Activation, Pronuclear Formation, Parthenogenesis

Question 1

When is the oocyte activated to finally complete meiosis?

Answer 1

At fertilization (before the fusion of the pronuclei)

Question 2

Oocyte activation involves what ion for how long?

Answer 2

1. Ca2+ ion flux affect many factors re:the egg (important cytoplasmic rearrangements, embryogenesis)
2. Responses are both early (in seconds) and late (in minutes) and involve metabolic processes during fertilization to form a zygote

Question 3

During fusion of sperm and oocyte, which organelles come from which gamete?

Answer 3

1. Oocytes contribute membrane, cytoplasm, cell organelles (esp mitochondria) and macromolecular matrix.
2. Sperm contributes chromosomes and centrosome (with centrioles AND pericentriolar materials to help karyokinesis and cytokinesis).

Question 4

Oocyte activation consists of what four processes?

Answer 4

1. Release of cortical granules to block polyspermy.
2. Activation of oocyte metabolism (protein synthesis, DNA replication, and cytoplasmic movements)
3. Pronuclear formation
4. Subsequent mitotic cleavages

Question 5

What triggers the repetitive release of Ca2+ ions by the oocyte and why is it important?

Answer 5

Sperm; the intracellular calcium ion release is the hallmark of mammalian fertilization and its mechanism is still unknown

Question 6

What is one possible way in which sperm triggers calcium release by the oocyte?

Answer 6

Via a signal transduction cascade (meaning that stored information may be able to drive embryonic events)

Question 7

What are two hypothesis describing possible mechanisms for calcium release from the oocyte endoplasmic reticulum?

Answer 7

1. “Receptor hypothesis” = spermatozoa binds to a receptor and activates tyrosine kinase (or a G-protein-linked receptor) and then activates phospholipase C (no one knows how) in order to trigger the IP3 pathway (and diacylglycerol).
2. “Cytoplasmic factor hypothesis” = cytoplasmic factor is released from the fusing spermatozoa which may act on the Ca2+ channels to release it into the cytoplasms (note that sperm-specific protein to generate repetitive Ca2+/egg activation hasn’t been found for sure).

Question 8

Sum up the “Receptor hypothesis” for oocyte calcium release in 8 steps:

Answer 8

1. Sperm binds to oocyte receptor, activating tyrosine kinase.
2. Phospholipase C is activated (mechanism unknown).
3. Phosphodiinitosol (IP3) is split into inositol triphosphate and diacylglycerol.
4. IP3 releases Ca2+ into cytoplasm after binding to the receptor and opening the channels for Ca2+ in the endoplasmic reticulum.
5. Diacylglycerol activates protein kinase C.
6. Protein kinase C activates a protein that swaps Na+ for H+ ions which raises the pH of the egg (alkalic cytoplasm stimulated protein synthesis).
7. After alkalizing the cell and releasing the Ca2+ cell division (via degrading cyclin and inactivating MAP kinase) can occur. DNA synthesis, RNA translation are initiated in the late response and new protein synthesis now uses mRNA’swhich were already stored in the oocytes to produce new proteins and begin the next step of embryogenesis.
8. Oocyte metabolism is officially activated.

Question 9

What are three possible mechanisms for phospholipase C activation prior to embryogenesis?

Answer 9

1. Bindin receptor in oocyte plasma membrane has tyrosine kinase activity, which can activate phospholipase C.
2. The binding receptor may instead activate a cytoplasmic tyrosine kinase.
3. An activated tyrosine kinase in the SPERM’s plasma membrane activates the oocyte pathway after the sperm-oocyte membrane fusion.

Question 10

What are some important details/differences about the “Cytoplasmic Factor hypothesis” re: oocyte activation?

Answer 10

1. the factor from SPERM is released into the egg cytoplasm (based on prior studies finding something called “oscillin”)
2. This reacts to release the Ca2+
3. Process is delayed when sperm factor isn’t found** (wth does this mean)
4. Calcium ions are either directly released from oocyte endoplasmic reticulum, OR soluble activator pathways will activate phospholipase C to initiate Ca2+ release.

Question 11

What are the two distinctive type of activations that occur post-fertilization?

Answer 11

1. Diploid genetic constitution of zygote is ensured.

2. Developmental program is initiated.

Question 12

How are oocytes activated following ICSI?

Answer 12

1. Sperm is microinjected into oocyte at metaphase II (bypassing procedures of sperm-oocyte interaction).
2. Most oocytes are fertilized and activated and form embryos, which suggests that signaling for oocyte activation can be triggered by sperm without plasma membrane interactions.
3. Sperm factor hypothesis (II) may be responsible for the initiation of oocyte activation post-ICSI.

Question 13

What three experiments indicate the involvement of inisitol triphosphate in oocyte activation?

Answer 13

1. Found to appear at site of sperm entry (indicates correlation).
2. When injected into egg it releases calcium (sufficient to activate oocyte).
3. When its production is blocked no oocyte activation occurs (indicates its necessity).

Question 14

How long do the calcium oscillations take place and when do they end?

Answer 14

Anywhere from several minutes to several hours; end when pronuclear formation occurs.

Question 15

What have mammalian studies shown to occur when calcium oscillations have too high or too low amplitudes?

Answer 15

Pronuclei fail to form.

Question 16

What have mammalian studies shown to occur when calcium oscillations only occur for a short period of time?

Answer 16

Oocytes do not complete meiosis II.

Question 17

What have mammalian studies shown to occur when calcium oscillations have too high frequency and amplitude?

Answer 17

Abnormal oocyte activation and cell death.

Question 18

What is one important difference in pH in vitro and in vivo that may affect oocyte activation?

Answer 18

Ability to regulate intracellular pH is poor until 7 hours after oocyte activation, which means eggs fertilized in vitro may have difficulty maintaining proper pH for the first few hours in culture (could affect both embryo development as well as overall viability).

Question 19

New studies suggest what four things about phospholipase C zeta?

Answer 19

1. It’s introduced into the oocyte after membrane fusion.
2. It plays a role in oocyte activation.
3. It’s linked to some cases of male infertility (with a defect in its expression and activity).
4. It is suggested that it can be used as a therapeutic agent to rescue oocyte activation deficiency or as a diagnostic biomarker of oocyte activation ability in sperm samples.
   (taken from 2012 studies)

Question 20

How do the sperm and oocyte pronuclei form?

Answer 20

1. Sperm pronucleus forms by decondensation of chromosomes and reformation of nuclear membrane.
2. Oocyte pronucleus migrates from oocyte surface after completing meiosis to meet male pronucleus in center of cell.

Question 21

How do male and female pronuclei fuse?

Answer 21

1. They migrate to each other via microtubule asters (usually from the sperm’s centriole) which takes up to 12 hours in mammals
2. DNA synthesis can occur during migration or after fusion in mammals.
3. Nucelar membranes contact and break down and mitotic devises are assembled for new chromosome condensation.
4. Chromosomes immediately arrange on metaphase place for first cleavage division (which involves a special series of events that differs per species).

Question 22

When is a fertilized embryo labeled a “zygote”?

Answer 22

Upon completion of pronuclei fusion (diploid genes must be ensured and developmental program must be started).

Question 23

Which gamete (male or female) in humans donates the centrioles which pull each pronucleus together during syngamy?

Answer 23

sperm

Question 24

In mammals, defective aster formation (of microtubules from centrioles) during pronuclei syngamy can create fertility problems caused by which gender?

Answer 24

Male, because centrioles (asters) come from sperm.

Question 25

Which gamete (male or female) in mice donates the centrioles which pull each pronucleus together during syngamy?

Answer 25

Oocyte centrioles (in sea urchins, there is both but paternal is dominant usually).

Question 26

What is syngamy?

Answer 26

The fusion of male and female pronuclei during fertilization (third step following polyspermy prevention and cytosol stabilization).

Question 27

How long after fertilization does syngamy occur?

Answer 27

15 hours

Question 28

What happens as the sperm enters the oocyte prior to syngamy?

Answer 28

Microtobules condense around the sperm and the oocyte completes its second meiotic division. Then the spermatic nuclear envelop dissolves and its DNA uncoils in order for syngamy to occur (and its genetic makeup migrates towards the center of the oocyte)

Question 29

What is a human dispermic egg and how many centrioles will it have?

Answer 29

An egg fertilized by two sperm and it will have FOUR centrioles (one from each sperm).

Question 30

Explain the five morphological signs that an egg is undergoing fertilization.

Answer 30

1. Two polar bodies are clearly visible. 2. Two pronuclei exist (male is larger). 3. Sperm tail is visible although very small. 4. Sperm nucleus successfully decidedness because of the glutathione from the oocyte cytoplasm, which breaks the disulfide bridges.and will later help in the microtubule activity during the combination of pronuclei. 5. Oocyte shrinks because it releases water and its membrane is separated from zone, making the previtelline space more clear.

Question 31

When is the oocyte's second polar body extruded?

Answer 31

After sperm penetration and fusion activates the oocyte (and it subsequently finishes metaphase II).

Question 32

What kind of protein binds to and brings the male and female pronuclei together during syngamy?

Answer 32

Asper microtubules (like little highways).

Question 33

What marks the end of syngamy?

Answer 33

The male and female pronuclei membranes break down and each sex's homologous chromosomes are fully formed.

Question 34

The first mitosis following fertilization begins with what step?

Answer 34

Metaphase (then anaphase and telophase to form a 2-cell embryo).

Question 35

The first mitotic division forming a zygote is driven by coding from which parent?

Answer 35

Maternal mRNA previously stored in the oocyte.

Question 36

Define parthenogenesis.

Answer 36

Successful embryonic development (whether it survives or not) with both sets of genes contributed by one gender.

Question 37

What living things can reproduce via parthenogenesis?

Answer 37

1. Invertebrates: rotifers, nematodes, flatworms, and many insects like honeybees (to make drones). 2. Vertebrates: rarely in fish, salamanders, lizards, and some birds. 3. Experimental: frogs and sea urchins and mice have been experimented on in the lab and artificial treatments have been made to provoke sperm-like responses in the oocytes.

Question 38

How is parthenogenesis different than gynogenesis and androgenesis?

Answer 38

1. Gynogenesis involves sperm in egg activation but doesn't include male DNA in embryo (some salamanders). 2. Androgenesis also involves sperm for egg activation but the embryo doesn't include female DNA (some plants use this and used in vitro).

Question 39

What are two reasons why parthenogenesis fails in mammals?

Answer 39

1. Oocytes lack the centrosomes donated by sperm during fertilization. 2. Imprinting (some maternal and paternal genes are silenced).

Question 40

Attempts to create parthenogenetic mice led to what discovery?

Answer 40

Imprinted genes--that homologous chromosomes between males and females are not identical (are marked as male or female).

Question 41

Spontaneous parthenogenesis in mammals (including humans) can cause what pathological issue and why?

Answer 41

It can be followed by the appearance of a teratoma or teratocarcinoma and is indicative of a Mos deficiency in oocytes.

Question 42

What are the characteristics of an ovarian cystic teratoma?

Answer 42

1. MRI reveals mass attached to ovary. 2. Teratoma can contain ex, meso and endodermal tissue with variable differentiation. 3. In one cray-cray Japanese case a young woman had a dead little baby growing off her ovary (including brain and teeth and thyroid and blood vessels).

Question 43

What are six ways parthenogenesis (aka royal screw ups) can be caused in vitro?

Answer 43

1. Electric or temperature shock. 2. Exposure to certain enzymes. 3. Exposure to certain chemicals. 4. Exposure to alcohol. 5. Exposure to the calcium ion ionophore. 6. Aging of the oocytes - -These stimuli are thought to induce calcium transients which mimic the effects of sperm fusion.

Question 44

How did one Japanese study succeed in producing a fertile mouse lacking paternal DNA?

Answer 44

1. One success in 460 attempts, 2. Genetically altered one of the maternal genes (H19) to mimic male epigenetics. 3. Conclusion: both male and female genomes are needed for normal development.

Question 45

What are the two-part early steps to oocyte activation?

Answer 45

1. Sperm binding and/or fusion: Na+ influx --< membrane potential change --> fast block to polyspermy. 2. Sperm binding and/or fusion: Kinase stimulation --> Phospholipase C activation --> IP3 production and DAG production.

Question 46

What are the two late steps following the formation of IP3 and DAG production for oocyte activation?

Answer 46

2. IP3 Production --> Ca2+ release --> a. degradation of cyclin and inactivation of MAP kinase to restore mitotic cell cycle, b. activation of NAD+ kinase to convert NAD+ to NADP+ for membrane biosynthesis, c. cortical granule exocytosis for slow block to polyspermy as well as formation of hyaline layer, and d. stimulate protein synthesis, DNA replication, and cytoplasmic movements. 2. DAG Production --> Protein kinase C activation --> Na+/H+ exchange --> Increase in intracellular pH --> Stimulation of protein synthesis, DNA replication, and cytoplasmic movements.