Practical 3 07

Practical 3

Meiosis

Gametogenesis

Chromosomal sex determination

• Drosophila type

• Male – XY

• Female - XX

Sexual reproduction

2n

2nmeiosis

meiosis

n

n

Gametes (haploid)

sperm

oocyte

fertilization 2n

n + n = 2n zygote

♂

♀Life cycle:

haploid phase (gametes)

diploid phase (somatic cells)

Recommended website

• http://genetics.gsk.com/chromosomes.htm#chromo

Written test

• 8 minutes

• Don't forget to put down your name, your

group and the test version.

• In multiple choice questions more than 1

statement could be correct.

• Don't write anything on the question sheet!

Meiotic division

1st meiotic division

2nd meiotic division

2n

n n

n n n n

First meiotic division

• Heterotypic or reduction• Prophase

– Leptotene– Zygotene– Pachytene– Diplotene– Diakinesis

• Metaphase• Anaphase• Telophase

Leptotene• Beginning of prophase• Chromosomes can't be

distinguished. • Condensation of

chromosomes begins.

Zygotene• Attachment of homologous

chromosomes – the chromosomes

begin to synapse.

• Assemblage of chromosomes is

enabled by synaptonemal complex.

Pachytene• Bivalents or tetrades are present.

– 1 bivalent = 2 chromosomes

– 1 tetrade = 4 chromatids

– bivalent = tetrade

• Crossing-over between homologous

chromosomes is possible.

Crossing-over

• Reciprocal exchange of chromatid segments

between chromosomal homologues

Importance of the crossing-over• New combinations of alleles of genes that are

located on the same chromosome. • New allele combinations lead to higher variability

of the offspring (important in evolution). • This increases the potential for genetic variability

between members of the same species.

Chromosomes X and Y• are attached in

pseudoautosomal regions (PAR) during the 1st meiotic prophase

X

Y

SRY – sex determining region – is located on chromosome Y in the neighborhood of PAR.X

Y

Task A:

• An infertile man with azoospermia had been examined in cytogenetic lab. He has no other striking phenotypic features.

• The cytogenetic analysis revealed abnormal finding because his cells contained two X chromosomes while chromosome Y is missing.

• Explain this surprising result.

Crossing-over in PAR

• Nonreciprocal crossing-over can result in translocation of the SRY gene from the chromosome Y to chromosome X

Task A – result

male

female

Normal combination of gonosomes X;Y translocation

„XX-male“

„XY-female“XX

XY

Hermaphroditism – possible consequence of sex chromosome

abnormalities

Prophase of the 1st meiotic division

next periods

Pachytene

Diplotene• Condensation of chromosomes

continues

• Homologous chromosomes are divided

• Disassembling of synaptonemal complex

• At the end of diplotene homologues are connected only in crossover sites. These sites are called chiasmata (singular: chiasma).

Diakinesis• Final period of the 1st meiotic

prophase. • Chiasmata are transferred to

terminal chromosomal regions.

• Under specific staining chromosomes could be well distinguished under the microscope.

• Nuclear membrane disappears.

Terminal phases of meiotic division

prophase

metaphase + anaphase

2nd meiotic division

(homeotypic)

1st meiotic division

(heterotypic)

Separation of double-chromatid

homologous chromosomes

Metaphase I

Metaphase I

Anaphase I X Anaphase II

Gametogenesis

• Spermatogenesis – sperm development

• Oogenesis – oocyte development

n n

1st meiotic division

2nd meiotic division

n n

primary spermatocyte

secondary spermatocyte

sperm

n n

2nSpermatogenesis

n n

1st meiotic division

2nd meiotic division

n n

primary oocyte

secondary oocyte

mature oocyte

nn

2n

polar bodies

polar body

Oogenesis

A schedule of oocyte development

Prophase I

fetus childhood – dictyotene mature ovary

fertilization

Spermatogenesis X Oogenesis

Task B: Risk of chromosomal nondisjunction defects

• Compare possible risk of chromosomal

abnormalities for two couples that visited

the genetic counselling clinic:

– Couple 1: female (21 years), male (38 years)

– Couple 2: female (36 years), male (32 years)

• Which family has higher risk of numerical

chromosomal aberrations?

Task B – result

• Oogenesis lasts much longer

than spermatogenesis – risk of

chromosomal nondisjunction

defects is age-related.

• The second couple has higher

risk due to 36 years old female.

• In pregnant females older than

35 years prenatal examination

of the fetus is recommended.

35 years … risk 1/365

Age-related risk of the Down syndrome child

Mother´s age

Practical tasks

Task 1: Observation of meiosis in the grasshopper testis

• The slide was prepared from the testis of the grasshopper. After removing from the individual the tissue was fixed with methanol + glacial acetic acid (3 : 1) and stained with the Giemsa-Romanowski solution. After dehydration the tissue was mounted in the Canada balsam.

• Find the spermatocytes on the slide using 10x objective lens.

• Change the objective magnification into 40 or 45x and observe different phases of meiosis. For their determination use adjacent photos.

Task 2: Disjunction of the sex chromosomes during meiosis

• Redraw the scheme of disjunction of the sex chromosomes during meiosis of the male to your materials and fill it.

• In the first scheme (A) distinguish the chromosomes according to their shape and number of chromatids.

• In the second scheme (B) designate the chromosomes using letters X or Y no matter of the number of chromatids.

XYA B

Task 3: Changes of nuclear DNA concentration during the cell cycle

• Contrast the two types of cell division (mitosis X meiosis) by completing the following graphs below.

• Redraw the picture to your materials and fill curves demonstrating changes in the DNA content per one cell:– during the cell cycle followed with mitosis– during the cell cycle followed with meiosis.

• (Value „C“ on y-axis represents DNA content per one cell in G1 phase.)

G1 G2S M G1

C

2C

G1 G2S M1 M2 G1

Cell cycle + mitosis Cell cycle + meiosis

Nuclear DNA content

Task 3: Changes of nuclear DNA concentration during the cell cycle

Results

Task 3: Observation of meiosis in the grasshopper testis

Task 4: Disjunction of the sex chromosomes during meiosis

XY

X Y

XX YY

Scheme A Scheme B

Task 5: Changes of nuclear DNA concentration during the cell cycle

G1 G2S M G1

C

2C

G1 G2S M1 M2 G1

Cell cycle + mitosis Cell cycle + meiosisNuclear DNA

Assisted reproduction

• A group of methods that allows reproduction of

infertile couple that can't be therapeuted by

common medical treatment (e.g. using drugs of by

psychological intervention)

• The assisted reproduction could be also used for

prevention of genetic defects in families with

normal fertility but with increased risk of genetically

conditioned disorder for the offspring.

Assisted reproduction methods

• Intrauterine artificial insemination

– application of sperm to the uterus of the

female.

• In vitro fertilization and embryo transfer

– fertilization is performed outside the organism

of the mother

Artificial insemination

• homologous – the

female is fertilized with

sperm of her husband;

• sperm of anonymous

donor are applied – the

donor and the couple

remain unknown to

each other

In vitro fertilization

• Application of gonadotropins (= fertility drugs) induce maturation of numerous oocytes.

• The oocytes are removed from ovaries by biopsy. In a special dish they are mixed with sperms of the husband.

• After short term cultivation in medium with nutrients 3 embryos are transferred to the female's uterus.

ICSI (intracytoplasmatic sperm injection)

ICSI protocol

Preimplantation diagnostics• Genetic or other examinations of oocytes or

embryo before transfer to the mother's • We use

– Polar body– 1 – 2 cells from 8-cell embryo– More cells from blastocyst

Presentation

Ethical issues of assisted

reproduction

Next seminar

• In vitro cell cultivation

• Recommended websites:

– http://www.research.umbc.edu/~jwolf/method5.htm

– http://homepages.gac.edu/~cellab/chpts/chpt12/intro12.html

• Be careful when handling cell cultures!

• Test: previous topics (only seminars)

See you next week!