Research Interests

Fertilization not only rescues a sperm and an egg from assured death (if they don't unite they will die), but in doing so it generates a cell able to produce an entire organism: it is totipotent (Fig. 1). The goal of the Perry laboratory is to explain how this transformation occurs.

 

The egg-to-embryo transition is rapid and nearly 100% efficient, and although fundamental it is poorly understood in any species.

 

We are addressing this gap in our knowledge using the mouse as a model system; the mouse was selected because its genetics and embryology place it superbly to contribute to human biomedical applications.

 

The egg-to-embryo transition integrates different intracellular process, so if we wish to understand how the transition is achieved, we need to understand each of them. Two are now listed, with the methods we use to study them and potential applications of this work.

Cell cycle resumption

 

The unfertilized egg is like an athlete on the blocks awaiting the sound of the starting pistol. This is effectively because the cell is arrested in the cell cycle - the internal engine that drives it.

 

The signal to start the race is supplied by the sperm during fertilization; it causes the egg cell cycle to resume and changes the activities of multiple pathways.

 

We showed in 2006 that mouse eggs contain a protein, Emi2, that is necessary to arrest the cell cycle and prevent it from racing ahead to begin development without a sperm (a process called parthenogenesis that in mammals is doomed - parthenogenetic embryos naturally die early in development). We are working out the molecular details of how Emi2 causes this arrest and is inactivated during fertilization and how, if at all, this is related to other pathways that are critical in the egg-to-embryo transition.

 

Embryo chromatin building and remodelling

 

Chromatin describes the combination of genetic material - DNA - and all of its accessories, especially proteins. Rather than being decorative fashion items, these accessories are critical to the function of DNA, dictating which genes are active at any given time, and which are not. One unusual feature of fertilization is that the proteins associated with a large portion of the DNA (the chromosomes contributed by the sperm) are almost entirely removed and replaced by new ones from the egg (Fig. 2). Chromosomes from the maternal side that were already in the egg are also altered, but not as extensively.

 

What this all adds up to is that sperm and egg chromatin is dismantled or modified during fertilization and replaced with new chromatin that can function in a totipotent embryo. We are working to discover precisely what chromatin changes take place and how. Our hypothesis is that the changes are archetypal: all increases in cellular potency (the range of commitment options open to a given cell) borrow from some of the chromatin changes occurring during fertilization. We argue that this has implications for multiple processes, including carcinogenesis and induced pluripotency, discussed below.

 A fundamental transformation establishes totipotency, but its nature remains obscure. We would like a better understanding of the mechanisms that drive the transformation ("What happens here?"). Such an understanding is expected to facilitate the safe manipulation of cellular processes involved in biological regeneration.

 Mouse eggs that have been injected with a sperm head. a. Hoffman modulation microscopy 6h and b, immunofluorescence following sperm head injection at the times indicated, showing DNA (left) and the chromatin protein, histone H1. Black arrowheads show where the chromosomes lay, just under the egg surface; white arrowheads show uneven chromatin colonization by H1. The paternal genome is highly depleted of protein after 40 min. Bar = 5 um.

Application of our work

 

Major technological advances are apparently not possible without some minimal understanding of the underlying natural processes; the jet engine is one example. The natural process that increases cellular potency most radically - from death to totipotency - occurs safely and highly efficiently when sperm and egg combine at fertilization. If we understood which molecular processes and entities were critical in the egg-to-embryo transition, we might be able to introduce them - or some of them - to turn other specialized cells such as skin cells into embryo-like cells.

 

This is part of the dream of regenerative medicine. For example, a diabetic patient with no functional pancreatic beta cells might one day donate some skin cells that may be caused to produce embryo-like cells using approaches based on our work; new pancreatic cells can then be derived from these embryo-like cells so that they can be transplanted back to the diabetic patient with less fear of rejection, since they donated the original cells. It must be emphasized that as of 2011 this is a distant dream, but we think that the dream will not be realized without a sound model of the establishment of totipotency. Today, no such model exists.

 

Methods

 

Ours is one of the few labs anywhere to combine molecular, cellular and embryological methods to study early mammalian embryos. A unique type of micromanipulation called piezo-actuated micromanipulation ('piezo') is indispensable for delivering relatively large cargoes (eg sperm heads) into mouse eggs, minimizing cellular trauma and allowing >95% survival; conventional injection typically kills the eggs and is impracticable. Piezo permits synchronized injection of living or 'dead' sperm, co-injection of multiple sample types, such siRNA, cRNA or protein plus sperm. It has been applied to genetic modification such as transgenesis and all somatic cell nuclear transfer cloned mice bar one have been generated with piezo. These and multiple other applications of piezo are routine in our laboratory.

 

The experimental manipulation of sperm, eggs and embryos in this way is critical if we are to discover how the egg-to-embryo transition is achieved. We can determine the outcome of this manipulation in a number of ways. Image analysis of transgenic fluorescent marker proteins or antibodies against wild-type proteins is by laser scanning confocal and other fluorescence microscopy and can reveal details of molecular architecture in the nanometer range. It can also be performed on living mouse embryos, that develop normally for the first 3.5 days or so. Ultimately, we can even determine whether the manipulation interferes with the birth of healthy offspring following embryo transfer.

Figure 1.