhythmic actomyosin contractions associated with cytoplasmic movements. At their maximum speed, these cytoplasmic movements have vectors directed towards the protrusion formed above the site of sperm entry that undergoes pulsations in synchrony with them. The speed peaks of the cytoplasmic movements correspond in timing with fertilization induced Ca2+ transients, and they depend on calcium oscillations. Finally, we show that analysis of the interval between peaks provides a novel non-invasive and very rapid way of assessing the vitality of the embryo and its ability to succeed in development.
The involvement of the actomyosin cytoskeleton in mediating cytoplasmic movements is evident from our findings that inhibition of MLCK or treatments that either stabilized or depolymerized F-actin diminished both speed peaks and the basal cytoplasmic speed. The movements also depend on Ca2+
transients, because they were prevented by chelating Ca2+
with BAPTA. One possibility is that Ca2+
-dependent kinases such as Ca2+
/calmodulin-dependent kinase II or conventional protein kinase C participate in triggering the movements as these enzymes are known both to regulate the cytoskeleton11
and respond to Ca2+
. However, whereas the amplitude of Ca2+
oscillations are similar throughout the whole post-fertilization period (except for the first Ca2+
spike that is usually bigger than subsequent ones31
), the amplitude of the speed peaks increases at onset of FC formation and decreases on FC regression. Moreover, the Ca2+
transients evoked by parthenogenetic activation are not sufficient to promote fast, oscillatory cytoplasmic movements suggesting that further fertilization-associated factors must enable the movements to take place.
The coincidence of speed peaks with actomyosin-mediated rhythmic movements of the FC suggests that these are manifestations of the same process. This is further borne out by studies of FC formation after sperm injection. As FC formation requires interaction of chromatin with cortical proteins35
, it does not occur when sperm is injected deep into the egg. Accordingly, eggs fertilized in this way showed much weaker speed peaks than in zygotes with FCs. The fact that speed peaks, although weak, were still present, could be because sperm chromatin was positioned close enough to the cell surface to cause some reorganization of the cortical actomyosin. Although insufficient to produce a well-defined FC, this would still be able to enhance weak cytoplasmic movements. Alternatively, the speed peaks could be triggered by contractions of the cortical actomyosin accumulated above the maternally derived chromatin. Indeed, we frequently could see that a bulge formed above the set of maternal chromosomes pulsates in a way similar to the FC. It is also possible that low-amplitude speed peaks may be an effect of general contractility of the actomyosin cytoskeleton, not associated with the cortex. The last two possibilities could also explain why there are some low-amplitude speed peaks visible in the Sr2+
-activated parhenogenotes. High-speed movements were also not triggered, and FC motions were very much diminished in Sr2+
-activated eggs injected with heat-inactivated sperm heads. Thus, the sperm may well contribute proteins that facilitate FC motions and cytoplasmic movements that are inactivated by heat treatment. It is also possible that differences observed between normal zygotes and embryos injected with inactivated sperm followed by Sr2+
activation may be due to the altered characteristics of the Ca2+
transients themselves. The dynamics of typical Sr2+
peaks differ from those of Ca2+
peaks triggered by fertilization39
. Moreover, frequency of the Sr2+
oscillations was very high under our conditions, and this seemed to affect negatively generation of high-amplitude speed peaks.
As the timing and pattern of cytoplasmic movements mirror these of Ca2+
oscillations and depend on the integrity of the cytoskeleton, we find that they provide a powerful indicator of the embryo quality. Our data shows that embryos with low mean basal speed (below 10 nm s−1
; indicates poor quality of the actomyosin cytoskeleton), as well as embryos with very frequent speed peaks (interpeak interval below 10 min; reflecting frequent Ca2+
transients), rarely develop to the blastocyst stage in vitro
or to full term in vivo
. This result accords with findings that both functional actin cytoskeleton and correct pattern of Ca2+
transients (especially the total time of Ca2+
elevation) are crucial for development13
. Unfortunately, these factors cannot be used in research and medical facilities to select good quality embryos as their assessment involves invasive procedures using fluorescent dyes and harmful irradiation. Thus, current practice in the in vitro
fertilization clinic is to assess the viability of embryos on day 3 from their morphology and growth at day 5, because of unreliability of the 3-day assessment44
. A considerable advance may be offered by imaging and monitoring of a series of parameters over the first two days of development47
. Our present findings suggest that, in future, it might be possible to assess the vitality of the embryo and its ability to succeed in development by using non-invasive imaging over a much shorter period of time, just 2 h after the time of fertilization.
In conclusion, we have identified the importance of the sperm in triggering a dynamic oscillatory behaviour of the actomyosin cytoskeleton at fertilization. The ensuing movements have a pattern and timing that provides a powerful method of assessing an egg's ability to achieve its full developmental potential. As such, our method has considerable potential in finding practical application in the assisted reproduction clinic.