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PLNT 3140 Introductory Cytogenetics - 2024

Making Sense of Meiosis

Learning Objectives

Why Meiosis?

Meiosis is the physical basis of Mendel's laws of independent segregation and independent assortment. As we will see, it is  also an elaborate, metabolically costly, and sometimes faulty mode of reproduction. Considering the costs involved, what are the benefits?

A. Sexual reproduction creates genetic diversity

  1. Independent segregation of chromosomes

  2. Because each gamete receives chromosomes at random from one parent or the other,  there is a 50/50 chance of getting a given copy of any chromosome. Put another way, there are 2 possible outcomes for getting either the maternal or paternal chromosome. For a genome of n chromosomes, there are 2 n possible gametes from a single parent. For example, each human parent, with 23 chromosome pairs, can produce 2 23 =  8.4 x 10 6 possible gametes. Since each chromosome carries a unique combination of alleles for thousands of loci, progeny are always unique.
     
  3. Genetic recombination

  4. The unique combination of alleles contained on a given chromosome would be fixed if recombination didn't occur. Given a large enough population, recombination will occur between homologous chromosomes  at all loci, so that an almost limitless number of combinations of alleles, at different loci, can be tested by evolution.

B. In organisms with a haploid stage, meiosis may provide a "genetic cleansing" mechanism to eliminate deleterious alleles.

Flowering plants and fungi go through haploid stages as part of their life cycles. In plants, this is the gametophyte generation. Metabolically-active haploid cells must carry out all fundamental cellular functions. Since only one copy of each gene is present, there is strong selective pressure against deleterious alleles in the haploid state.


C. The problem: production of haploid gametes requires some mechanism to ensure that each gamete gets a complete set of chromosomes.


In mitosis, the two homologues for each chromosome can replicate and segregate independently and you'll always come out with a balanced set of chromosomes in each daughter cell. The strategy is simple: just attach a spindle fiber to each centromere from pole to pole, and as long as one chromatid migrates to each pole, each daughter cell will be complete. But in meiosis, that wouldn't work. In the figure, a diploid with two sets of chromosomes, I and II, has just undergone a round of DNA replication, such that there are now two copies of each homologue. A single reduction division, as in mitosis, would still result in two balanced diploid cells. The problem arises in the second division. The figure at right shows one possible outcome for a meiosis in which spindle fibers randomly attached to the kinetochore for one copy of each chromosome, during the second meiotic division. There is no orderly way to undergo a second cell division and be sure that each cell gets one and only one copy each of chromosomes I and II. This is why chromosome pairing (synapsis) is neccessary in meiotic prophase I . By keeping all homologues for a chromosome together,  a balanced segregation can occur. At the same time,  synapsis provides an opportunity for genetic recombination to occur.

It's not just enough to keep chromosome pairs together. In order for crossing-over to work, chromosomes must be aligned gene by gene along their entire length. If recombination occurred between non-homologous parts of a chromosome pair, one chromosome would gain extra genes, and the other chromosome would lose those genes. Therefore, synapsis ensures that each copy of the chromosome will have exactly the same gene complement.
 

Meiosis I

In a nutshell:  The diploid cell contains two homologues of each chromosome, one parental and one maternal. These replicate in S phase, resulting in two identical sister chromatids. Prophase I begins with pairing of both homologues, resulting in a tetrad containing 4 homologous chromosomes. Crossing over occurs here. As chromosomes continue to condense, the sites of crossing over become visible as chiasmata. Chromosomes begin to separate as they further condense, pushing chiasmata to the chromosomal termini. As in mitosis, chromosomes are aligned between the centrosomes in metaphase I. One kinetochore forms per chromosome pair, rather than one per chromatid. Consequently, chromatid pairs migrate to opposite poles,  such that both chromatids at a given pole are derived from a single parent. In telophase 1, cytokinesis divides the two diploid cells, and in most species, the chromosomes remain condensed and a nucleus does not re-form. In prophase 2, centrosomes divide again, pulling bivalent chromosomes to the center of the cell. Kinetochores divide, and chromosomes migrate to the poles (anaphase 2), followed by telophase 2, in which haploid nuclei form.

Click here for Meiosis summary

Premeiotic Interphase

Premeiotic interphase can result in an increase in nuclear volume 3 to 4 times that of mitotic nuclei, in preparation for the creation of 4 daughter cells.

Meiotic prophase I


Early prophase I

Late Prophase I
Meiotic prophase I in Rye anthers
Images from Dept. of Plant Science, University of Manitoba
Click here for a figure summarizing meiotic prophase I, from Hank Bass' Lab at Florida State University

1. Leptotene (leptonema): chromosomes begin coiling - During leptotene, there are many independent coiling events occurring simultaneously along the length of each chromosome. The chromosomes appear distinct as long slender threads with many bead-like structures scattered along the length. These bead-like chromomeres are localized aspects of late leptotene coiling that later spreads along the length of the pachytene chromosomes. These are the result of many independent coiling events happening simultaneously along the entire length of the chromosome.

Remember that prior to prophase I,  DNA replication has already occurred. That means that each thin 'thread' contains two identical chromatids, coiled together. The individual chromatids cannot be resolved at this point. 

Chromosomal termini can be seen to be attached to the inner surface of the nuclear membrane at "attachment plaques".  Through a "homology searching" mechanism that is still unknown, the ends of homologous chromosomes migrate together on the nuclear membrane,  making it possible for synapsis to begin at the termini.


2. Zygotene (zygonema): chromosomes pair along their entire length - Think of zippering. Zygotene is characterized by the synapsis of homologous chromosomes. (Remember, each chromosome has two identical chromatids, so at synapsis there are 4 homologous chromatids.) During zygotene, the synaptonemal complex forms between the homologues, apparently aligning them gene by gene.

right - Zygotene in pollen mother cell meiosis of Lilium regale. Arrows - sites of synapsis
from Rickards GK (1965) The Cell Nucleus. Tuatara 13:43.
http://nzetc.victoria.ac.nz/tm/scholarly/tei-Bio13Tuat01-t1-body-d5.html#Bio13Tuat01-fig-Bio13Tuat01_059a


The observation that telomeres appear to cluster together at the beginning of meiotic prophase I (ie. leptotene) suggests that synapsis may be physically coordinated for all chromosomes at a single location. One simple hypothesis is that by bringing all chromosome ends together into a small bundle, it is easier for the homology-searching mechanism to bring both homologues of each chromosome together.

Click here for a Quicktime movie showing a single zygotene nucleus from maize, in which telomeres hybridized with FITC dye (green) appear to cluster together in a "bouquet" formation. Chromosomal DNA is visualized using DNA-specific DAPI stain (red), and heterochromatic knobs stained with Rhodamine (white).
From Bass lab web site: http://bio.fsu.edu/~bass/images2.html.



3. Pachytene (pachynema): crossing over occurs - Pachytene is defined as the phase at which chromosome pairing is complete and crossing over occurs.


Human chromosomes at pachytene. Note that the X and Y chromosomes remain unsynapsed except at one terminus. Chromosome painting probes highlight chromatin from the X (magenta) and Y (chromosomes). PAR - Pseudoautosomal region, which is shared by both X and Y chromosomes. Pairing can only take place at the PAR, because only those sequences are homologous, between the X and Y chromosomes.

From: http://www.sciencedirect.com/science/article/pii/S0006291X06013349
http://dx.doi.org/10.1016/j.bbrc.2006.06.040

 
Structure of synaptonemal complex of the nematode worm, C. elegans.:One role of the Pairing Centers in C. elegans is to promote synapsis, which holds matching chromosomes together during recombination. Fluorescent stains make the proteins of the synaptic "zipper" visible under the microscope
 from http://www.lbl.gov/Science-Articles/Archive/LSD-worm-meiosis.html


Meiosis can proceed in the absence of the synaptanemal complex but there is no recombination. The synaptanemal complex brings paired chromosomes into stable alignment during which crossing over can occur. 

Pachytene is the stage at which crossing-over produces genetic recombination. Crossing over appears to occur within  recombination nodules. There is a stable association between homologues, pairing is completed and bivalents formed. When crossing over occurs there is a breakage and reunion of chromatin strands. DNA synthesis inhibitors cause an increase in chromosome breakage, implying that DNA repair is necessary during crossing over.
DNA synthesis inhibitors cause an increase in chromosome breakage. What does this suggest about DNA repair during crossing over?

4. Diplotene (diplonema): pairing is undone 


Chromosomes undergo desynapsis, which is accompanied by further condensation of chromatin. Chromatids composing the bivalents begin to separate at one or more points, including  the centromeres. The individual chromatids may be visible due to contraction and dissolution of synaptonemal complex. The diplotene bivalent consists of two distinct pairs of chromatids which appear to be attached solely at the chiasmata (singular: chiasma ), the X shaped attachments between the chromosomes.

Diplotene in Rye anthers

Image from Dept. of Plant Science, University of Manitoba

The chiasmata are the visible consequence of recombination events which occurred at pachytene. As would be expected of crossover products, each chiasma involves one sister chromatid from one chromosome and one from the other chromosome.

The number of chiasmata is dependent on the species and the length of the chromosome. The longer the chromosome, the more chiasmata. For example,  up to 12 chiasmata per chromosome are seen in Vicia faba.

Terminalization - As the centromeres move apart, the chiasmata slide over the cross-over points along the chromatids that are involved in the exchange toward the distal portions. (The physical process is analogous to pulling apart two pieces of rope that are tangled together.) As they reach the chromosome ends they become arrested there forming terminal chiasmata, locked at the telomeres. The purpose appear to be holding homologues together until metaphase congression is completed. Extreme tension is exerted on the terminal chiasmata, because the chromosomes are being pulled in opposite directions by the spindle fibers, and the terminal chiasmata are all that hold them together.

In animal oocytes, chromosome decondensation can occur at this point, allowing RNA synthesis. Oocytes produce RNA required for production of proteins during the first few rounds of cell division after fertilization. After RNA synthesis is completed, oocytes can halt at diplotene for years before completing meiosis.
What might be the purpose of terminalization?

In animal oocytes, chromosome decondensation can occur at this point, allowing RNA synthesis. Oocytes produce RNA required for production of proteins during the first few rounds of cell division after fertilization. After RNA synthesis is completed, oocytes can halt at diplotene for years before completing meiosis.

5. Diakinesis: chromosomes remain paired only at termini -  Chromosome condensation continues, and all four chromatids comprising the tetrad are now distinct. Each bivalent is clearly seen to contain 4 separate chromatids, with each pair of sister chromatids linked at their centromere, while non-sister chromatids that have crossed over are linked by chiasmata. This stage is good for counting as the chromosomes are short and thick, well dispersed in the cell. It is easy to get a good spread but this is a short stage. As diakinesis proceeds, the bivalent chromosomes shorten and thicken to become almost spherical so that the double nature of each half of the bivalents is undetectable. Rod or ring bivalents are evident. The nuclear membrane disassembles and the bivalents attach themselves by their centromeres to the rapidly formed spindle.

Diakinesis in Rye anthers
Image from Dept. of Plant Science, University of Manitoba
 

A summary of the stages of synapsis and desynapsis.
From: http://users.path.ox.ac.uk/~pcook/students/11mtmebm.html

Go over the stages of meiotic prophase I. What would you look for under a microscope to distinguish among stages?


C. Metaphase I


Attachment of spindle fibers from one pole or the other to any given kinetochore is a random process. Only one spindle can be attached to each kinetochore. Consequently, bivalents are arranged at random with respect to which spindle pole is related to the maternal or paternal centromeres. This is the physical basis for the genetic concept of random assortment which determines the meiotic segregation and distribution of maternal and paternal chromosomes to the two daughter cells. It forms the basis of Mendel's Law of independent assortment of non-linked loci.

from http://www.wikihow.com/Image:Coin-Toss-1.jpg

This file is licensed under Creative Commons Attribution 2.0 license.
There is a short prometaphase which ends when the bivalent chromosomes are arranged on the metaphase plate of the spindle.

The chromosomes are short and thick. Each bivalent has two kinetochores. The two sister chromatids are attached to each centromere of the bivalent. Each of the two centromeres is joined to the nearer pole by spindle fibres. The two halves of the bivalent are joined by one or more chiasma and the shape of the bivalent is determined by the number and positions of chiasmata in each arm. (That is, we don't see a perfect 'X' because of the terminal chiasmata.)

Metaphase I in Rye anthers
Image from Dept. of Plant Science, University of Manitoba






As metaphase continues, the centromeres are pulled towards opposite poles, chiasmata terminalize. There is resistance to separation caused by the remaining chiasmata, causing the chromosomes to appear stretched and thin. This is a  very different appearance to the chromosomes at metaphase in mitosis in which the sister chromatids are held together and located on the equatorial plate.


Two tetrad chromosomes,
              stretched into an O shape by being pulled to opposite
              poles

In meiosis, the centromeres of the homologous chromosomes are oriented on the long axis of the spindle equidistant from the equator and the terminal chiasmata are located on the equatorial plate.

Equilibrium is established when all bivalent chromosomes have reached equatorial plate until all chromosomes yield to tension of spindle fibres from opposite poles.

This is a good stage for observing chromosome numbers and such conditions of chromosome pairing as univalents, bivalents, trivalents, quadrivalents etc.

D. Anaphase I

During anaphase I, the arms of dyad diverge as the chromatids appear to repel each other. The chiasmata slip off the ends of the chromosomes and the poleward-moving chromatids are bound together at only one point, the centromere. The dyads appear as a double V if chromosome is metacentric or acrocentric and as a single V if telocentric. If no crossover has taken place, the separation is purely reductional. If crossover has taken place, each separating dyad carries part of its homolog, resulting in an equal division of the exchanged chromosomal material to each daughter cell. Chiasmata exchanged at meiosis will redistribute chromosome material from both parents to the daughter cells.

Errors in Anaphase I  have serious consequences for the cell. Any chromosomal connections between the two separating chromosomes form "bridges" often accompanied by fragments of chromosomes that lack a centromere (ie. acentric) and therefore do not move to either pole, lagging on or near the metaphase plate. Another aberration of anaphase I is nondisjunction - the two homologs do not separate and/or both go to the same pole.

If no crossover has taken place, the separation is purely reductional. If crossover has taken place, each separating dyad carries part of its homologue, resulting in an equal division of the exchanged chromosomal material to each daughter cell. Chiasmata exchanged at meiosis will redistribute chromosome material from both parents to the daughter cells.

Anaphase I is shorter in duration than Metaphase I. The function of the stage is to evenly distribute the partners of homologous chromosomes to the daughter nuclei and this reduces the number of chromosomes by half. If the somatic chromosome number is 2n= 14, the number is reduced to the gametic chromosome number n=7. Somatic counts at mitosis stated as 2n and a chromosome count in meiosis is stated as n.

Aguiar-Perecin, Margarida L.R. de, Fluminhan, Antonio, Santos-Serejo, Janay A. dos, Gardingo, José R., Bertão, Mônica R., Decico, Maria Juliana U., Mondin, Mateus. (2000). Heterochromatin of maize chromosomes: structure and genetic effects. Genetics and Molecular Biology, 23(4), 1015-1019. https://dx.doi.org/10.1590/S1415-47572000000400047

E. Telophase I


This stage is similar to mitotic telophase as the chromosomes assemble at the poles. Nuclear membranes and nucleoli may develop and eventually two daughter nuclei with diploid chromosome number are produced. The chromatids are still widely separated from each other and show no relational coiling.

Telophase I in Rye anthers

Image from Dept. of Plant Science, University of Manitoba

 
Telophase in mitosis and meiosis is difficult to tell apart because the characteristics are so similar. Which stage would you choose to examine in order to determine whether a cell was in mitosis or meiosis, and why?

Interkinesis

Interkinesis is defined as the period between telophase I and prophase II. During interkinesis, the chromosomes do not synthesize new DNA and there is no duplication of chromosomes. The chromosomes are already prepared for the second division each consisting of two chromatids only held together by the centromere. Therefore, despiralization, uncoiling and hydration of chromosomes are not necessary. The length of time between meiosis I and meiosis II may vary greatly between species.


Meiosis II

Prophase II.

This second meiotic division is very similar to a mitotic division. Prophase II differs from Prophase I in appearance as the sister chromatids arms are widely separated from each other. This makes the dyad arms look like crosses and the shortness of the partially coiled chromosomes makes it easier to count chromosomes.
 
 

Prophase II in Rye anthers
Image from Dept. of Plant Science, University of Manitoba

Metaphase II
Anaphase II

Metaphase II in Rye anthers
Image from Dept. of Plant Science, University of Manitoba

Anaphase II in Rye anthers
Image from Dept. of Plant Science, University of Manitoba

Telophase II

 

Telophase II in Rye anthers

Image from Dept. of Plant Science, University of Manitoba

Gametogenesis is a specialized form of meiosis

Evolution is constantly reinventing the mechanisms of sexual reproduction. We will only discuss one example here. In this example, we will detail one of the many variants of the process of gametogenesis across the higher eukaryotes, as found in flowering plants.

Microsporogenesis - male meiosis in flowering plants

The primary function of meiosis in sexually-reproducing species is to produce gametes. This process, called gametogenesis, varies in details among the higher eukaryotes. However, almost all gametogenic processes involve asymmetrical cell division in some way. The examples we've just seen are from gametogenesis of rye anthers, which you might recall is the 'male' gamete in plants. This process produced four similarly sized cells. Certainly they contain different genetic information (that's the whole point of meiosis!), but they are functionally and structurally similar, if not mostly identical. Once meiosis is finished, each haploid cell, called a microspore, develops further into a pollen grain.

Once the meiotic division is complete, each microspore develops a sperm nuclei and a nuclei for pollen tube formation.

Megasporogenesis - female meiosis in flowering plants
After going through meiosis, the 4 resulting cells do not each form an ovum. Instead, three of the cells are designated as polar bodies, and the primary nucleus undergoes further divisions. One nucleus from each pole known as polar nuclei migrates to the middle of the embryo sac and the two nuclei fuse to give rise to a secondary nucleus or polar fusion nucleus with 2n chromosome constitution. At the micropylar end of the embryo sac, one nucleus differentiates into an egg cell and the remaining nuclei become synergids, part of the egg apparatus. The other three nuclei are called the antipodal nuclei. During pollination in higher plants, the tube nucleus of the pollen grain directs the growth of the pollen tube down the style, through the micropyle and into the nucellus. The two sperm are released into the nuclear sac. The end result, after fertilization, is an ovum with 2N chromosomes, surrounded by a 3N endosperm.



Image displayed from: http://philschatz.com/biology-book/resources/Figure_32_02_07.jpg

In humans, the process is similar in some ways and different in others. Instead of microsporogenesis and megasporogenesis, we have spermatogenesis and oogenesis, respectively. The relative sizes of the gametes are similar to plants: human ova are approximately 10,000 times larger than human sperm. In developing those gametes, we see again the theme of asymmetrical cell division.

Actually, spermatogenesis, like microsporogenesis, produces four functional gametes after meiosis. Oogenesis is where we see asymmetrical cell division. Similar to megasporogenesis, we also see the formation of polar bodies and only one ovum per meiosis. Most of the cytoplasm stays with the ovum, with only a small amount of cytoplasm going to the polar bodies. Although the fate of the polar bodies is generally to disintegrate through apoptosis, in some species the polar bodies may play other roles.
Displayed from https://old-ib.bioninja.com.au/higher-level/topic-11-animal-physiology/114-sexual-reproduction/gametogenesis.html

Why might be the purpose of creating polar bodies?

Summary