S4: Fertilisation and the Luteal Phase

Question 1

Describe ejaculated sperm

Answer 1

• During ejaculation, the sperm exit the epididymis and get mixed with secretions from the seminal vesicles and the prostate.
• This coagulates which makes the semen thick and gloopy, this keeps the sperm and semen together, preventing everything separating. This helps target the whole ejaculate at the entrance to the cervix.
• After a little while, the ejaculate starts to liquefy.

Question 2

Why is it easier for sperm to pass into cervix to get into uterus during the ovulation part of the menstrual cycle in women?

Answer 2

• If the woman has ovulated, there will be more blood flow to the cervix, the cervical os will be more open and there will be more oedema causing the cervical mucus to be thinner.
• The cervical mucus is thin when oestrogen is high around ovulation and this makes it easy for sperm to swim up.

Question 3

Why is it harder for sperm to pass into cervix after ovulation? e.g. how does it aid implantation but not fertilisation

Answer 3

• After about 6/7 days post-ovulation, progesterone reaches its highest levels, this causes the cervical mucus to thicken to prevent sperm entering as well as any pathogens.
• This is because by 7 days, the blastocyst would have implanted and if the oocyte hasn’t been fertilized it is now too late for fertilisation so we don’t want sperm entering into the uterus.
• However the high progesterone is good for implantation, if there has been fertilisation, we now have a receptive differentiated endometrium to accept the egg and we have a plugged cervical os to prevent sperm entering or pathogens.

Question 4

How does seminal fluid affect ejaculated sperm?

Answer 4

The seminal fluid contains factors that prevents the sperm undergoing capacitation/acrosome reaction too quickly. The sperms movement through the cervical mucus removes the seminal fluid as well as abnormally morphological sperm and cellular debris, so is like a filter ensuring only the best sperm get to the uterus. By the seminal fluid being removed it means they can then undergo capacitation.

Question 5

What are cervical crypts?

Answer 5

They are grooves in the cervix. Some sperm get stuck in these crypts and this forms a reservoir of sperm that over the next few hours/ days, sperm will leave the crypt and swim up to the uterus.

Question 6

How long does it take sperm to reach the oocyte?

Answer 6

Usually the sperm will reach the oocyte in about 24-48 hours, so will survive around this amount of time. However sperm has been found in the female even up to five days after intercourse.

Question 7

Describe the passage of sperm through the uterus to the egg

Answer 7

• The passage of the sperm through the uterus is not well understood, it may be that uterine (in uterus) or tubal (in uterine tube) cilia may have a role.
• Chemotaxis also seems to play a role, with chemoattractants being released from the cumulus oocyte complex that attracts the sperm, or it could be chemoattractants from the ovary. Progesterone may act as an attractant.
• As the sperm undergo capacitation they become hyperactivated, their tail beats more forcefully with increased frequency and amplitude. This hyperactivated state of the sperm is thought to be mediated by influx of Ca2+ into the sperm via CatSper channels

Question 8

What are CatSper channels?

Answer 8

These CatSper channels are voltage gated cation channels om sperm. It appears they are set by pH, as the sperm gets closer to the egg, the environment becomes more alkaline and this opens the CatSper channels Ca2+ rushes in and the sperm become hyperactivated. They require this hyperactivation to give them the push and accelerates them to get to the egg and this occurs during capacitation of sperm.

Question 9

Describe capacitation

Answer 9

• A series of changes involving biochemical rearrangement of the surface glycoproteins and changes in membrane composition in sperm.
• Partly achieved by removing the sperm from seminal fluid (that contains factors inhibiting capacitation) and also when the sperm enters into the uterus and uterine tubes the uterine/tubal fluid may contain factors that promote capacitation (decapitation factors).
• Takes place over 4-18 hours and must occur before acrosome reactions can occur. Both capacitation and acrosome needed for sperm to fertilise an egg.

Question 10

Describe acrosome reaction

Answer 10

• The acrosome reaction occurs when the sperm get right next to the egg, it is where the acrosome (holding the bag of enzymes) on the sperm head fuses with the egg releasing enzymes that cut through the outer layers of the cumulus that surround the oocyte.
• Acrosin is a digestive enzyme that is bound to the inner acrosomal membrane and digests the zona pellucida so the sperm can enter into the oocyte. This is the true acrosome reaction.

Question 11

What in the female is occuring at the same time as capacitation and acrosome reaction?

Answer 11

• We have ovulation occurring at same time.
• The sustained high oestrogen at the end of the follicular phase causes switch to positive feedback leading to the LH surge. The LH surge causes meiosis I to complete, the 1st polar body is extruded and you’re left with the secondary oocyte.
• Basement membrane breaks so blood pours into the middle.
• In ovulation, the dominant follicle is quite hugely inflamed due to LH. The cumulus oocyte complex (oocyte now haploid) is extruded out along with some fluid and this is caught by the fimbriae of the uterine tube. Prostaglandins are important in mediating this.
• A mixture of theca and granulosa cells are left behind.

Question 12

What is the luteal and secretory phase?

Answer 12

• The luteal phase refers to the follicle and secretory phase refers to the endometrium, but both are the same stage after ovulation, the second half of the cycle.
• The theca/granulosa mix gets lutenised to the corpus luteum. The corpus luteum makes the endometrium secretory and receptive to implantation.

Question 13

Describe progesterone mechanism in secretory phase

Answer 13

• By the middle of the secretory phase progesterone reaches its peak and suppresses the cilia and secretions in the uterine tubes because the oocyte has already passed so we get rid of these cilia and its too late for fertilisation.
• The high progesterone after about 6/7 days post-ovulation causes the cervical mucus to become thick and viscous again, to prevent further sperm penetration by plugging cervical os in case there has been fertilisation. This prevents more sperm and pathogens entering that would harm the embryo.
• Together high progesterone makes the perfect environment for implantation of an embryo and shifts away from conditions of fertilisation as it is too late after 6/7 days because the oocyte/embryo will be ready to implant.

Question 14

Describe oestradiol mechanism in luteal phase

Answer 14

The corpus luteum does release some oestradiol where it helps maintain the endometrium in the luteal phase. However it plays a lesser role in the luteal phase and its main role is in the follicular phase causing proliferation of the endometrium.

Question 15

Describe the formation and function corpus albicans (demise of corpus luteum)

Answer 15

• If fertilisation doesn’t occur the corpus luteum has an inbuilt finite lifespan of 14 days, so after 14 days it dies. This tells the body that fertilisation didn’t occur and an embryo didn’t implant.
• Eventually it becomes small white scar tissue called the corpus albicans.
• Once the corpus luteum has degenerated it stops producing progesterone and this fall in progesterone means the endometrium cannot be maintained and is shed and this is the start of the next menstrual cycle.
• At the same time the low progesterone causes FSH (and LH) to rise, this is the intercycle rise in FSH required for the next cycle that recruites new antral follicles.

Question 16

Describe role of hCG and LH receptors in pregnancy

Answer 16

• If there is a pregnancy we obviously don’t want the endometrium to be shed, it needs to be maintained for about 8 weeks until the newly formed placenta has developed and keeps the endometrium maintained for the rest of pregnancy.
• We prevent the endometrium shedding when there is fertilisation by the embryo trophoblasts releasing hCG, a glycoprotein similar in structure to LH. It binds to LH receptors on the corpus luteum and stops it from dying and maintains it keeping progesterone levels high and keeping the endometrium nice, thick and receptive.

Question 17

What is the hormone that facilitates and looks after the embryo if fertilisation has occurred?

Answer 17

Progesterone

Question 18

Describe what the menstrual cycle achieves (its purpose)

Answer 18

• Selection of a single follicle and oocyte each month.
• Regular spontaneous ovulation. By spontaneous it means that the human female ovulates every month regularly regardless of whether she is mating or not. Many animals only ovulate when they mate.
• Ensures there is a correct number of chromosomes in the oocyte. Meiosis I occurs as ovulation and meiosis II completes at fertilisation.
• Cyclical changes in the cervix and uterine tubes occurs to enable efficient egg transport and sperm access.
• Controls preparation of the endometrium of the uterus to receive by the corpus luteum releasing progesterone.
• It also allows support of the implanting of the embryo and endometrium by the corpus luteum releasing progesterone.
• If no fertilisation occurs, the corpus luteum dies and progesterone falls, the endometrium sheds and a new cycle has started.

Question 19

Describe the oocyte structure at ovulation

Answer 19

• First polar body is present as first meiotic division is resumed and completed at ovulation.
• Near ovulation the granulosa cells secrete the corona radiata and the granulosa become a loose complex that surrounds the egg.
• The cumulus oophorous are a cell layer that used to be granulosa cells. They protect the egg and secrete mucus.
• Underneath the cumulus cells is the corona radiata, this is a secretion of extracellular matrix that is made from a couple of layers of granulosa cells that just cover the oocyte. The main component of the corona radiata is hyaluronic acid (found in many ECMs).
• Cortical granules are important in fertilisation.
• Under the corona radiata is the zona pellucida. The zona pellucida is a glycoprotein layer secreted by the egg (oocyte) first while it is in the follicle. This is done by cytoplasmic projections from the oocyte secreting these proteins. At the LH surge these projections are withdrawn. The zona pellucida contains many important glycoproteins involved in binding sperm (e.g. ZP3).
• Underneath the zona pellucida is a small fluid filled space called the perivitelline space, this space undergoes changes at fertilisation and this helps block more than one sperm entering into the egg. It prevents polyspermy.

Question 20

What is redundancy of zona pellucida?

Answer 20

It is difficult to tell the roles of different zona pellucida individual proteins as the sperm may bind multiple proteins, this is redundancy. Also note that each species sperm binding proteins are specific, a mouse sperm cannot even bind/adhere to the ZP of a human egg. There is species specific fertilisation. This is why inter-species breeding doesn’t happen.

Question 21

Describe sperm binding (triggers of acrosome reaction)

Answer 21

• By the time the sperm reaches the egg it has undergone capacitation and is able to undergo the acrosome reaction.
• Some factors still unknown cause (probably chemotaxis factors from egg) cause the acrosome to burst releasing hyaluronidase, this digests through the cumulus cells and corona radiata.
• ## This allows the sperm to reach the zona pellucida and bind to ZP3 (adhesion molecule between zona pellucida an sperm) and possibly other proteins. This triggers the acrosome reaction.

Question 22

Describe sperm penetration in fertilisation of egg and the cortical reaction

Answer 22

• The sperm membrane then fuses with the oocyte plasma membrane (they are both phospholipid) and enters into the cell with the sperm nucleus falling into the oocyte cytoplasm. The tail of the sperm usually doesn’t enter, although in IVF we can just inject the entire sperm into the egg and cause fertilisation. In this case the oocyte discards the male mitochondria as all mitochondria DNA is maternal.
• When the sperm head fuses with the oocyte plasma membrane and the nucleus falls in it triggers a huge spike in calcium in the egg. This is believed to occur via the Phospholipase C pathway. The sperm has a special PLC called PLC-zeta, we believe when it gets into the oocyte it triggers the PIP pathway (PIP2–> DAG + IP3) that causes rise in Ca2+.
• This results in cortical granules that lay just under the oocyte membrane to release enzymes which is the cortical reaction.
• Mucopolysaccharides are released into the perivitilline space, they also release proteases that cleave adhesion molecules that the sperm binds to (thus nothing for other sperm to bind to).
• Finally peroxidases cause cross-linkage formation in the ZP making it very hard and preventing polyspermy (digestion from other sperm).

Question 23

stages of what occurs to ejaculated sperm for fertilisation

Answer 23

Capitation –> sperm binding –> acrosomal reaction –> sperm penetration –> cortical reaction of egg

Question 24

What is syngamy?

Answer 24

Syngamy is the fusion of two cells or their nuclei in reproduction and this is what occurs between the sperm and egg nuclei.

Question 25

Describe syngamy

Answer 25

1. After meiosis I the oocyte is haploid and contains 23 chromosomes, but each chromosome is bound to its identical sister chromatid.
2. At fertilisation the sperm binds to the ZP, penetrates it and fuses with the oocyte plasma membrane. This causes the increase in Ca2+ via PLC-zeta that then triggers the cortical reaction and also the completion of meiosis II and the expulsion of the second polar body. So the oocyte is now truly haploid. The sperm nuclear membrane breaks down and the chromatin decondenses and the chromosomes separate (remember sperm is also haploid)
3. 4-7 hours after fusion of sperm and egg, the two sets of haploid chromosomes become surrounded by distinct membranes forming pronuclei. Each haploid pronuclei starts to synthesise/replicate its DNA in preparation for the first mitotic division.
4. Then the two pronuclei fuse and the chromosomes become aligned on the mitotic metaphase spindle, the chromosomes move to their appropriate positions down the equator. So that each daughter cell will receive the chromosomes with their homologous pairs (i.e. so each has full 46, diploid number, 23 from dad, 23 from mum).
5. Mitosis is then complete and the one cell zygote has become a two cell embryo (identical replication).

Question 26

What is the first sign of fertilisation in IVF?

Answer 26

In IVF, the sperm and eggs are mixed and left over night in a nutrient medium. The next day if you see the two pronuclei in the egg, this is the first sign of fertilisation. Some people argue that this is actually the start of life.

Question 27

Describe early differentiation of embryo

Answer 27

• After three days the developing embryo contains 6-8 cells and is called a morula.
• After about 5 days after fertilisation we have approximately 100 cells and the embryo has started to differentiate. The embryo now called a blastocyst has an inner cell mass, a fluid filled cavity called the blastocoele and the periphery is surrounded by trophoblast that will become the placenta. The ICM cells will become everything of you! These are pluripotent cells.