Kickstart Summer Programme
22 nd July 2010
Cell Biology Practical
Wellcome Trust Centre for Cell
Biology.
University of Edinburgh.
1.- BRIEF INTRODUCTION TO MEIOSIS.
Sexually reproducing organisms need a special kind of cell division called meiosis to produce sperm and eggs (gametes). During meiosis one original cell divides twice to generate four daughter cells with half the number of chromosomes (haploid). In this way when a sperm and an egg fuse during fertilization the normal (diploid) number of chromosomes is restored (sperm and egg each contributing half).
Meiosis is also an essential process in the generation of genetic diversity: during the first meiotic division the chromosomes exchange segments through the process of recombination, and afterwards the chromosomes are distributed randomly through division. The combination of these two processes ensures that each resulting gamete has a unique combination of genes. Genetic diversity will give the species an adaptive advantage.
Abnormalities in meiosis lead to embryos with the wrong number of chromosomes (aneuploid). In humans this normally leads to early death of the embryo. In some instances however, aneuploid individuals survive and live to adulthood. An example of this is Down’s Syndrome.

2.- WHAT YOU WILL SEE TODAY: MEIOSIS IN MALE FLIES.
In male flies meiosis occurs in the context of a complex developmental process called spermatogenesis. We will observe this process using phase contrast microscopy.
In the apex of the testis there is a proliferation centre, in which germ line stem cells divide asymmetrically to generate the primary gonial cells.
Each primary gonial cell undergoes four mitotic divisions, giving rise to a cyst of
16 primary spermatocytes.
Proliferation center: gonial mitosis
Young primary spermatocyte cyst
These primary spermatocytes enter a growth period in which their nuclear volume increases 25-fold and in which they synthesize and accumulate all the
RNA and proteins required for the formation of sperm.

When primary spermatocytes enter meiosis the nucleolus disappears and the nuclear membrane outline becomes fuzzier:
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Cyst entering meiosis I
Mature primary spermatocyte nucleolus
During meiosis we can visualize the microtubule spindle because the mitochondria associated with it make it dark under phase contrast optics. This is an example of a cyst in meiosis I (telophase):
Daughter nuclei
(white)
Meiotic spindle
microtubules
(black)
The result of the two meiotic divisions is a 64-spermatid cyst. Each spermatid contains a haploid nucleus (white sphere) and a mitochondrial derivative (black sphere). They all should be the same shape and size. This is a good diagnostic stage, as any mistakes in meiosis will result in abnormalities in the sizes and ratios of nuclei/mitochondrial derivatives.

After meiosis the spermatids differentiate into sperm:
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Comet stage spermatids nucleus (white)
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mitochondrial derivative
Sperm bundles
(black)
In some instances we will be able to see mobile sperm in the slides:
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3.- Materials.
Plastic Petri dishes.
Phosphate Buffer (PBS) or saline solution (0.7% NaCl) .
Dissecting Forceps.
Glass slides and coverslips (20x20 mm).
3MM Whatmann paper.
Dissecting microscope with cold light source.
Phase contrast microscope equipped with 40x dry objective.
4.- METHOD.
1 Select male pharate adults in saline solution on a Petri dish.
No sex combs
(female)
Sex combs
(male) o Drosophila males have distinctive structures on the first pair of legs called sex combs (arrows) which will be used to distinguish them from the females).

-
2
3
Remove pupal case and membrane surrounding fly.
Dissect out testes.
4
5
Place tissue in a drop of saline solution on a clean slide
Cut testis open using dissecting forceps
6
7
Place glass coverslip on top trying to avoid air bubbles.
Squash gently using little pieces of filter paper to absorb the saline solution by capillarity. o Control the degree of squashing under the microscope using phase contrast optics (40x objective). If you oversquash, start all over again…
8 Try to identify the different stages of meiosis described in the previous section of the handout. You are observing living cells now, this is meiosis in action!
9 Warning: this is a temporary preparation, the tissue will not last for long!