Exercise 9: The Cell Cycle, Mitosis, Meiosis and Gametogenesis
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OBJECTIVES:
 Understand the major events involved in the cell cycle.
 Learn about the process of cellular division in plant and animal cells.
 Compare and contrast mitosis and meiosis.
 Understand the difference between male and female gametogenesis.
 Learn how to examine a karyogram.
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INTRODUCTION:
The Cell Cycle
Eukaryotic cells undergo a series of growth and division events, referred to as the cell
cycle (Fig. 1). The duration of the cell cycle is specific to cell type and organism. In general, the
cell cycle consists of three main phases: Interphase, Mitosis (M) and Cytokinesis (C). The first
stage, Interphase, is considered the non-dividing or growth portion, and is subdivided into Gap 1
(G1), Synthesis (S), and Gap 2 (G2), of which G1 and G2 are the main growth stages.
Specifically, during G1 (the normal state of a cell), the cell grows and generates the enzymes
necessary for DNA replication that takes place during the S phase. In G2, the cell synthesizes
proteins, carbohydrates and lipids, which all function to increase the cell’s size, and the
chromosomes prepare to condense in preparation for the M phase.
Question:
Interphase is sometimes referred to as a “resting stage.” Why is this inaccurate?
The cell cycle is controlled by a series of checkpoints (Fig. 1), namely the G1/S, G2/M
and spindle checkpoints. The G1/S checkpoint, determines if the cell should continue into the S
phase or if it should enter a resting state (G0 = Gap 0 phase), which is important for cell types
that divide infrequently and/or cells that are terminally differentiated (e.g. nerve cells). This
checkpoint is followed by the G2/M checkpoint, which serves as a control mechanism to
prevent damaged cells from entering the M phase. Once the cells are committed to mitosis, the
role of the spindle checkpoint is to ensure that all chromosomes are attached to the mitotic
spindle during metaphase; if any chromosome is not attached, the cell will not be able to proceed
into anaphase. In addition, DNA damage checkpoints located in G1, S and G2 ensure that DNA
is not damaged before allowing the cell to proceed to mitosis. For example, the p53 protein,
which plays a key role in the G1 checkpoint, monitors the integrity of DNA during this stage. If
the DNA is healthy (i.e., no mutations) p53 will allow the cell to progress onwards through the
cell cycle. On the other hand, if p53 detects DNA damage, then it will stop the cell in G1 either
for repair or for destruction. If any of these checkpoints are nonfunctional or mutated, control of
the cell cycle is lost and cancer develops.
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G2/M Checkpoint
G1/S Checkpoint
Spindle Checkpoint
Figure 1. The cell cycle and its associated checkpoints
Questions:
a. How might you use the knowledge of the cell cycle checkpoints to prevent, diagnose, and
treat cancer?
b. What problems may occur as a result of having a mutated p53 protein?
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Cellular Division: Mitosis vs. Meiosis
The genetic material (DNA) of all eukaryotic organisms is housed within the cell’s
nucleus and is passed on from generation to generation. While a cell is in interphase, the DNA
exists in an extended form called chromatin (Fig. 2) that repeatedly folds on top of itself,
condensing into visible chromosomes when the cell is ready to divide (i.e., entering the M phase
of the cell cycle). In somatic (body) cells, chromosomes exist in pairs and are called homologous
chromosomes. Each homologue within the pair is referred to as a sister chromatid and is joined
to the other by the centromere (Fig. 3). In eukaryotic organisms, the number of chromosomes
present differs between species (Table 1) but most eukaryotes are diploid (2n), meaning they
have 2 pairs of chromosomes.
Figure 2. Cell as it appears during Interphase
Figure 3. A pair of sister chromatids
Table 1. Chromosome numbers vary across species
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Eukaryotic cells divide either by mitosis or meiosis. Mitosis is the process in which a
diploid parental cell is divided into 2 identical daughter cells, also diploid in number. In contrast,
meiosis involves the division of a diploid parental cell into 4 daughter cells, all of which are
haploid (n) in number. Mitosis occurs in somatic (body) cells, while meiosis takes place only in
the germ cells, i.e., cells of the reproductive organs (testes and ovaries).
Mitosis (Fig. 4) is a nuclear event comprised of 4 stages, Prophase, Metaphase, Anaphase
and Telophase. Usually following nuclear division, the 2 newly generated daughter cells are
separated from each other through the process of cytokinesis (division of the cytoplasm). During
cytokinesis, animal cells form a cleavage furrow or indentation on the periphery of the cell that
pulls the plasma membrane inward, dividing the cell into two parts. Plant cells, in contrast, are
unable to divide using the cleavage furrow since they possess a rigid cell wall. Instead, they
generate a cell plate at the center of the cell that grows outward to split the cell into two.
Figure 4. Mitosis
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Meiosis, on the other hand, occurs only in germ cells, i.e., those cells destined to become
gametes. The stages of Meiosis I are Prophase I, Metaphase I, Anaphase I and Telophase I
(Fig. 5) and of Meiosis II are Prophase II, Metaphase II, Anaphase II and Telophase II (Fig.
6). Meiosis I involves the separation of homologous pairs of chromosomes which are further
separated into sister chromatids during Meiosis II.
Figure 5. Meiosis I
Figure 6. Meiosis II
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During Prophase I of Meiosis, two pairs of homologous chromosomes form a tetrad through
synapsis and exchange genetic material via the process of crossing over (Fig. 7). In this
process, the genetic material is neither gained nor lost. Instead, new combinations of alleles arise,
thereby increasing genetic variation.
Figure 7. Crossing over between homologous pairs of chromosomes
In today’s lab, you will examine the cell cycle. You will then consider the role of the
different phases of the cell cycle to understand the significance of each step in the production of
healthy cells and the possible consequences of mistakes during cell division. Finally, you will
learn how to examine karyotypes, which are used to determine the number of chromosomes in a
species as well as for the diagnoses of birth defects and genetic abnormalities.
TASK 1 - Cycling Through the Cell Cycle
A) Identify the Stages of Mitosis
1. Examine a prepared slide of the whitefish blastula on high power.
2. Complete Table 2, making sure to draw examples of each phase of mitosis.
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Table 2:
Stage of Mitosis
Description of Events
Drawings of Stages
Prophase
Metaphase
Anaphase
Telophase
Questions:
a. Why are cells from a blastula used to examine mitosis?
b. How fast do you think cells divide when an embryo is forming compared to the normal
growth of an animal?
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c. How does cytokinesis differ between plant and animal cells?
B) Onion Root Tip Preparation
1. Using a scalpel cut the terminal 4mm of an onion root tip and add it to a small tube
containing 100µL of Carnoy's fixative.
2. Place the tube in a 60°C water-bath for 15 min to soften the tissue.
3. After 15 min, remove the onion tip from the fixative with forceps. Rinse the tip 2-3 times
with an ice cold 70% ethanol solution to remove any residual acetic acid from the
fixative. Note: Acetic acid reduces the ability to stain the chromosomes.
4. Place the root tip on a clean microscope slide and add a drop of Hydrochloric acid (HCl).
Using a dissecting microscope remove the very end of the tip. Keep this portion and
discard the remaining tissue.
5. With a dissecting needle, attempt to macerate/crush the tissue into small pieces.
6. Add one drop of Aceto-orcein stain to the crushed tissue.
7. Gently warm the slide by passing it over an ethanol lamp (see figure below). DO NOT
BOIL!!! Heating the slide will speed up the staining process and allow some of the
Hydrochloric acid in the stain to soften the tissue.
http://www.microscopy.fsu.edu/optics/intelplay/polsamples.html
8. Allow the slide to sit for 1 min to cool down. In the meantime, smear a small amount of
Mayer's albumen onto a coverslip and allow it to dry. Make sure to set the coverslip face
up on your table so that you will know which side contains the albumen.
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9. Place the slide on a piece of paper towel. Using forceps lower the coverslip (albumen side
down) over the stained tissue.
10. Place the end of the paper towel over the coverslip and, with your thumb, press down
onto the coverslip (do not press down so hard that the slide breaks, or you will have to
repeat steps 1-9 again). The act of squashing separates the cells from each other, making
the chromosomes more visible.
C) Cellular Replication
1. Using your prepared onion root tip slide, count the number of cells in each phase of the
cell cycle (i.e., interphase and each stage of mitosis) in the high power field of view.
Move the slide and repeat 3 times for an approximate total of 100-200 cells, record your
results in Table 3.
2. Assuming that an onion root tip cell takes 14 hours (840 minutes) to complete the cell
cycle, the time that an onion cell spends in each stage of the cell cycle can be calculated
using the following formula:
Time for each stage = Number of cells at each stage
Total number of cells counted
Table 3:
Stage of Cell
Cycle
Number of Cells
FOV 1
FOV 2
FOV 3
FOV 4
Interphase
Prophase
Metaphase
Anaphase
Telophase
9
x
840 minutes
Time Spent in Each Stage
Total
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TASK 2 – Meiosis and Gametogenesis
Gametes (sperm and eggs) are haploid reproductive cells that are formed by the process
of gametogenesis. In mammals and many other vertebrates, gametes and gametogenesis differ
between males and females; males produce sperm through the process of spermatogenesis (Fig.
8) while females produce eggs via oogenesis (Fig. 9).
Sperm is produced in the seminiferous tubules of the testes. Within the seminiferous
tubules, spermatogonia constantly replicate mitotically throughout the life cycle of males. Some
of the spermatogonia move inward towards the lumen of the tubule and begin meiosis. At this
point, they are called primary spermatocytes. Meiosis I of a primary spermatocyte produces
two secondary spermatocytes, each with a haploid set of double-stranded chromosomes.
Meiosis II separates the strands of each chromosome and produces two haploid spermatids that
mature and differentiate into sperm cells via spermiogenesis.
In females, oogenesis occurs in the oocytes of the ovaries. Unlike spermatogonia,
oocytes are not produced continuously. Oogonia, which are produced during early fetal
development, reproduce mitotically to produce primary oocytes. In humans, the ovaries of a
newborn female contain all the primary oocytes that she will ever have. At birth, primary oocytes
begin meiosis I, but are arrested in prophase I. At puberty, circulating hormones stimulate
growth of the primary oocytes in the follicles (surrounding tissue) each month. Just before
ovulation, the oocyte completes meiosis I producing a Graafian follicle which contains the
haploid secondary oocyte. Meiosis II proceeds but is not completed until fertilization occurs.
Figure 8. Spermatogenesis
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Figure 9. Oogenesis
Questions:
1. Why do gametes have only half the number of chromosomes as the original parent cell?
2. Would evolution occur without the events of meiosis and sexual reproduction? Why or
why not?
Procedure:
1. Examine prepared slides of sperm from humans, rats, and guinea pigs and draw what
you see in the space provided below.
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Magnification: _________ Magnification: _________ Magnification: _________
2. Examine a cross section of a monkey’s seminiferous tubules and draw what you see
in the space provided below. Locate the spermatogonia, primary spermatocytes,
secondary spermatocytes, spermatids and mature sperm and label each of these cells
on your drawing.
Magnification: _________
3. Examine a cross section of cat ovary and draw what you see in the space provided
below. Locate and label the developing follicle with the egg inside on your drawing.
Magnification: _________
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4. Examine the slide of a mature follicle (Graafian follicle) and draw what you see in the
space provided below.
Magnification: _________
5. Compare Mitosis and Meiosis in Table 4:
Table 4:
Mitosis
Purpose of process
Location
Number of cells
generated per cycle
Number of nuclear
divisions per cycle
Ploidy (n or 2n) of
daughter cells
Daughter cells genetically
identical to parent?
Pairing of homologues
Occurrence of crossing
over
Questions:
1. Why is meiosis referred to as reduction division?
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Meiosis
2. If a species has 24 chromosomes in the nucleus prior to meiosis, what number will
each cell have after meiosis is complete?
3. How do sperm and eggs differ in size? (Hint: consider size and the quantity of each
gamete). Explain a possible reason for these differences.
4. What would happen if females produced 100’s or 1000’s of eggs during each cycle?
What if males were born with a limited number of sperm?
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TASK 4 - Karyotype Analysis
Karyotyping is the process scientists use to visualize a complete set of chromosomes to detect
any possible abnormalities such as deletions, translocations or insertions. Karyotype analysis is
performed when the chromosomes are highly condensed, i.e. in metaphase (halted in this phase
with the addition of colichicine). A normal human karyotype should consist of 22 autosomal
pairs, listed from largest (chromosome 1) to smallest (chromosome 22), and 1 pair of sex
chromosomes; XX if female and XY if male (Fig. 10). Known abnormalities that result from
variations in normal chromosome structure or number in humans are listed in Table 5.
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Figure 10. Normal human karyotype
Table 5
Abnormality/disorder
(alternative name)
Down syndrome
(Trisomy 21)
Turner syndrome
(Gonadal dysgenesis)
Cri du chat
(cry of the cat)
Edwards syndrome
(Trisomy 18)
Patau syndrome
(Trisomy 13)
Klinefelter syndrome
Symptoms
Cognitive disabilities, characteristic physical
features, congenital heart disease
Gonadal dysfunction, characteristic physical
features, congenital heart disease
Abnormalities including problems with the larynx
and nervous system, resulting in a characteristic
infant cry that sounds like a meowing kitten
Mortality rate – 50% die within the first 2 months
of life. Three times more common in boys than
girls. Birth defects include several organ
abnormalities, including heart and kidneys
Mortality rate- 80%. Birth defects, including
severe neurological problem and heart defects
Infertility, language impairment, characteristic
physical features
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Karyotype
3 copies of
chromosome 21
1 copy of the X
chromosome
Truncated
chromosome 5
3 copies of
chromosome 18
3 copies of
chromosome 13
Extra X
chromosome in
males
Above: karyotype of Edward’s syndrome in male.
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Procedure:
A fellow scientist was assigned the task of performing karyotype analysis for 2 infants,
but he needs a second opinion before informing the parents. The karyotype for each infant is
presented below. Record your findings for both in the tables provided.
Karyotype #1:
http://www.ratsteachgenetics.com/Genetics_quizzes/Lecture%207/7q4.jpg
CH #
1
2
3
4
5
6
Remarks
CH#
7
8
9
10
11
12
Remarks
CH#
13
14
15
16
17
18
17
Remarks
CH#
19
20
21
22
23
24
Remarks
Karyotype #2:
https://ccr.coriell.org/images/karyotype/gm18241-xyy.jpg
CH #
1
2
3
4
5
6
Remarks
CH#
7
8
9
10
11
12
Remarks
CH#
13
14
15
16
17
18
Remarks
CH#
19
20
21
22
23
24
Remarks
Question:
Based on the karyotypes provided, do these babies have detectable problems in their
chromosomes? If yes, use that information to diagnose what disease/genetic abnormality
the child has.
Infant Number One Diagnosis: ________________
Infant Number Two Diagnosis: ________________
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