Frog Oocytes and Early Embryos Facilitate Biochemical Characterization of the Cell Cycle Machinery

Biochemical studies require the preparation of cell extracts from many cells. For biochemical studies of the cell cycle, the eggs and early embryos of amphibians and marine invertebrates are particularly suitable. These organisms typically have large eggs, and fertilization is followed by multiple synchronous cell cycles. By isolating large numbers of eggs from females and fertilizing them simultaneously by addition of sperm (or by treating them in ways that mimic fertilization), researchers can obtain extracts from cells at specific points in the cell cycle for analysis of proteins and enzymatic activities.

To understand how X. laevis oocytes and eggs can be used for the analysis of cell cycle progression, we must first lay out the events of oocyte maturation, which can be recapitulated in vitro. So far, we have discussed mitotic division. Oocytes, however, undergo a meiotic division (see Figure 19-35 for an overview of meiosis). As oocytes develop in the frog ovary, they replicate their DNA and become arrested in G2 for 8 months, during which time they grow in size to a diameter of 1 mm, stockpiling all the materials needed for the multiple cell divisions of the early embryo. When stimulated by a male, an adult female’s ovarian cells secrete the steroid hormone progesterone, which induces the G2-arrested oocytes to mature and enter meiosis. As we will see in Section 19.8, meiosis consists of two consecutive chromosome segregation phases known as meiosis I and meiosis II. Progesterone triggers oocytes to undergo meiosis I and progress to the second meiotic metaphase, where they arrest and await fertilization (Figure 19-5). At this stage the cells are called eggs. When fertilized by sperm, the egg nucleus is released from its metaphase II arrest and completes meiosis. The resulting haploid egg nucleus then fuses with the haploid sperm nucleus, producing a diploid zygote nucleus. DNA replication follows, and the first mitotic division of embryogenesis begins. The resulting embryonic cells then proceed through 11 more rapid, synchronous cell cycles, generating a hollow sphere of cells called the blastula. Cell division then slows, and subsequent divisions are non-synchronous, with cells at different positions in the blastula dividing at different times.

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FIGURE 19-5 Progesterone stimulates maturation of Xenopus oocytes. (a) Step 1: Progesterone treatment of G2-arrested Xenopus oocytes surgically removed from the ovary of an adult female causes the oocytes to enter meiosis I. Two pairs of synapsed homologous chromosomes (blue) connected to meiotic spindle microtubules (green) are shown schematically to represent cells in metaphase of meiosis I. Step 2: Segregation of homologous chromosomes and a highly asymmetric cell division expels half the chromosomes into a small cell called the first polar body. The oocyte immediately commences meiosis II and arrests in metaphase II to yield an egg. Two chromosomes connected to spindle microtubules are shown schematically to represent egg cells arrested in metaphase of meiosis II. Step 3: Fertilization by sperm releases eggs from their metaphase arrest, allowing them to proceed through anaphase of meiosis II and undergo a second highly asymmetric cell division that expels one chromatid of each chromosome into a second polar body. The resulting haploid female pronucleus fuses with the haploid sperm pronucleus to produce a diploid zygote. Step 4: The zygote undergoes DNA replication and the first mitosis. Step 5: The first mitosis is followed by 11 more synchronous divisions to form a blastula. (b) Micrograph of Xenopus eggs.
[Part (b) © MICHEL DELARUE/ISM/Phototake.]

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The advantage of using X. laevis to study factors involved in mitosis is that large numbers of oocytes and eggs can be prepared that are all proceeding synchronously through the cell cycle events that follow progesterone treatment and fertilization. This makes it possible to prepare sufficient amounts of extract for biochemical experiments from cells that were all at the same point in the cell cycle. It was in this system that the cyclin-CDK complexes that trigger mitosis and the oscillatory nature of their activity were first discovered. This activity was called maturation-promoting factor (MPF) because of its ability to induce entry into meiosis and oocyte maturation when injected into G2-arrested oocytes.