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In this article we will discuss about:- 1. Meaning of Meiosis 2. Occurrence of Meiosis 3. Genetics 4. The Spindle Apparatus 5. Forces Required for Chromosome Movement 6. Regulation of the Cell Cycle.
Meaning of Meiosis:
The division which takes place in cells of the germ line is called meiosis (Fig. 6.2). It results in four products of a parent cell with half the amount of genetic material because DNA is duplicated only once and there are two cell divisions, meiosis I and II.
Prophase I is of very long duration and consists of five sub-stages. Leptotene shows very long, thin thread-like chromosomes. Zygotene marks the initiation of pairing of homologous chromosomes. By some force of attraction identical partners are drawn towards each other.
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Condensation and shortening of chromosomes is visible. By pachytene pairing is complete and stabilized. Due to the intimate nature of pairing or synapsis and continued shortening of chromosomes, thick ribbon-like bivalents are formed. Pairing is exact so that chromomeres and centromeres of homologues lie against each other.
The number and arrangement of beadlike chromomeres, position of centromeres and arm length are distinct for each bivalent and allow mapping of pachytene chromosomes. The nuclear membrane and nucleolus disappear. At diplotene a force of repulsion between the paired chromosomes tends to draw them apart.
They are held together at positions of chiasmata where genetic crossing over or exchange of segments takes place. Continued condensation of chromosomes through diakinesis gives rise to the short thick cross-shaped configurations of chromosomes.
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By Metaphase I maximum condensation has been achieved giving rise to short rod-like bivalents which move towards the centre of the cell and align in the equatorial plane. Their centromeres become attached to spindle fibres running to opposite poles and homologues separate. Due to separation of homologues, half the number of chromosomes will reach one pole and one half of the other pole. Consequently Metaphase I is referred to as the reductional division.
The beginning of separation of homologous is also the beginning of Anaphase I. When an entire set of chromosomes reaches either pole we call it Telophase. The nuclear membrane and nucleolus are reorganised to form two daughter nuclei. The newly formed nuclei go through a short rest period or interkinesis before entering the second meiotic division.
Prophase II of meiosis is initiated by condensation of chromosomes and is completed rapidly as in mitosis. Metaphase II shows the equatorial alignment of rod like chromosomes which have reached their maximum limit of contraction.
The centromere of each chromosome now divides longitudinally. The daughter centromeres are attached to spindle fibres and separate to the poles as in mitosis. Thus in meiosis, metaphase II is an equational division.
Anaphase II and telophase II are completed as in meiosis I and in mitosis. At the end of meiosis II four products are formed each with half the amount (haploid) of genetic material that was contained in the original parent nucleus.
Occurrence of Meiosis:
In sexually reproducing diploid plants meiosis takes place in the anther and the ovule. One or more specialised cells of the germ line known as archesporium divide mitotically to produce the sporogenous cells. In the case of male, sporogenous cells multiply and increase in number. After a certain stage they stop dividing, are ready to enter meiosis and are called microspore mother cells.
The four products of meiosis are united in a tetrad but later separate as uninucleate microspores. Thickenings deposited on the microspore wall produce uninucleate pollen grain which soon becomes bi-nucleate and a large vegetative cell and a small generative cell are organised.
In many plants pollen grains are shed in this stage. The nucleus of the generative cell divides usually after pollen germination has begun on the stigma resulting in two male gametes. One male gamete fertilises the egg to form a zygote: the second male gamete fuses with the secondary nucleus in the embryo sac and gives rise to the endosperm which provides nutrition to the growing embryo.
Similarly in the case of female, the sporogenous cell in the ovule enlarges to become a megaspore mother cell which divides meiotically to form a tetrad of four megaspores. Usually three megaspores degenerate and one enlarges into the embryo sac. By three mitotic divisions the nucleus of the megaspore forms 8 nuclei (haploid) of which one organises as the egg.
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In the male in higher animals (mammals and man) the spermatogonial cells in the testis increase in number by mitosis. When they are ready to divide meiotically they are called primary spermatocytes. During meiosis II they are called secondary spermatocytes. After meiosis they organise into the elongated spermatids which finally produce sperms after undergoing some changes (head, middle piece and tail are formed; some histone proteins are changed; motility is acquired).
In the female of mammalian species including humans, and in contrast to the males meiosis takes place during the embryonal life of an individual. During the first few months after conception in human beings, the primary oocytes in the foetal ovary start undergoing meiosis.
After meiosis I the primary oocyte produces a secondary oocyte which alone will produce the functional ovum, and a small first polar body. The polar body gives rise to two more polar bodies: eventually all three polar bodies degenerate.
A unique feature of ovarian meiosis is that it stops at about metaphase II stage in the secondary oocyte. In this condition the secondary oocytes (about 400 in number) remain suspended for 40 to 50 years of life after birth. At puberty ovulation starts when one oocyte gets released from the ovary wall each time and is discarded.
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If an oocyte gets a chance to meet a sperm, then before fertilisation it completes the remaining stages of meiosis (anaphase II to telophase II). Out of the four resulting cells three are polar bodies which will degenerate; the remaining cell enlarges and functions as ovum during fertilisation.
Genetics of Meiosis:
There are two special features of meiosis: production of haploid gametes containing recombined genetic material of the two parents; the process of genetic exchange and recombination in homologues. Genetic events in meiosis have also been studied from mutants. Mutations affecting meiosis lead to abnormalities in genetic recombination or in chromosome segregation.
Many such mutants have been isolated in the lower eukaryotes like yeast, Neurospora, and a few higher plants. The work on yeast mutants has shown that recombination is regulated at two levels: control of overall frequency of crossing over; and controls which influence the frequency of crossing over only in particular regions. Some mutations are known to affect the subsequent stages of chromosome segregation. There are also mutations which suppress initiation of meiosis.
Detailed studies of meiotic mutants have been carried out in Drosophila. Meiosis is unusual in normal Drosophila males due to absence of crossing over. The females show crossovers in all chromosome pairs except number IV. Mutations in male meiosis affect chromosome segregation. One such effect is nondisjunction due to which chromosomes fail to disjoin and move to opposite poles either at meiosis I or II.
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Most meiotic mutants of D. melanogaster interfere with meiosis I either in male or in female, but not in both. This implies that control of meiosis I is different in male and in female flies. Some mutations affecting meiosis I in Drosophila males have been investigated. The mutant segregation distorter (SD) is due to a dominant gene on chromosome II.
The heterozygous males (SD/±) transmit the mutant SD gene to 50% of the progeny; the homozygous (SD/SD) males are sterile. Another mutant in males called recovery disrupter (RD) is due to an X-linked gene. It causes fragmentation of Y- bearing sperm resulting in excess of female flies in the progeny.
A few more mutations in males such as mei-S8 and mei-081 cause nondisjunction at meiosis in Drosophila females such as c(3) G, and mei-218 either reduce recombination or interfere with chromosome segregation. Those that affect recombination also cause increased nondisjunction of all chromosomes.
The Spindle Apparatus of Meiosis:
Electron microscopy has shown that spindle fibres are made up of bundles of parallel filaments called microtubules. The microtubules are assembled from cytoplasmic proteins namely α and β tubulin, and have an outside diameter of 24 nm, a central lumen of 15 nm across, and variable length.
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In cross section a microtubule appears circular in outline, the circle itself being composed of about 13 smaller circles. These small circles represent cross sections of long strands called protofilaments which are assembled from the globular protein subunits, the α and β tubulin present in the cytoplasm. One molecule each of α and β tubulin become associated to form a dimer.
The dimers are arranged in linear order to form protofilaments (Fig. 6.3). Each dimer appears to have a specific binding site for colchicine and another for vinblastine, both of which inhibit spindle formation by preventing the assembly of microtubules.
The microtubules are supposed to be present in the form of a cytoplasmic network in a resting cell. During cell division they become organised as spindle fibres. They are disassembled after cell division. It seems that for normal mitosis there must be a state of equilibrium between microtubules and free subunits of tubulin present in the cytoplasm. Low temperatures disturb the equilibrium and dissociate microtubules.
The alkaloid colchicine obtained from the corms of a liliaceous plant Colchicum autumnale binds to tubulin and prevents formation of spindle fibres. Due to the resulting failure of chromosome movement, cell division becomes arrested at metaphase. Such cells may either degenerate, or the duplicated chromosomes may form a nucleus which is polyploid.
The effect of colchicine can be reversed by depriving the cells of colchicine. A few other chemicals such as nitrous oxide, acenaphthene, chloral hydrate, vinblastine and podophyllotoxin have the same effect as colchicine.
Forces Required for Chromosome Movement in Meiosis:
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The forces powering chromosome movement have not been understood. A variety of different molecular motors have been identified in a wide variety of species. All of the motors thought to be involved in chromosome movement are microtubule motors that include some kinesin-related proteins and cytoplasmic dynein.
Kinesin and motor proteins dynein move along microtubules in opposite directions, kinesin toward the plus end and dynein toward the minus end. Kinesin trans-locates along microtubules in only a single direction, toward the plus end. The kinesin molecule is 380 kDa in weight, consists of two heavy chains of 120 kDa each, and two light chains, 64 kDa each.
The heavy chains have long a-helical regions wound around each other in coiled coil manner. The amino-terminal globular head domains of the heavy chains are the motor domains of the molecule. They bind to both microtubules and ATP, the hydrolysis of ATP providing the energy required for movement.
The tail portion of the kinesin molecule consists of the light chains in association with the carboxy-terminal domains of the heavy chains. This portion of kinesin is responsible for binding to other cell components, such as organelles and membranous vesicles that are transported along microtubules by the action of kinesin motors.
Dynein is a very large molecule, up to 2000 kDa, consisting of two or three heavy chains, complexed with various light polypeptides. In dynein too, the heavy chains form globular ATP-binding motor domains that are responsible for movement along microtubules. The light chains of this molecule bind to organelles and membranous vesicles. All members of the dynein family move toward the minus ends of microtubules.
Regulation of the Cell Cycle in Meiosis:
Most eukaryotic organisms duplicate cells by following a more or less similar cell cycle. Since diverse organisms follow a similar pattern for cell duplication, it implies that the cell cycle is under genetic control. Disruption of the genetic controls leads to uncontrolled cell proliferation, as seen in malignancy.
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Regulation of the cell division cycle involves extracellular signals from the environment as well as internal signals that exert their effect on processes during different cell cycle phases (G1, S, G2 and M). In addition, cellular processes such as cell growth, DNA replication and cell division must be coordinated for progression of cell cycle. This is accomplished by a number of control points that check and regulate progression through the different phases of cell cycle.
An important cell cycle regulatory point occurs late in phase G1 and controls progression from G1 to S. This regulatory point was first found in yeast (Saccharomyces cerevisiae), where it is referred to as START (Fig. 6.4). After passing START, cells become committed to go through one cell division cycle. In yeast, the passage through START is controlled by external signals such as availability of nutrients.
If there is shortage of nutrients, yeast cells become arrested at START and enter a non-dividing resting stage. START is also the point at which cell growth is coordinated with DNA replication and cell division. Budding yeasts which produce progeny cells of different sizes, cell size is monitored by a control mechanism. Accordingly, each cell must reach a minimum size before it can pass through START.
In eukaryotes, cell proliferation is regulated at the G1 phase of cell cycle called restriction point. In contrast to yeasts, the passage of mammalian cells through cell cycle is regulated by extracellular growth factors that signal cell proliferation, instead of availability of nutrients. When the appropriate growth factor is present, cells pass the restriction point and enter S phase.
Once it has passed through the restriction point, the cell becomes committed to proceed through S phase and complete the cell cycle, even in absence of further growth factor stimulation. Progression through cell cycle stops at the restriction point if appropriate growth factors are not available in G1.
Thus cells become arrested at a quiescent stage called G0 (G zero). Cells in G0 are metabolically active, but have reduced rates of protein synthesis. It has been noted that many animal cells remain in G0 unless induced to proliferate by appropriate growth factors or other extracellular signals. A good example is afforded by skin fibroblasts that remain arrested in G0.
When they are required to repair damage resulting from a wound injury, they are stimulated to divide. The proliferation of fibroblasts is signalled by platelet-derived growth factor released from blood platelets during clotting.
An example of cell cycle control in G2 is provided by vertebrate oocytes. Oocytes can remain arrested in G2 for very long periods of time, until they are triggered by hormonal stimulation to proceed to M phase. In human female, oocytes become arrested in G2 during fetal life for several decades until stimulated to complete the meiotic cell cycle.
Mechanisms for Regulation of Cell Cycle:
Studies on molecular mechanisms that control the progression of mammalian cells through the division cycle have revealed that the cell cycle is controlled by a set of protein kinases which are responsible for triggering the major cell cycle transitions.
Cdc2 and Cyclin:
Studies on frog oocytes indicated that the oocytes that are arrested in G2 phase of cell cycle could be stimulated to enter into the M phase of meiosis by hormonal stimulation. Later investigations showed that oocytes arrested in G2 could be induced to enter M phase by microinjection of cytoplasm taken from oocytes that had been hormonally stimulated.
Thus, a cytoplasmic factor present in hormonally stimulated oocytes would allow oocytes that had not been exposed to hormone, to progress from G2 to M. This cytoplasmic factor was called maturation promoting factor (MPF). Further studies showed that MPF is not specific to oocytes, but appeared to act as a general regulator of the transition from G2 to M in somatic cells as well.
Studies on temperature-sensitive mutants of yeast Saccharomyces cerevisiae that were defective in cell cycle progression also contributed to understanding of regulation of cell cycle. These mutants called cdc (cell division cycle mutants) were remarkable as they showed growth arrest at specific points in the cell cycle.
For example, the mutant designated cdc28 replicates normally at the permissive temperature, but at the non-permissive temperature there is arrest of cell cycle at START, thus indicating that the cdc28 protein is necessary for passage of cells through the regulatory point START in G1.
Further investigations on cdc2 brought to light two important points:
First, that cdc2 encodes a protein kinase, indicating the role of protein phosphorylation in cell cycle regulation.
Second, a gene related to cdc2 was identified in humans and shown to function in yeasts, implying that the cell cycle regulatory activity of this gene is conserved.
Studies on protein synthesis in sea urchin embryos provided further insights into cell cycle regulation. After fertilisation, sea urchin embryos go through rapid cell divisions. However, the entry of embryonic cells into M phase requires new protein synthesis.
Two proteins were then identified that accumulate during interphase and are degraded at the end of each mitosis. These proteins were called cyclin A and cyclin B. It seemed that cyclins might be able to induce mitosis by controlling entry and exit from M phase. It was then demonstrated that cyclins control G2 to M transition.
Subsequent investigations on MPF, the regulator of cell cycle gave interesting results when it was shown that MPF is composed of two subunits, cdc2 and cyclin B. Cyclin B is required for the catalytic activity of the cdc2 protein kinase. Thus, MPF activity is controlled by the periodic accumulation and degradation of cyclin B during cell cycle progression.
Further studies have demonstrated regulation of MPF by phosphorylation and dephosphorylation of Cdc2 protein. Cyclin B synthesis takes place in S phase, and it forms complexes with Cdc2 protein throughout S and G2.
During this time, Cdc2 is phosphorylated at two regulatory positions, one on threonine-161 (required for Cdc2 kinase activity), the other of tyrosine-15 catalysed by a protein kinase called Weel that inhibits Cdc2 activity resulting in accumulation of inactive Cdc2/cyclin B complexes during S and G2. The transition from G2 to M takes place by activation of the Cdc2/cyclin B complex brought about by dephosphorylation of threonine- 14 and tyrosine-15 by a protein phosphatase called Cdc25.
After activation the Cdc2 protein kinase phosphorylates a variety of target proteins for entry into M phase. Furthermore, Cdc2 activity also stimulates degradation of cyclin B by ubiquitin-mediated proteolysis. Degradation of cyclin B inactivates Cdc2, causing the cell to exit mitosis, undergo cytokinesis and enter interphase.
The Cyclins:
The characterisation of the Cdc2/cyclin complex have provided insights into the regulation of the cell cycle. Both Cdc2 and cyclin B are found to be members of large families of related proteins, with different members of these families controlling distinct phases of the cell cycle.
In the case of yeasts, Cdc2 controls passage through START and entry into mitosis. By associating with distinct cyclins, Cdc2 is able to phosphorylate different substrate proteins required during specific phases of the cell cycle.
In higher eukaryotes, cell cycles are controlled not only by multiple cyclins, but also by multiple Cdc2-related protein kinases. Referred to as Cdks (cyclin-dependent kinases). The original member of this family, Cdc2, is known as Cdk 1 with others following up to Cdk8. Members of the Cdk family associate with specific cyclins to accomplish progression through different stages of the cell cycle.
Briefly, progression from G1 to S is regulated by Cdk2 and Cdk4 in association with cyclins D and E; Cdk4 and Cdk6 control progression through restriction point in G1 in association with cyclins D1, D2, and D3; Cdk2 and cyclin E complexes are required for G1 to S transition as well as initiation of DNA synthesis in S; Cdk2 with cyclin A control progression of cells through S phase; Cdc2 complexed with cyclin B plays a role in transition from G2 to M.
The activity of Cdks during cell cycle is regulated by at least 4 molecular mechanisms. The first level of regulation involves formation of Cdk/cyclin complexes. Second, activation of Cdk/cyclin complex by phosphorylation of a Cdc threonine residue at position 160. The third involves inhibitory phosphorylation of tyrosine residues near the Cdk amino terminus.
Fourth, binding of inhibitory proteins called Cdk inhibitors or Ckls to Cdk/cyclin complexes. The combined effects of these multiple mechanisms of Cdk regulation accomplish control of cell cycle progression in response to both checkpoint controls and to the large number of extracellular stimuli that regulate cell proliferation.
The D-Type Cyclins:
The proliferation of animal cells is regulated by a variety of extracellular growth factors that control progression of cells through the restriction point in late Gl. In absence of growth factors, cells are not able to progress through the restriction point, become quiescent or enter G0. When stimulated by growth factor, the cells can re-enter the cell cycle. The study of D-type cyclins has provided an important link between growth factor signalling and cell cycle progression.
Cyclin-D synthesis is induced in response to growth factor stimulation and continues as long as growth factors are present. Growth factors must necessarily be present through G1 to allow complexes of Cdk 4, 6/cyclin D to drive cells through the restriction point.
But if growth factors are removed prior to G1, the concentration of D-type cyclins falls rapidly. The cells are then unable to pass through G1 and S, become quiescent and enter G0. Thus, D-type cyclins play a role in growth factors control of progression of cells through G1.
The insights gained on growth factors and cyclin D led to a most important finding that defects in cyclin D regulation contribute to the loss of growth regulation that is characteristic of cancer cells. Indeed, many human cancers have been found to arise as a result of defects in cell cycle regulation, whereas many other cancers result from abnormalities in the intracellular signalling pathways activated by growth factor receptors.
Therefore, mutations which result in the continuous unregulated expression of cyclin D1 lead to development of several human cancers, including lymphomas and breast cancers. Furthermore, a key substrate protein of Cdk4, 6/cyclin D complexes is found to be frequently mutated in a variety of human tumors.
This protein is called Rb since it was first identified as the product of a gene responsible for retinoblastoma, a childhood eye tumor. It was later found that if Rb is rendered nonfunctional due to mutation, it could result in a variety of human cancers, besides retinoblastoma. Rb seems to be the prototype of a tumour suppressor gene, a gene whose inactivation leads to development of tumors.
Subsequent investigations have revealed that Rb plays a significant role in coupling the cell cycle machinery to the expression of genes required for cell cycle progression and DNA synthesis. Rb’s function is regulated by changes in its phosphorylation as cells traverse the cell cycle. For example, Rb becomes phosphorylated by Cdk 4, 6/cyclin D complexes as cells pass through the restriction point in G1.
When Rb is under phosphorylated (present in G0 or early G1), Rb binds to members of the E2F family of transcription factors, which regulate expression of several genes involved in cell cycle progression, such as gene encoding cyclin E. Details of Rb’s action indicate that Rb acts as a molecular switch that converts E2F from a repressor to an activator of genes required for cell cycle progression.
Inhibitors of Cell Cycle:
Agents that cause damage to DNA result in arrest of cell cycle. Cell contacts between cancerous cells in culture arrest division (contact inhibition). A number of extracellular factors inhibit cell division, frequently mediated by regulators of the cell cycle machinery, usually by induction of Cdk inhibitors. For example, arrest of cell cycle in response to DNA damage is mediated by protein p53.
Protein p53 a transcriptional regulator that stimulates expression of the Cdk inhibitor known as p21. The p21 inhibits several Cdk/cyclin complexes. Following DNA damage, p53 induces synthesis of p21 and brings about cell cycle arrest. Protein p21 can also directly inhibit DNA replication.
A well characterised extracellular inhibitor of animal cell proliferation is the polypeptide factor TGF-β, that inhibits the proliferation of a variety of types of epithelial cells by arresting cell cycle progression in G1. This action of TGF-β is mediated by induction of Cdk inhibitor p 15 which blocks Cdk4 activity. Then Rb is not phosphorylated and the cell cycle becomes arrested in G1.
Checkpoints in Cell Cycle:
The coordination between the different phases of the cell cycle depends on a system of checkpoints as well as feedback controls that prevent entry into the next phase of the cell cycle until events of the previous phase have been completed. To ensure that incomplete or damaged chromosomes are not replicated and passed on into daughter cells, there are several cell cycle checkpoints.
One well defined checkpoint occurs in G2 which prevents initiation of mitosis until DNA replication is completed. The G2 checkpoint senses un-replicated DNA, then generates a signal that leads to cell cycle arrest. Thus the G2 checkpoint prevents the initiation of M phase before completion of S phase. The cells remain in G2 until the entire genome has been replicated.
The checkpoint then releases inhibition of G2, allowing the cell to progress into mitosis, and completely replicated chromosomes are passed on into daughter cells. Progression into the cell cycle is also arrested at the G2 checkpoint in response to DNA damage caused by radiation. In this case, arrest in G2 allows time for repair of damage.
DNA damage can also arrest cell cycle progression at a checkpoint in G1, that allows repair of damage to take place before the cell enters S phase. Thus, replication of damaged DNA is prevented. In eukaryotes, arrest at the G1 checkpoint is mediated by the action of a protein called p53 which is rapidly induced in response to damaged DNA (Fig. 6.5). The gene encoding p53 is frequently mutated in human cancers.
Mutations in this gene lead to loss of function of p53 that prevents G1 arrest, in response to DNA damage. Thus, damaged DNA is replicated and passed into daughter cells without being repaired. Through damaged DNA the daughter cells inherit an increased frequency of mutations and instability of the genome which contribute to cancer development. Mutations in p53 gene are most common genetic alterations in human cancers.
The third cell cycle checkpoint occurs toward the end of mitosis. This checkpoint ensures alignment of chromosomes on the mitotic spindle, so that a complete set of chromosomes is distributed into the daughter cells.
If one or more chromosomes fail to align correctly on the spindle fibres, the checkpoint leads to arrest at metaphase. The chromosomes do not separate at the equator until a complete complement of chromosomes has been organised for accurate distribution to the two poles.
