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Knowledge Co-Creation - Profiles of researchers

Elucidating the Molecular Mechanism of Organisms’ Fertilization

Toshiyuki Mori
Assistant Professor, Waseda Institute for Advanced Study

A fascination for molecular and cellular biology

I went to university in the middle of the genetic research boom of the 1990s, a period when molecular biology was in the limelight. My interest in biology began in high school, and I was always more attracted by the microscopic world of cells than the macro world of ecosystems, so I too became fascinated with molecular biology and cytogenetics. For my graduation research, I had no hesitation in joining a cellular biology laboratory where I engaged in research into the molecules functioning in the generative cells of plants, the area of specialization of my supervisor Dr. Ichiro Tanaka (currently a professor at Yokohama City University). Taking the trumpet lily (Lilium longiflorum) as his main research material, Dr. Tanaka had been researching the process of pollen development and generative cell differentiation. During its development process, a pollen grain forms a generative cell within, and this generative cell differentiates into sperm cells by dividing into two (Figure 1).

Figure 1 Pollen development and typical fertilization process in plants
The two sperm cells formed by cell division during pollen development (top diagram) are delivered after pollination to an ovule where one fuses with an egg cell and the other with a central cell (bottom diagram). The fertilized egg becomes the embryo that will grow into a new plant, while the fertilized central cell develops into endosperm tissue (This is the tissue that nourishes the embryo such as the white grain in the rice plant).

At that time, Dr. Tanaka, who had already developed a technique for isolating generative cells from pollen grains, often said, “There must be something specific in generative cells concerned with reproduction.” But predictions of this something were very vague. On his suggestion to “search for a new gene expressed mainly in generative cells,” to find this something, I conducted my graduation research. This was the starting point of my research, since when I have been constantly researching the same theme with the same methodology.

My initial research into several (non-specific) genes that I found was not very productive, but I did become rather skillful during the course of my work at discovering efficient methods of gene searching. I conducted my doctoral program at the University of Tokyo, where finally I found a new gene that is expressed exclusively in pollen generative cells, which I named GCS1 (GENERATIVE CELL SPECIFIC 1, a gene that is specific to pollen generative cells)*. I found that the product of the GCS1 gene, that is, GCS1 protein, was a molecule bound to the cell membrane and, as expected, localized on the surface of pollen generative cells and sperm cells (Figure 2).
*In biological protocol, gene names are written in italics (GCS1) and protein names in ordinary characters (GCS1).

Figure 2 Observation of GCS1 protein expression
When the fusion protein of GCS1 and GFP (green fluorescence protein), i.e. GCS1-GFP, was expressed in Arabidopsis, GCS1 was found to be localized on the surface of sperm cells. The left picture shows a luminous GCS1-GFP molecule inside a pollen grain. As the diagram on the right shows, both sperm cells express GCS1.

First discovery in the world of a protein that controls fertilization

Next, to find out how GCS1 works, I conducted a function analysis with Arabidopsis, a plant widely used in experiments. When I investigated the effect on reproduction by GCS1 gene mutation, I found that seed formation after fertilization was dramatically reduced by half compared with wild strains. The fact that this single gene mutation led to such an obvious effect on seed formation meant the GCS1 molecule should play an important role in fertilization. Experiments in which mutated strains were crossed with wild strains also confirmed that the cause of the halved seed numbers was on the male side (pollen side).

So why is seed production inhibited? The reason itself reflects the direct role of GCS1. When the behavior of GCS1 mutant sperm cells (gcs1 sperm cells) labeled with red fluorescent protein was traced, I found that sperm cells inside an ovule were excluded to the outer edge of the egg cell (Figure 3). In other words, gcs1 sperm cells are unable to fuse with egg cell or central cell. This observation data was conclusive proof that the GCS1 protein is required for the fusion of sperm cells with female gametes (egg and central cell), that is, fertilization.

Figure 3 Inhibition of fertilization in an Arabidopsis GCS1 mutant
Using the pollen from a GCS1 mutant, I pollinated a strain whose egg cell membrane was GFP-labeled, and then observed the inside of the ovule. The gcs1 sperm cells had been labeled beforehand with red fluorescent protein, or RFP, so their behavior could be traced. As a result, I observed the gcs1 sperm cells coming very close but being unable to fuse with the egg cell. The left picture shows an entire ovule into which gcs1 sperm cells have entered. The right picture is a magnification of the area indicated by an arrow in the left picture, showing that none of gcs1 sperm cells could fertilize with the egg cell.

In 2006 I organized these results in my thesis and announced them in the international science journal Nature Cell Biology. It was the first discovery worldwide of a gene involved in the fertilization of plants, so it was even reported by a number of mainstream newspapers and gained spotlight. I had by this time completed my PhD and become a postdoctoral researcher. How difficult and time-consuming is it to achieve a significant result from a step-by-step search? Having thought I would be no good as a researcher unless I had succeeded by the age of 30, I was fortunate enough to be able to announce my results two months before turning 31.

Expression also found in malarial parasites

Another interesting thing about GCS1 is that its expression can be seen not only in plants but also in protists (algae, amoebae, malarial plasmodia, etc.) and animals. Although it is not present in mammals or fungi, we do know that arthropods such as insects, cnidarians such as sea anemones, and porifers all have the GCS1 homologs. This has led people worldwide to think that GCS1 may have been, in the very distant past, a factor in the fertilization of eukaryotes, the common ancestors of plants, animals, and protists.

Happily, I received a request from Dr. Makoto Hirai (then Assistant Professor on the Department of Infection and Immunity at Jichi Medical University), who had read the newspaper announcements in 2006 and wanted to study malarial plasmodia GCS1. An effective vaccine to eliminate malaria has yet to be found, and the disease continues to claim more than a million human lives every year. Dr. Hirai proposed “a plan to eradicate malarial plasmodia by targeting its GCS1 to inhibit their fertilization.” As a result of our joint research we quickly started, it was revealed that GCS1 is also expressed in the malaria parasite sperm and that it controls fertilization in the same way as in plants.

At that time, competing researchers from overseas were also engaged in similar research, which meant there was stiff competition with the announcement of results. Even over the New Year’s holiday I was writing up the manuscript at home, and in 2008 I somehow managed to have it published in the prestigious American academic journal Current Biology. This has raised hopes for the eradication of not only malarial plasmodia but also many other protozoa that have the GCS1 gene.

Searching for the next fertilization factors following gcs1

In recent years, there has been increased competition worldwide to find the next fertilization factors after gcs1. Since gcs1 is present on the male side, it must have a partner on the corresponding female side, and factors connected to gcs1 could still possibly be found on the male side. Of course I am also conducting research in these areas. Most of these are still confidential, but when I joined a team of overseas and other Japanese researchers a few years ago in the search for a new fertilization related gene, only I was fortunate enough to be able to locate one possible candidate, of which I am now performing a vigorous analysis.

I have a lot of ideas for research that I’d like to do. Gcs1 is a completely new protein and so its functional structure has not been discovered. How does gcs1 work on a molecular level? It is vital that we identify its detailed function, such as whether it plays a role in breaking the membrane on the female side. I aim to continue researching the phenomenon of fertilization from a variety of angles.

Toshiyuki Mori
Assistant Professor, Waseda Institute for Advanced Study

Graduated from the Faculty of Liberal Arts and Science, Yokohama City University in 1998 and completed his master’s program in the Graduate School of Integrated Science, Yokohama City University in 2000, before going on to gain a PhD (Doctor of Science) in 2003 from the School of Science, the University of Tokyo. Having been a postdoctoral fellow of the Japan Society for the Promotion of Science (at Rikkyo University), special postdoctoral researcher at RIKEN, and researcher at Advanced Science Institute of RIKEN, he took up his current post in 2011.
He received the Botanical Society of Japan’s Botanical Society Award for Young Scientists in 2006, the Japanese Society of Plant Morphology’s Hirase Award in 2006, and the Young Scientists’ Prize, the Commendation for Science and Technology by the Minister of Education, Culture, Sports, Science and Technology in 2013.