Scientists have found the molecule that triggers the start of human life. John Parrington tells the story.
Scientists have found the molecule that triggers the start of human life. John Parrington tells the story.
In a well-known fairy tale a beautiful princess is woken from a deep sleep by a kiss from a handsome prince. While this may be the stuff of bedtime stories, scientists now believe that the real-life Sleeping Beauty might conceivably be the human egg. Until disturbed by the sperm, the female ovum lies effectively dormant, in a state of suspended animation in which all of its metabolic processes are switched off and the cycle of cell division is arrested. The prince among sperm that first encounters the quiescent egg acts as a wake-up call and begins embryo development. For over a century scientists have been trying to fathom how egg activation takes place. But only recently have we finally begun to identify the molecular components that mediate this fundamental event.
The first person to study egg activation was physiologist Jacques Loeb. He believed that all living phenomena would ultimately be explained in terms of physical chemistry. This was a bold and controversial idea at a time when many scientists still believed that life must involve some kind of mysterious ’vital source’ that could not be reduced to mere molecules.
Loeb’s beliefs led him to try and trigger embryo development by artificial means. He achieved this in 1899 when, working at the Wood’s Hole Marine Biology Laboratory in the US, he showed that sea-urchin eggs could be activated to develop merely by changing the chemical composition of the sea water that bathed them, and without the need for a sperm. Loeb’s discovery made him a celebrity, prompting newspaper headlines such as ’Scientist nears secret of life’. Even though the experiments were carried out on sea urchins, this did not stop speculation that it would soon be possible to create human beings in this way.
Loeb was ahead of his time: it was 70 years later before sperm was shown to induce egg activation by triggering the release of one particular element: calcium.
Calcium’s role as a cellular messenger is vitally important in processes like muscle contraction, hormone secretion and the transmission of messages in the brain. Changes in calcium concentration inside a cell are translated into cellular responses by proteins that ’sense’ these changes and modify their activity as a result. This was first realised in 1974 when researchers, led by Rick Steinhardt and Ted Chambers, triggered activity in sea-urchin eggs with a divalent ionophore, a chemical that selectively allows release of calcium.
But by far the most convincing demonstration of calcium’s central role in egg activation came in 1977 when Lionel Jaffe and his team injected aequorin, a jellyfish protein that glows in the presence of calcium, into a fish egg. This allowed them to visualise directly changes in calcium concentration within the egg. What they saw was quite spectacular: when the sperm fuses with the egg, a wave of calcium spreads through it like a forest fire. We now know calcium is released from the egg’s own internal stores.
Since then, similarly dramatic calcium ’signals’ have been observed in eggs from many more organisms. In mammals, the pattern of calcium release is particularly interesting. After the first calcium wave comes a series of further surges, often referred to as ’calcium oscillations’. After each oscillation, calcium is pumped back into the internal stores, which are then ready for the next cue to release their contents. Although aequorin is still used, most scientists studying calcium signals now use fluorescent indicator probes, which glow much more brightly.
The demonstration of the central role of calcium signalling was a major step forward in our understanding of egg activation. But it left one important question unanswered - the identity of the molecular components that trigger the calcium signal. Until recently, that question remained unsolved and bitterly controversial. Initially, it was thought that the activating species was a protein on the outer membrane of the sperm, which would activate a receptor on the egg’s surface. The receptor would then trigger the release of calcium from the egg’s internal stores. This seemed a plausible idea since hormones, the chemical messengers in our blood, transmit signals by attaching to receptor proteins on the surface membranes of cells. Yet problems in identifying such a transmitter in eggs led some to doubt the theory.
Others argued that egg activation was not triggered by an interaction between proteins at the egg surface but rather by a soluble ’sperm factor’, released into the egg after fusion. In 1990 Karl Swann at St George’s Hospital Medical School, London, provided evidence for such an entity. He showed that an extract of hamster sperm injected into a hamster egg caused calcium oscillations identical to those seen at fertilisation. The sperm factor appeared to be some kind of protein.
While it was one thing to show the existence of the sperm factor, identifying it turned out to be far from straightforward. Together with biochemist Tony Lai, who was based at the National Institute for Medical Research in London, Swann and I spent several years trying to isolate the sperm factor without success.
One problem was the nature of the assay that we were using. Micro-injection of mouse eggs is far from trivial and is very laborious, so we turned to a so-called ’cell-free’ assay: the sea urchin egg homogenate. Developed by Hon Cheung Lee of the University of Minnesota, US, the homogenate consists of mashed sea-urchin eggs in which the internal calcium stores remain intact enough to allow the different calcium signalling pathways to be explored.
Working at University College London, with new team member Keith Jones, we were able to use the homogenate assay to find one of the first important clues about the identity of the sperm factor protein. The factor had similar properties to a family of proteins called phospholipase Cs (PLCs), which were already known to be involved in calcium signalling. Eleven variants of PLC were known but, despite looking extensively, we could not prove that one was the sperm factor. And while PLCs are found in most cells of the body, the sperm factor appeared to be a completely new type, peculiar to sperm.
In the end, the human genome project helped us. In particular, a new DNA technology called Expressed Sequence Tags (ESTs) has made it possible to identify novel signalling proteins that are found only in particular tissues. EST technology works because, although our cells all contain the same genes, those which are turned on or ’expressed’ vary between different parts of the body.
An EST database reflects only those genes that are expressed. So each EST can be traced to the tissue from which it was isolated. New genes, specific to particular functions, can be identified. When Lai heard that we had shown the sperm factor to be a potentially novel PLC found only in sperm, he looked to mice EST databases to find such an entity. He identified a new isoform: PLC zeta, which only appeared in ESTs derived from testes. When we injected PLC zeta protein into a mouse egg it triggered calcium oscillations identical to those seen at fertilisation. The egg also began to develop into an embryo as if it had been fertilised.
These findings were published in the journal Development in 2002, more than a decade after the quest for the sperm factor had first begun.
With the discovery of PLC zeta, scientists can now address a whole series of questions. So far PLC zeta has only been discovered in mice, men and other mammals. What remains to be demonstrated is whether similar proteins are involved in triggering egg activation across the animal kingdom. There have been intriguing hints that this is so. Most recently my laboratory has shown that sperm extracts from tilapia, a common farmed fish, have PLC activity similar to that found in mammals. Remarkably, PLC zeta from tilapia triggers calcium oscillations when injected into mouse eggs, suggesting the possibility of a universal mechanism for egg activation.
Studying PLC zeta in mice could improve our understanding of how infertility arises in humans. Currently infertility affects more than 10 per cent of couples. Some forms of male infertility are thought to be due to the inability of sperm to activate the egg. Could this be because their PLC zeta is mutated in some way? Or could it be due to a failure in manufacturing PLC zeta properly in the sperm? Either way, the discovery of PLC zeta may eventually lead to new ways to diagnose and even treat infertility.
John Parrington is a lecturer in the department of pharmacology at the University of Oxford.
- P. J. Pauly, Controlling life: Jacques Loeb and the engineering ideal in biology. New York: OUP, 1987.
- D. Gerhold and C.T. Caskey, Bioessays, 1996, 18, 973.
- J. Parrington, J. Andrology, 2001, 22, 159.
- C. M. Saunders et al, Development, 2002, 129, 3533.
- K. Coward et al, Biochem. Biophys. Res. Comm., 2003, 305, 299.
- John Parrington’s group at Oxford university is currently engineering mice whose sperm contains the phospholipase C (PLC) zeta protein ’tagged’ with a green fluorescent marker. This will let them follow PLC synthesis and determine where it is localised within the sperm. This is crucial in determining exactly what happens to it during fertilisation.
- Sperm themselves are very sensitive to calcium, which regulates the finely coordinated events leading to fertilisation. Early activation of PLC zeta could therefore be catastrophic.
- A similar approach could reveal how PLC zeta works once it is inside the egg, and tell us about the mechanisms that regulate its action. Researchers have known for some years that the calcium oscillations triggered at fertilisation stop after several hours and start again just as the fertilised egg is about to divide.
- This raises the question: what is turning the oscillations off and on?
- John Carroll and his team from University College London suggest an answer: the trigger for the calcium oscillations must be trapped inside the nucleus.Therefore once the nucleus is formed, the egg’s calcium stores cannot be instructed to release their contents.
- To test this idea, Carroll and his team treated a mouse egg with a drug that prevents proteins from entering the nucleus. When the egg was fertilised the oscillations continued, instead of stopping as they would normally. So it does look as if the source of the oscillations is being sequestered by the nucleus. It will be interesting to see whether a fluorescently tagged PLC zeta also behaves in such a way.