Lab #6: The Cell Cycle, Mitosis, Meiosis and Gametogenesis ...

GENERAL BIOLOGY LAB 1 (BSC1010L)
Lab #6: The Cell Cycle, Mitosis, Meiosis and Gametogenesis
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
• Learn how to examine a karyogram
• Perform karyotype analysis
• Understand the difference between male and female gametogenesis
READINGS:
• Exercise 14: pgs 149-156
• Exercise 15: pgs 159-165
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INTRODUCTION:
The Cell Cycle:
All eukaryotic cells undergo a series of growth and division events, collectively referred to as
the cell cycle (Fig 1). Duration of the cell cycle is specific to the organism as well as the cell
type. In general, the cell cycle consists of three main phases: Interphase, Mitosis (M) and
Cytokenesis (C). The first stage, Interphase, is considered the non-dividing or growth portion of
a cell’s life cycle and is subdivided into Gap 1 (G1), Synthesis (S) phase, and Gap 2 (G2). G1
and G2 are considered the main growth phases. During G1 (the normal state of a cell), the cell
grows and generates the enzymes necessary for DNA replication to take place during the S phase
while in G2, the cell synthesizes proteins, carbohydrates and lipids which all function to increase
the cell’s size. Also during this time, the chromosomes prepare to condense in anticipation of
entry into M phase.
Question:
Interphase has sometimes been called a “resting stage.” Why is this inaccurate
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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 (G 0 = Gap 0 phase), which is important for some cell
types that divide very infrequently and/or for cells that are already 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 the DNA is not damaged before allowing the cell to proceed into mitosis. If any of these
checkpoints are nonfunctional or mutated, control of the cell cycle is lost and cancer develops.
G1/S Checkpoint
G2/M Checkpoint
Spindle Checkpoint
Figure 1. The cell cycle and its associated checkpoints
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Question:
How might you use the knowledge of the cell cycle checkpoints to prevent, diagnose, and treat
cancer
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). However, when the cell is ready to divide
(i.e., it enters the M phase of the cell cycle), the DNA repeatedly folds on top of itself,
condensing into visible chromosomes. The chromosomes exist in pairs and are called
homologous chromosomes. Each homologue within the pair is called 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 but most eukaryotes are diploid (2n), i.e., they
have 2 sets of chromosomes. For example, human cells possess a total of 46 chromosomes (23
chromosomes per set) while canine cells possess 78 chromosomes (39 chromosomes per set).
Figure 2. Cell as it appears during Interphase
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Figure 3. A Pair of Sister Chromatids
Eukaryotic cells, depending on the type (somatic vs. germ cell), divide by either mitosis
or meiosis. Mitosis is the process in which a diploid (2n) 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). Mitosis occurs in somatic cells,
which are all cells of the body excluding the reproductive cells (i.e. eggs and sperm). Meiosis, on
the other hand, only occurs in the germ cells, which are cells of the reproductive organs (i.e.
testes and ovaries).
Mitosis is comprised of 4 stages, Prophase, Metaphase, Anaphase and Telophase
(although some authors describe Prometaphase as a distinct phase). Following nuclear division,
cytokenesis (division of the cytoplasm) begins. The primary function of this process is to
completely separate the 2 newly generated daughter cells from each other. In animal cells a
cleavage furrow or indentation in the middle area of the cell develops and divides the cell into
two parts. Plant cells, in contrast, are unable to divide using the cleavage furrow since they
possess a cell wall. Instead they generate a cell plate at the center of the cell that splits the cell
into two.
Meiosis, on the other hand, occurs only in germ cells, i.e., those destined to become the
gametes. This process is referred to as a reduction division since the 4 daughter cells generated
from the division of the diploid parental cell are haploid. The stages of Meiosis I are Prophase I,
Metaphase I, Anaphase I and Telophase I and of Meiosis II are Prophase II, Metaphase II,
Anaphase II and Telophase II (Fig 4a and b). Meiosis I involves the separation of homologous
pairs of chromosomes that are then separated into sister chromatids during Meiosis II.
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MEIOSIS I
Figure 4a. Meiosis I
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MEIOSIS II
Figure 4b. Meiosis II
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In general, Meiosis I is very similar to Mitosis except that (1) Prophase I involves
synapsis (forms a tetrad) and crossing over (Fig 5) occurs between homologous pairs of
chromosomes and (2) the homologous pairs of chromosomes are separated during Anaphase I.
Figure 5. Crossing Over between Homologous Pairs of Chromosomes
Gametogenesis:
Gametes are reproductive cells with haploid nuclei that result from meiosis and are
formed by gametogenesis. In mammals and many other vertebrates, gametes and gametogenesis
differ between males and females. Males produce sperm through the process of
spermatogenesis (Fig 6), while females produce eggs via oogenesis (Fig 7).
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 these spermatogonia move inward towards the lumen of the tubule and begin meiosis. These
spermatogonia are called primary spermatocytes. Meiosis I of a primary spermatocyte
produces two secondary spermatocytes, each with a haploid set of double-stranded
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chromosomes. Meiosis II separates the strands of each chromosome and produces two haploid
spermatids that mature and differentiate into sperm cells.
Figure 6. Spermatogenesis
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 will 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 consists of the
secondary oocyte. Each secondary oocyte contains a haploid set of double-stranded
chromosomes. Meiosis II proceeds but is not completed until fertilization occurs.
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Figure 7. Oogenesis
Review the basic stages of spermatogenesis and oogenesis on pages 162-165 and answer the
questions that follow.
Questions:
1. Why do gametes only have half the number of chromosomes as the original parent cell
Why is this important
2. Would evolution occur without the events of meiosis and sexual reproduction Why or
why not
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In today’s lab, we will examine the cell cycle, mitosis and meiosis. We will then consider
the role of the different phases in the cell cycle to better understand the significance of each step
in the production of healthy cells without DNA damage. We will also consider the consequences
when specific aspects of cell division fail to function properly. Finally, we will learn how to
examine karyotypes which are used to determine the number of chromosomes in a species as
well as for the diagnosis of birth defects or genetic abnormalities.
TASK 1: Cycling Through the Cell Cycle
A) Stages of Mitosis
1. Examine a prepared slide of the whitefish blastula on high power.
2. Complete Table 1, making sure to draw examples of each phase of mitosis.
Table 1:
Stage of Mitosis Description of Events Drawings of Stages
Prophase
Metaphase
Anaphase
Telophase
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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
c. How does mitosis differ between plant and animal cells
B) Time for Cellular Replication
1. Using the prepared onion root tip slide, count the number of cells in each phase of the cell
cycle (i.e., interphase and mitosis) in the high power field of view. Repeat 3 times for an
approximate total of 100 cells and record your results in Table 2.
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 x 840 minutes
Total number of cells counted
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Stage of Cell
Cycle
Number of Cells
FOV 1 FOV 2 FOV 3 FOV 4 Total
Time Spent in Each Stage
Interphase
Prophase
Metaphase
Anaphase
Telophase
TASK 2: Effect of Colchicine on Mitosis
Colchicine, a product of the plant Colchicum autumnale (common name = Meadow
saffron), is well documented for its use in the treatment of gout, cirrhosis, and psoriasis, among
other disorders (Ben-Chetrit and Levy, 1998). This compound is known to interact with tubulin,
a component of the spindle fibers. During metaphase, the chromosomes attach to the mitotic
spindle via their kinetochore and oscillate at the equatorial region under high tension. Colchicine
decreases this tension, therefore suspending the chromosomes in metaphase (Jordan and Wilson,
2004).
Based on the information above, propose a null and alternate hypotheses about the stages
of mitosis that you expect to see in the onion cells treated with colchicine.
Ho:
Ha:
Procedure:
1. Each student in the group will prepare an onion root tip slide as follows:
a. To a watch glass add 5-10 drops 1M HCl.
b. Using a scalpel cut the terminal 4mm of an onion root tip grown in a dilute
(0.05%) colchicine solution and add it to the acid to soften the tip. Leave the tip in
the acid for about 5 minutes.
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c. Add 1 drop of acetocarmine stain to a clean slide.
d. Once softened, cut the root tip in half and add one half (2mm) to the slide with the
acetocarmine stain.
e. Use the scalpel to chop the tip into numerous pieces and then crush the pieces
with a glass rod.
f. Apply a cover slide to the slide and then gently warm by passing the slide over the
ethanol lamp. DO NOT BOIL!!!
g. Invert the slide onto a clean piece of tissue/paper towel and push down firmly
with the thumb to flatten and disperse the cells.
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h. Examine the slide using the high power objective
i. Count the number of cells observed in metaphase in 1 high power field of view.
Repeat this step for an additional 2 high power field of views.
j. Seal the cover slip to the slide by coating the edges with clear nail polish.
k. Examine chromosome morphology using the oil immersion lens (See Procedure
below) by selecting cells in anaphase. Count the number of chromosomes that you
see at one pole since this represents the diploid number for the species. Record the
number from each cell in the Table 2. Make sure to move the focus from the top layer
of the cell downwards as you scan for the chromosomes.
Working with the Oil immersion lens: (Adopted from Dolphin, 2005)
CAUTION: Oil immersion lenses allow you to approach 1000X magnifications with an
increase in resolution. However, the distance from the lens surface to the slide is very
small, and it is quite easy to push the lens through the slide, possibly breaking the slide and
ruining the lens.
Procedure:
1. Focus first on the object on the slide by proceeding from the scanning (4X) to
high-power (40X) objectives as you have done before. Now you are ready to
try the oil immersion lens.
2. Do NOT touch the focus knobs or the stage knobs. Turn the turret to swing the
high-power (40X) objective out of the way. Place a single drop of immersion
oil on the slide right over where the light is coming through the stage, and
rotate the oil immersion lens into place. The lens will actually contact the oil
drop, making a column of oil from the slide surface to the lens surface. This
column of oil prevents light scattering and improves resolution.
3. Now look through the oculars and open the substage diaphragm to increase the
light. The object on the slide should still be in the field of vision but will
probably be out of focus. Use the fine-adjustment knob to focus clearly.
Never use the coarse-adjustment knob!!!!
4. Once you have an oil immersion lens in place, do NOT swing the 40X
objective back into place. Because the objective focuses close to the slide, the
40X objective will get oil on it and it is difficult to clean the oil from the lens
surface.
5. When you have finished using the oil immersion objective, you must clean the
oil from its surface, as well as from the slide, using lens cleaner and lens paper.
Because oil immersion lenses require extra cleanup and the danger of breaking
slides is great, this is the only time during the semester that we will use them.
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Table 2:
Cell # # of Chromosomes Cell # # of Chromosomes
1 6
2 7
3 8
4 9
5 10
Questions:
1. Are any mitotic stages present that were not observed in the preserved onion root tip slide
Are there any mitotic stages that are completely absent
2. Compare the stages of mitosis that you observed in the colchicine-treated and untreated
onion cells (preserved slide). What do your results suggest about your hypothesis
3. Can you discern how many chromosomes are present in one cell at anaphase If so, how
many
4. Given colchicine’s properties, could this compound be used to prevent cancer Explain.
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TASK 3: Karyotype Analysis
Karyotyping refers to the process by which scientists are able to microscopically
visualize the complete set of chromosomes in an organism. Karyotype analysis is performed
when the chromosomes are the most highly condensed, i.e. in metaphase (halted by the addition
of colichicine), in order to determine the number of chromosomes present in the individual as
well as to detect the presence of any chromosomal abnormalities such as deletions, translocations
or the insertion of extra copies. A normal human karyotype should consist of 22 autosome pairs,
listed from largest (chromosome 1) to smallest (chromosome 22), and 1 pair of sex
chromosomes; XX if female and XY if male (See Fig 8). Known abnormalities that result from
variations in normal chromosome structure or number in humans include:
a. Downs Syndrome: Three copies of chromosome 21
b. Turner syndrome (in females one gains reduction in female characteristics, e.g.
ovaries don’t produce eggs): One copy of the X chromosome
c. Cri du chat (disease that affects larynx and nervous system, infants cry like a
cat): Chromosome 5 is truncated
d. Edwards syndrome (severe birth defects such as intestine growing outside the
body cavity): Three copies of chromosome 18
e. Patau Syndrome (severe birth defects including heart and nervous dysfunction):
Three copies of chromosome 13
Figure 8. Normal Human Karyotype
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Procedure:
A fellow scientist of BCBB Cytogeneics was assigned the task of performing karyotype
analysis for 2 newborn babies, but he needs a second opinion of the results before informing the
parents. The karyotype for each baby is presented below. It is your task to examine both
karyotypes (#1 and #2), record your findings in the tables provided and report them to your
colleague.
Karyotype #1:
http://www.ratsteachgenetics.com/Genetics_quizzes/Lecture%207/7q4.jpg
CH # Remarks CH# Remarks CH# Remarks CH# Remarks
1 7 13 19
2 8 14 20
3 9 15 21
4 10 16 22
5 11 17 23
6 12 18 24
CH = Chromosome
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Karyotype #2:
Chart for Infant Number Two:
https://ccr.coriell.org/images/karyotype/gm18241-xyy.jpg
CH # Remarks CH# Remarks CH# Remarks CH# Remarks
1 7 13 19
2 8 14 20
3 9 15 21
4 10 16 22
5 11 17 23
6 12 18 24
CH = Chromosome
Questions:
1. 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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TASK 4: Meiosis and Gametogenesis
Procedure:
1. Examine prepared slides of sperm from humans, rats, and guinea pigs. How do the sperm
from the three species compare
2. Examine a cross section of a monkey’s seminiferous tubules and draw what you see in the
space provided below. Try to locate the spermatogonia, primary spermatocytes, secondary
spermatocytes, spermatids and mature sperm.
3. Examine a cross section of cat ovary and draw what you see in the space provided
below. Try to locate the developing follicle with the egg inside.
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4. Examine the slide of a mature follicle (Graafian follicle) and draw what you see in the space
provided below.
5. Complete the table below:
Mitosis
Meiosis
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
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Questions:
1. Why is meiosis referred to as reduction division
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 the sizes of the oocytes differ as they move from the follicle stage towards the
mature Graffian follicle
4. How do sperm and eggs differ in size Why do you think this happens Why do you think
there is such a difference in the number of each produced 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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HOMEWORK:
Before coming to lab next week, make sure to read the Mendelian Genetics task sheet as well as
Chapter 17 (pgs. 177-185) in your lab manual.
References:
Ben-Chetrit, E and Levy, M (1998) Colchicine: 1998 Update. Seminars in arthritis and
rheumatism 28: 48-59.
Jordan, MA and Wilson, L (2004) Microtubules as a target for anticancer drugs. Nature reviews
4: 253- 265.
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