Preimplantation development and genetic diagnosis

Question 1

What is preimplantation development

Answer 1

• fetilisation marks the start of preimplantation development. during preimplantation development the embryo moves along the oviduct towards the uterus from the contraction of smooth muscle and sweeping of cilia, where the embryo implants

Question 2

How does fertilisation initiate development

Answer 2

• fusion of gametes initiates activation of egg metabolism and reactivation of meiosis
• fusion of sperm with egg leads to cortical granule release and activates signaling cascades that lead to the activation of the egg metabolism and the resumption of the second meiotic division

Question 3

What does fertilisation trigger

Answer 3

fertilisation triggers egg activation in matured oocyte
- a sperm-specific phospholipase C isoform has been proposed to be delivered into oocyte
- increased IP3 stimulates Ca2+ release from ER , which signals cortical granule exocytosis and resumption of meiosis II

• fertilisation also activates signaling pathways leading to release of Ca2+ from intracellular stores in the egg
• high calcium triggers more calcium to be released lasting 2-3 minutes followed by further spikes

Question 4

How does increased cytoplasmic calcium reactivate meiosis

Answer 4

• before fertilisation the egg is arrested in M phase by cytostatic factors (CFS) preventing the degradation of cyclin B component of M phase promoting factor
• CFS prevents anaphase-promoting complex
• increased Ca2+ concentrations leads to activation of CALMODULIN which activates CALMODULIN DEPENDENT KINASE II (CaMKII)
• CaMKII inhibits CFS so anaphase-promoting complex is released causing cyclin B to breakdown
• this allows exit of metaphase II and resumption of the cell cycle - activation of kinases leads to activation of signaling pathways important of embryo development

Question 5

What is cell cleavage

Answer 5

• a series of mitotic divisions while embryo travels in oviduct towards uterus

compaction: at 8-16 cells stage binding is tighter, tight junctions form, cells are polarised and cell division occurs with some cells remaining at surface and others are inner cell mass

the embryonic genome is developed by maternal factors initially but rapid degeneration of maternal mRNA causes embryonic mRNA to be produced

Question 6

How does the blastocyst form

Answer 6

after compaction the blastocyst forms
- outer cells: trophoblast cells
- inner cells: inner cell mass
cavitation: formation of BLASTOCOEL, outer cells pump Na+ inside embryo causing water to enter embryo

blastocyst hatches from zona pellucida - enzymes form a hole in the zona pellucida

Question 7

What occurs in preimplantation development

Answer 7

• after MORULA forms further cell division cause fluid-filled blastocyst, outer layers form part of placenta, inner cell mass forms embryo
• blastocyst implants into uterine lining around five days after fertilisation
• placenta formed by maternal and embryonic cells and is cite of gas exchange and nutrient/waste between maternal and embryonic blood

Question 8

What is gastrulation

Answer 8

• leads to a fundamental reorganisation of the embryo
• around 3rd week

inner cell mass differentiation into epiblast and hypoblast
- epiblast: cells divide and migrate inwards forming three layers of germ cells (outer ectoderm, intermediate mesoderm and inner endoderm)

Question 9

What is organogenesis

Answer 9

• transformation of germ layers into all organ systems of the body
• 3rd to 8th week
• ectoderm: outer layer of skin and nervous system
• mesoderm: circulatory system, muscle and bone
• endoderm: lining of respiratory, digestive and lung tracts

Question 10

What is preimplantation genetic diagnosis (PGD) and its clinical applications

Answer 10

can be performed in combination with IVF, a biopsy is taken from embryo and genetically tested before deciding whether to transfer the embryo into the uterus

for
- detecting single gene defects
- detecting chromosomal abnormalities
- sexing

Question 11

How does PGD work for all of its clinical uses

Answer 11

single gene defects
- can test if a parent carries a known dominant mutation causing disease, or if both parents a carriers of a recessive disease-linked mutation
- can see if disease is transmitted to embryo

chromosomal abnormalities
- can test for translocation or trisomies normally seen in older women or after frequent miscarriages

sexing
- selecting the sex that cannot inherit certain sex-linked mutations

Question 12

What are the different biopsy approaches

Answer 12

• first polar body can be tested for gene mutations or aneuplodies before fertilisation
• after fertilsation second polar body can be tested (only tells about maternal genotype)

Question 13

What methods are used for genetic anaylsis

Answer 13

• PCR and DNA sequencing
• FISH (fluroescence in-situ hybridisation)

is now being replaced by new techniques

Question 14

PCR and DNA sequencing for genetic analysis

Answer 14

PCR and subsequent sequencing of PCR product can be used to determine of embryo has inherited any single gene mutations

can also be used for sexing by amplifying parts of Y chromosome

Question 15

How does FISH work for genetic analysis

Answer 15

a DNA or RNA piece complementary to genomic sequence is labelled
- directly with fluorophore
- indirectly with hapten molecule that will be later detected

probe and genomic DNA are denatured and combined, allowing probe to bind to complementary genomic sequence

FISH can also test for gender and chromosomal abnormalities