A Tribute to the Xenopus laevis Oocyte and Egg

Donald D. Brown Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21210 Address correspondence to: brown{at}ciwemb.edu .

Next Section When I was asked to reflect as part of the celebration of the 100-year anniversary of the Journal of Biological Chemistry I recalled a talk by Seymour Cohen at the Federation meetings in Atlantic City about 1960. He began by thanking bacteriophage for providing him with so many wonderful research problems. I decided in the same spirit to pay homage to the Xenopus laevis egg and oocyte, two states of a cell that has played and continues to play a central role in most of the disciplines of modern biology including biochemistry and molecular biology. Many of us owe a debt of gratitude to this cell. The egg is the most important and interesting cell in the repertoire of any organism. In X. laevis this single cell has a diameter of about 1.3 mm. The oocyte is permeable to small molecules, but after it undergoes meiosis and becomes an unfertilized egg it is impermeable to these same molecules. The oocyte is an active site of RNA and protein synthesis but not DNA replication, whereas the unfertilized egg is poised to replicate every 10 min after it is fertilized. During the cleavage period the embryo is transcriptionally silent. An oocyte can be cultured for days, but once ovulated the egg degenerates rapidly if it is not fertilized. The oocyte is the largest single cell in the body yet it has many of the same structures as somatic cells. These compartments are so exaggerated in size that they are accessible to cell biologists for manipulation and visualization. The huge oocyte nucleus is called a germinal vesicle (GV). The premeiotic tetraploid chromosomes are expanded into a “lampbrush” configuration so that they can be studied with a light microscope. The X. laevis egg and its possibilities for experimental manipulation were introduced to modern biology in the late 1950s by John Gurdon (Fig. 1), then a graduate student in the laboratory of Michail Fischberg in the Department of Zoology at Oxford University. Gurdon's thesis was to reproduce with the South African “clawed toad” X. laevis the famous nuclear transplantation experiments that Briggs and King had perfected with the American leopard frog, Rana pipiens (1). Embryonic nuclei were injected into eggs whose own nuclei had been removed or destroyed. These early cloning experiments addressed the question of whether a differentiated and specialized somatic cell nucleus was still capable of expressing its full genetic repertoire. Briggs and King found that nuclei from R. pipiens embryos older than gastrula never promoted normal development, suggesting that the genetic material might change in cells as they specialize during embryonic development. This result was challenged by John Gurdon's demonstration that nuclei from X. laevis embryos are more permissive. Even a small fraction of nuclei derived from differentiated intestinal epithelial cells supported normal development (2). This crucial scientific question of whether irreversible changes occur in the genome of highly specialized cells was addressed more precisely by experimental manipulation than by biochemistry or genetics. The concepts behind these experiments have influenced the stem cell field. The topic of whether the genome is altered in specialized somatic cells was revisited just this year by the same strategy of nuclear transplantation. Postmitotic nuclei from olfactory neurons support the development of normal fertile mice when transplanted into enucleated mouse eggs (3). View larger version: Download as PowerPoint Slide F ig . 1. John B. Gurdon. Photo taken in 2000.

Previous Section Next Section Control of Genes Expressed in Oocytes A developing amphibian embryo requires no organic nutrients from its rearing medium. The entire development from the single-cell egg to a feeding tadpole of hundreds of thousands of cells occurs using materials stored in the egg. The unique composition of the oocyte and egg has led to many important discoveries that invariably can be traced to this storage function. In the 1950s and early 1960s there were reports that eggs of many species had much more DNA than expected for a single cell. The first person to investigate this by isolating and characterizing the “egg DNA” from R. pipiens and X. laevis was Igor Dawid (Fig. 2). He came to the Department of Embryology of the Carnegie Institution of Washington in Baltimore in 1963 as an independent investigator after completing his postdoctoral training in biochemistry at MIT. Dawid found that an egg contains orders of magnitude more high molecular DNA than the amount expected of a single cell (4). Using the new DNA hybridization technology that had been developed at the Department of Terrestrial Magnetism of the Carnegie Institution (5), Dawid showed that egg DNA is a select group of sequences rather than representative of the entire frog genome. The first reports had appeared that some cytoplasmic organelles contain DNA, and Dawid demonstrated that the extra egg DNA is mitochondrial DNA (6). Because a single X. laevis egg cell has the same abundance of mitochondria as about 100,000 somatic cells the quantity of mitochondrial DNA exceeds the amount of nuclear DNA in an X. laevis egg by several hundred-fold. These discoveries led to the realization that an individual's mitochondria are maternally inherited. View larger version: Download as PowerPoint Slide F ig . 2. Igor B. Dawid. Photo taken in 1999. In addition to mitochondria each mature X. laevis oocyte accumulates the same amount of ribosomes as hundreds of thousands of somatic cells. With the discovery of mRNA in 1960 it became clear that RNA was the direct gene product, and therefore changes in RNA synthesis, not protein content, better reflect gene expression. In 1963 Yankofsky and Spiegelman (7) demonstrated that the two large ribosomal RNAs (rRNA) in Escherichia coli were encoded separately in the bacterial DNA. Because they are so abundant in eukaryotic cells these two rRNAs became the first gene products that could be studied in animal cells. Jim Darnell (8) found that the two large rRNAs were derived from the same high molecular weight precursor. The question of how a single cell, the oocyte, could synthesize the RNAs for so many ribosomes became a focus of my own interest in differential gene expression for a decade. By the time that I graduated from medical school I had decided that ultimately I would study how embryos developed. The field was related to medicine but completely unexplored. Following 1 year of internship I had the good fortune to be chosen by Seymour Kety, who directed the laboratory on schizophrenic research at the National Institute of Mental Health, to be in the first class of “research associates” at the National Institutes of Health. This marvelous program that was designed to train newly minted M.D.s in research provided me with 2 years of experience learning biochemistry. Before leaving NIH for an exciting year in the Monod laboratory at the Pasteur Institute I needed to find a laboratory where I could begin my future studies in embryology. Entirely by accident I discovered a small research unit located on the campus of the Johns Hopkins Medical School in Baltimore whose faculty studied embryonic development. The Department of Embryology of the Carnegie Institution of Washington had specialized for 50 years in the descriptive anatomy of human embryos and reproductive biology. I arrived there as an independent fellow in the fall of 1960 indoctrinated with the new operon model of gene expression control determined to study the biochemistry of development. Frog eggs were large and plentiful, and they develop synchronously after fertilization. They seemed perfect for biochemistry. In 1962 I read about a mutation in the frog X. laevis that had been discovered in the Fischberg laboratory (9). This mutation altered the number of nucleoli. The homozygous embryos had no visible nucleoli and died at the time that a normal embryo begins to accumulate new ribosomes. Bob Perry (10) and Edstrom and Gall (11) had shown by cytochemical experiments that nucleoli contain high GC RNA characteristic of rRNA. In 1964 John Gurdon and I found that the homozygous anucleolate mutant of X. laevis cannot synthesize either the 18 or 28 S ribosomal RNA molecules (12). This discovery confirmed the nucleolus as the site of rRNA synthesis. The molecular explanation of the mutation came 2 years later from the work of Max Birnstiel (Fig. 3) who developed a method to isolate the rRNA genes (rDNA) from the bulk of the X. laevis genomic DNA (13). He reasoned from the known base composition of rRNA that rDNA should have a higher GC content than the average 40% GC genomic DNA of X. laevis. Sueoka, Marmur, and Doty (14) had established the relationship of GC content to the density of DNA when banded to equilibrium by centrifugation in CsCl. Wallace and Birnstiel (13) purified the high GC rDNA by repeated cycles of centrifugation. In doing so they demonstrated that the anucleolar mutant lacks rDNA and is therefore a deletion mutation. From the amount of radioactive rRNA that hybridized with genomic DNA they estimated that there are several hundred copies of 18 and 28 S RNA genes per haploid complement of X. laevis DNA. These repeated genes must be clustered because a deletion removes all of them. Birnstiel's purification of the X. laevis rDNA genes was the first isolation of a specific gene from any organism, and it occurred 10 years before cloning. Max Birnstiel (15) and I (16) have reviewed the research on purified genes that preceded the recombinant DNA era. X. laevis oocytes played a prominent role. View larger version: Download as PowerPoint Slide F ig . 3. Max L. Birnstiel. Photo taken in 2000. An oocyte is a tetraploid cell, but each X. laevis germinal vesicle contains about 1500 rather than the expected 4 nucleoli. At a 1966 meeting on the nucleolus in Montivideo, Oscar Miller showed his spectacular pictures of genes in the act of transcribing RNA. He had unraveled these “Christmas tree” structures from the nucleoli of X. laevis oocyte nuclei (17). Joe Gall (Fig. 4), who attended the same meeting, and I realized that these structures must be extra chromosomal tandemly repeated rRNA genes. Soon thereafter Igor Dawid and I (18) and Joe Gall (19) independently showed that the rRNA genes just like the nucleoli are present in a 1000-fold excess in oocyte GVs, a phenomenon that we named specific gene amplification. We had identified a novel regulatory mechanism by which a single cell could ramp up its synthesis of a normal cytoplasmic structure, the ribosome. In those days before cloning, X. laevis oocytes became a major source of purified rDNA to study rDNA structure (20). Joe Gall and Mary Lou Pardue (21) studied the early stages of rDNA gene amplification in immature oocytes by hybridizing radioactive rRNA to oocyte sections on slides. This method called “in situ hybridization” remains an essential tool of modern molecular biology. View larger version: Download as PowerPoint Slide F ig . 4. Joseph G. Gall. Photo taken in 1996. Following the discovery of rDNA gene amplification in oocytes it was logical to look into the genes that encode other RNA components of the ribosome. Along with one molecule of 18 and 28 S RNA there is a molecule of a 5.8 S and a 5 S RNA in each ribosome. Birnstiel (22) demonstrated that the 5.8 S RNA is cleaved from the same precursor as 18 and 28 S RNA, thus accounting for its stoichiometry in the ribosome. However, the 5 S RNA genes are not linked to the rDNA genes in X. laevis (23). Although there are several hundred rRNA genes in the X. laevis genome we found tens of thousands 5 S RNA genes arranged in tandem and distributed on many chromosomes. In fact 5 S DNA comprises 0.7% of the X. laevis genomic DNA. Its unusual nucleotide composition facilitated the purification of 5 S DNA by density gradient methods from X. laevis genomic DNA (24). The repeat length is a mere 700 base pairs. In the summer of 1973 I prepared 32P-labeled cRNA from genomic 5 S DNA by transcribing the separated strands with E. coli polymerase and then spent 2 months with George Brownlee in Cambridge helping him sequence the ribonuclease-generated 32P-labeled oligonucleotides by the two-dimensional separation method that George had developed with Fred Sanger. From these chromatograms we pieced together the essential features of a repeat of 5 S DNA (25). By 1970 it had been determined that the 5 S RNA stored in oocytes differs by several nucleotides from that found in somatic cell ribosomes (26). We found that each repeat of 5 S DNA has one oocyte-specific 5 S RNA gene, one partial gene that George named a “pseudogene,” and an AT-rich spacer. Each spacer consists of varying numbers of a degenerate AT-rich 15-base pair nucleotide repeat. Nina Fedoroff in collaboration with the Brownlee laboratory subsequently sequenced an entire repeat from genomic 5 S DNA (27, 28). This was the first time that a full-length eukaryotic gene had been sequenced. Subsequently we isolated two other families of 5 S RNA genes from the genomic DNA of X. laevis, one of which was the much smaller somatic 5 S RNA gene family (29). The “dual” 5 S RNA gene system is another mechanism by which the oocyte synthesizes large amounts of a ribosomal component. The thousands of oocyte 5 S RNA genes are active in growing oocytes so that the ribosomes in oocytes contain oocyte-specific 5 S RNA. When embryogenesis begins the amplified rDNA genes are lost and the oocyte 5 S DNA is inactivated. At gastrulation the chromosomal rDNA genes and the smaller gene family encoding the somatic 5 S RNA genes begin to be expressed. In somatic cells the rate of ribosome synthesis correlates with the rate of protein synthesis. In growing oocytes most ribosomes are stored as monosomes for later use during embryogenesis. 5 S RNA accumulates in oocytes stored in cytoplasmic ribonucleoprotein particles before ribosome assembly. One of the proteins in this particle is none other than TFIIIA, the 5 S DNA-specific transcription factor that is stored in large amounts in oocytes (30). I will discuss TFIIIA later in this article.

Previous Section Next Section The Use of Oocytes for mRNA Translation In 1969 a 9 S RNA was isolated from rabbit reticulocytes and deduced to be the mRNA for globin (31). A cell-free lysate prepared by Jerry Lingrel synthesized recognizable globin for short incubation periods before the extract activity died. This was the first identification of a eukaryotic mRNA. In 1971 John Gurdon and his colleagues injected globin mRNA into the cytoplasm of X. laevis oocytes and demonstrated the synthesis of rabbit globin (32). The advantages of this in vivo method to assay protein synthesis were immediately clear. The cultured living oocyte synthesizes foreign proteins for days rather than minutes, and the protein accumulates in the oocyte cytoplasm. The ease with which the large oocyte can be injected and then cultured brought this in vivo translation assay to the attention of the burgeoning field of molecular biology. Injection of mRNA into oocytes has provided a successful method to clone genes from complex mixtures of mRNAs. In addition to efficient translation the protein product is transported to its expected location in the cell and then functions correctly. An early example of the power of oocyte molecular biology was its use in interferon research. When poly(A)+ RNA from cultured mammalian cells activated to produce interferon was injected into X. laevis oocytes 500 times greater titers of the active molecule were synthesized and secreted than by cultured cell extracts as judged by a sensitive bioassay based on the antiviral effects of interferon (33). Charles Weissmann and his colleagues (34) fractionated poly(A)+ RNA by size on a sucrose gradient and cloned interferon cDNA from the active mRNA fraction assayed by injection into X. laevis oocytes. This was the first application of expression cloning. Enzymes encoded by rare mRNAs have been expression-cloned in X. laevis oocytes using their enzymatic reaction as an assay. An example is the cloning of deiodinase type 1 (35), a selenocysteine-containing protein from rat liver, by Reed Larsen and his colleagues. Douglas Melton developed the strategy of injecting synthetic mRNA (36) and then antisense oligonucleotides (37) into fertilized eggs as an assay for the influence of a gene on development. This alternative to traditional forward genetics has made X. laevis second only to Drosophila as a model system to identify genetic pathways in embryogenesis. The resulting embryos are analyzed in a number of ways to determine the effect of overproducing a gene product, mutations of that product, or antisense oligonucleotides. Many genes crucial to embryonic development have been cloned first from X. laevis because their function can be determined by these mRNA injection assays. An extension of expression cloning by Richard Harland (38) has used the effect on embryogenesis as an assay to identify genes that influence development. Recently the Harland laboratory (39) has developed a screening method to identify large numbers of genes important for development based upon the observable phenotype caused by their overexpression in a developing embryo.

Previous Section Next Section The Use of Oocytes in Cell Biology The injection of labeled proteins into the oocyte cytoplasm results in their transport to the site in the cell where they normally reside (40, 41). Newly translated secretory proteins from many different species cross intracellular membranes of the oocyte (42, 43). Once genes could be cloned and mutated synthetic mRNA was injected into oocyte cytoplasm to delimit the signals on a protein for secretion (44) and nuclear localization (45). The localization of this stored mRNA in the egg is controlled by sequences in the 3′-untranslated region of the mRNA that were identified by an oocyte injection assay (46). Neurophysiologists and biochemists have taken advantage of the X. laevis oocyte to clone and investigate receptors and ion channel proteins. The oocyte has some endogenous receptors. For example, it contains muscarinic acetylcholine receptors but no nicotinic receptors. Injection of mRNA from the electric organ of the ray (47) or skeletal muscle of the cat (48) produces functional nicotinic acetylcholine receptors. The multisubunit functional protein is assembled in oocytes, glycosylated correctly, and sequestered into the plasma membrane. Peter Agre (49) developed a striking assay for his Nobel Prize-winning discovery of a water channel gene. He prepared synthetic mRNA from the previously unknown cDNA, injected it into X. laevis oocytes, and then watched them swell and rupture.

Previous Section Next Section The Use of Oocytes for Transcription An early indication that X. laevis eggs would be rich in the molecules needed for RNA transcription was Bob Roeder's quantification of the three isoforms of eukaryotic RNA polymerase that he had previously discovered in sea urchin embryos. One X. laevis egg contains 4 orders of magnitude more of these RNA polymerases than does one somatic cell (50). In 1977 Mertz and Gurdon (51) demonstrated that SV40 DNA injected into X. laevis oocyte nuclei produced SV40 mRNA, and this mRNA was translated into recognizable SV40 proteins. We had purified the oocyte-specific 5 S DNA from X. laevis genomic DNA and had been studying its structure. This prompted John Gurdon and me to collaborate on another of our transatlantic experiments. I sent John the 5 S DNA. He injected the DNA into oocyte nuclei with a radioactive precursor and mailed the radioactive extract back to me for electrophoretic analysis. The newly synthesized 5 S RNA was accurately initiated and terminated (52). Then Ed Birkenmeier (53) prepared an extract from hand-isolated germinal vesicles that was competent to transcribe accurately added 5 S DNA. By that time repeating units of 5 S DNA had been cloned by the new recombinant DNA technology. Having a fully characterized cloned gene and an oocyte extract that faithfully transcribed it allowed us to identify the DNA-controlling regions that specify accurate initiation and termination of 5 S RNA synthesis. As Shigeru Sakonju and Dan Bogenhagen deleted the spacer regions adjacent to the 5 S gene we were astonished that the gene kept making 5 S RNA in our in vitro oocyte nuclear extract. When Shige deleted into the 5′-end of the gene's coding region and recloned the construct it continued to make 5 S-like RNA. Two of us independently sequenced this construct to confirm this remarkable fact. Before specific RNA synthesis stopped, about one-third of the coding region had been replaced with plasmid sequences (54, 55). Deletions from the 3′ end altered termination as soon as one of the four Ts that encode the Us at the 3′ end of the mature 5 S RNA had been removed (56). However, these constructs continued to initiate transcription correctly. This set of experiments delimited a region of about 50 base pairs within the coding region of the 5 S RNA gene that we named the “internal control region.” Meanwhile Bob Roeder and his colleagues (57) purified a protein from X. laevis oocytes that bound tightly to 5 S DNA and was required for accurate transcription. Our laboratories joined forces to show that this protein, which Roeder had named TFIIIA, complexed specifically with the internal control region of the 5 S DNA gene (58). Roeder's group (59) cloned the cDNA for TFIIIA from oocyte mRNA. Aaron Klug (60) recognized in the sequence 9 repeating peptide regions that he predicted must complex zinc. This was the discovery of “zinc finger” transcription factors. Active TFIIIA protein was purified easily for biochemistry because of its accumulation in huge amounts in X. laevis oocytes within the RNP particle. TFIIIA has the unusual feature of binding specifically to the 5 S RNA gene and 5 S RNA (30). The gene encoding TFIIIA is transcribed from a different start site in oocytes than it is in somatic cells, undoubtedly using one or more oocyte-specific transcription factors (yet another mechanism devised for enhanced ribosome synthesis). For many years the simple dual 5 S RNA gene system provided novel insights into the molecular aspects of differential gene expression (61). In 1980 Grosschedl and Birnstiel (62) delimited regulatory elements in the sea urchin histone H2A gene by injecting mutant constructs into the X. laevis oocyte. Steve McKnight (63) originated the linker scanning method to mutate systematically the promoter region and assayed the transcripts after injecting the DNA constructs into the oocyte nucleus. In the early days of recombinant DNA technology the X. laevis oocyte played an important role in the new “reverse” genetics or as we called it “genetics by DNA isolation.”

Previous Section Next Section The Use of X. laevis Eggs for Cell Cycle Research In 1971 Masui and Markert (64) and Smith and Ecker (65) found that cytoplasm from an oocyte that had been induced to undergo meiosis with progesterone could induce GV breakdown in an untreated oocyte. The factor was named maturation promoting factor or MPF. Its identity and how it acted catalytically remained a mystery for decades, but it ushered in a new career for X. laevis oocytes and eggs, which was their role in unraveling the intricacies of the cell cycle. An oocyte is arrested in prophase of its first meiotic division and has not undergone DNA synthesis for months, yet hours after completing meiosis and fertilization it divides every 10 min throughout its cleavage stages. Laskey and Gurdon (66) found that eggs but not oocytes could replicate injected double-stranded DNA. The realization that eggs have stored nuclear components including a huge excess of histones (67, 68) led to the development of X. laevis egg extracts for the assembly of chromatin (69) and nuclei (70). X. laevis egg extracts initiate and complete efficient semiconservative DNA replication (71). Progress on the molecular details of the cell cycle including DNA replication and chromatin, nuclear, and mitotic spindle assembly in the past 10 years has been remarkable due in no small degree to the X. laevis egg and oocyte.

Previous Section Next Section Lampbrush Chromosomes and the Oocyte Nucleus The GVs of amphibian oocytes have expanded chromosomes that resemble the brushes used to clean the dirt from lamps in the days before electricity. The larger the genome the greater is the size of the loops of these “lampbrush” chromosomes. Two scientists often working together over a period of decades have elucidated the structure of these huge chromosomes. Joe Gall and H. G. (Mick) Callan began their cytogenetic experiments over 50 years ago with salamander lampbrush chromosomes. In 1987 (72) they published the first physical maps of the X. laevis lampbrush chromosomes. With the accumulation of X. laevis sequence data it is possible that these chromosomes can be mapped with the degree of accuracy previously reserved for the giant polytene chromosomes of Drosophila. Joe Gall has demonstrated that the GV contains the same structures as somatic nuclei but so exaggerated in number and size that they are especially suited for analysis (73).