GENERAL BIOLOGY LAB 1 (BSC1010L) Lab #7: 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. Understand the difference between male and female gametogenesis. Learn how to examine a karyogram. Perform karyotype analysis. ______________________________________________________________________________ INTRODUCTION: The Cell Cycle All eukaryotic cells undergo a series of growth and division events, collectively 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 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 cells 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 arrest the cell in G1 either for repair or for destruction (apoptosis = programmed cell death). 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

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?

Cellular Division: Mitosis vs. Meiosis The genetic material (DNA) of all eukaryotic organisms is housed within the cells 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 but most eukaryotes are diploid (2n), meaning they have 2 pairs of chromosomes. Chromosome number differs between organisms (Table 1), for example human cells possess a total of 46 chromosomes (23 pairs), while canine cells possess a much larger number (39 pairs).

Figure 2. Cell as it appears during Interphase

Figure 3. A pair of sister chromatids

Table 1. Chromosome numbers vary across species Eukaryotic cells, depending on the type (somatic vs. germ 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 cells, which are all cells of the body excluding the reproductive cells (eggs and sperm). Meiosis, on the other hand, 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 (although some authors describe Prometaphase as a distinct phase). 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

Meiosis, on the other hand, occurs only in germ cells, i.e., those cells destined to become 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 (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

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 todays lab, you will examine the cell cycle, Mitosis and Meiosis. 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.

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?

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 100L of Carnoy's fixative. 2. Place the tube in a 60C 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 a 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. DO NOT BOIL!!! Heating the slide will speed up the staining process and allow some of the HCl in the stain to soften the tissue.

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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. 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 as hard as you can (but not so hard that the slide breaks). The act of squashing separates the cells from each other, making the chromosomes more visible.

C) Time for 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. Repeat 3 times for an approximate total of 100 cells and 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: 11

Time for each stage = Number of cells at each stage Total number of cells counted

840 minutes

Table 3: Stage of Cell Cycle FOV 1 Number of Cells 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 used in the treatment of gout, cirrhosis, and psoriasis, among other disorders (BenChetrit and Levy, 1998). During metaphase, chromosomes attach to the mitotic spindle via their kinetochore and oscillate at the equatorial region under high tension. By interacting with tubulin, a component of the spindle fibers, colchicine decreases this tension, suspending the chromosomes in metaphase (Jordan and Wilson, 2004).

Develop a Hypothesis: Based on the information above, propose null and alternate hypotheses about the stages of mitosis that you would expect to see in cells treated with colchicine. Write your hypotheses (Ho and Ha) below.

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Question: Given colchicines properties, could this compound be used to treat cancer? Explain.

______________________________________________________________________________ TASK 3 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.

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Figure 8. 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 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.

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Figure 9. Oogenesis

Questions: 1. Why do gametes have only 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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Procedure: 1. Examine prepared slides of sperm from humans, rats, and guinea pigs and draw what you see in the space provided below. How do the sperm from the three species compare?

Magnification: _________ Magnification: _________ Magnification: _________

2. Examine a cross section of a monkeys 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: _________

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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: _________

4. Examine the slide of a mature follicle (Graafian follicle) and draw what you see in the space provided below.

Magnification: _________

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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 Meiosis

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?

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3. How do the sizes of the oocytes differ as they move from the follicle stage towards the mature Graafian follicle?

4. How do sperm and eggs differ in size? (Hint: consider size and the quantity of each gamete). Explain a possible reason for these differences.

5. What would happen if females produced 100s or 1000s of eggs during each cycle? What if males were born with a limited number of sperm?

______________________________________________________________________________ TASK 4 - Karyotype Analysis Karyotyping is the process by which scientists microscopically visualize the complete set of chromosomes in an organism to detect any possible chromosomal abnormalities such as deletions, translocations or the insertion of extra copies. 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

Abnormality/disorder (alternative name) Down syndrome (Trisomy 21) Turner syndrome (Gonadal dysgenesis) Cri du chat (cry of the cat)

Symptoms Cognitive disabilities, characteristic physical features, congenital heart disease Gonadal dysfunction, characteristic physical features, congenital heart disease Some abnormalities include 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

Karyotype 3 copies of chromosome 21 1 copy of the X chromosome Truncated chromosome 5

Edwards syndrome (Trisomy 18)

3 copies of chromosome 18

Patau syndrome (Trisomy 13)

3 copies of chromosome 13

Table 5: Chromosomal abnormalities that can be detected by karyotype analysis

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Procedure: A fellow scientist of BCBB Cytogeneics 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. It is your task to examine both karyotypes (#1 and #2), record your findings in the tables provided, and then report them to your colleague.

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

Remarks

CH# 19 20 21 22 23 24

Remarks

CH = Chromosome

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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

CH = Chromosome

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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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. ______________________________________________________________________________ LOOK AHEAD: Before coming to lab next week, make sure to read the Mendelian Genetics task sheet as well as Chapter 17 in your lab manual.

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