Fertilisation Flashcards
How do sperm gain their fertilising capacity?
- Interactions with the female reproductive tract. Sperm is deposited in the vagina, moves through the cervix, uterus and oviducts to the site of fertilisation. During this journey, sperm undergo a series of interactions and events that culminate allow it to undergo fertilisation the oocyte. This process is known as sperm capacitation.
- Sperm Capacitation = further and final maturation process which confers the following functional characteristics on to sperm and ultimately renders them capable of fertilisation:
1) A change to a state of hyperactivated motility = whiplash flagellar movements by the sperm crucial for the penetration of the oocyte vestments. Hyperactivated motility occurs in the female tract in response to secretions from the COC, follicular fluids, uterine fluids.
2) The ability to bind to the oocyte’s zona pellucida and afterwards undergo the acrosome reaction. There are also changes in the membrane properties of the sperm that render it more able to bind to the oocyte’s zona and membrane.
3) The capacity to fuse with the oocyte. The final part of sperm capacitation is the capacity to fuse with the oocyte and complete the fertilisation process.
By what four mechanisms do sperm navigate through the female tract?
1) Chemotaxis
- Chemical guiding mechanism.
- Sperm responding to a gradient of chemoattractant, e.g. steroid hormones from the COC (progesterone).
- Chemotaxis is a response to chemical gradient stimuli. It is the interaction mechanism that has the most robust data in support of it to confirm it is definitely occurring. There is a lot of data on thermotaxis too, but a lot is still yet to be understood (most of the data on thermotaxis just comes from one research group, so a lot of understanding and validating is required).
- Molecular and behavioural mechanisms yet to be fully understood.
2) Thermotaxis
- Temperature guiding mechanism.
- Female tract consists of different areas with marginal differences in temperature.
- Studies carried out on some mammalian species have shown that the fertilisation site in the oviduct is 1 to 2°C warmer than some other parts of the Fallopian tube.
- It is believed that there are certain regions in the female tract that are one to two degrees cooler than others. This is believed to be a guiding mechanism. However, a lot of the data has been in vitro; it is quite difficult to characterise thermotaxis in vivo.
- Sperm responding to changes in the extracellular temperature gradient.
3) Rheotaxis
- Ability to respond to fluid currents in the female tract environment.
- Rheotaxis is a response to direction of fluid currents and boundary following navigation. Related to follicular fluid, uterine and tubal fluid etc. A lot is still yet to be known in terms of the mechanisms behind Rheotaxis. The data in support of rheotaxis is not as robust as chemotaxis and there isn’t as much as thermotaxis.
4) Boundary-following navigation
- Takes advantage of the sperm’s ability to turn corners in response to surface boundaries
- Human sperm with preference to follow boundaries on the left or right hand side have been shown to possess higher DNA integrity than straight swimming sperm (Huang et al., 2014; Eamer et al., 2016).
- A new concept in terms of interaction mechanisms is boundary-following navigation. This was studied using a microfluidic chip; loaded sperm cells on a chip with a microscopic version of a maze and observed different motility patterns. Found that straight-swimming human sperm had lower DNA integrity (more DNA fragmentation) than sperm with preference to follow boundaries on the side. Sperm in the female tract are not just swimming in the vacuum; they are actually crawling along the surface of the female tracts to get to the site of fertilisation.
What is CatSper?
CatSper = Cation channel of sperm. This is the principle sperm calcium channel that is located in the flagella area. It is responsible for calcium influx. This calcium influx triggers downstream signalling and any interaction mechanisms at the molecular level.
- A lot of the stimuli that results in chemotaxis, thermotaxis, rheotaxis and boundary-following navigation mechanisms are mediated largely via the CatSper channel. The CatSper has been characterised to be responsive to a wide range of stimuli, e.g. progesterone, prostaglandins, pH, small organic molecules; this is being actively studied and the list is growing. It responds to changes in membrane potential, pH, steroids etc.
Outline the mechanism that brings about the changes associated with sperm capacitation.
- A lot of studies have been carried out over the last decades to put the pieces of evidence together with regards to the process. These are the key concepts understood so far.
- The sperm cell membrane can be seen with different ion channels and receptors. One of the key events that occurs during sperm capacitation is the loss of cholesterol from the cell membrane. This alters the phospholipid to cholesterol ratio in the cell membrane, affecting the availability of certain soluble proteins in the cell.
- When characterising cholesterol loss, sperm cells in media can be looked at in the lab. If the media is supplemented with a cholesterol acceptor, it would result in an increased proportion of sperm cells in that culture undergoing sperm capacitation (switch to hyperactivated motility).
- One of the most popular and well-characterised cholesterol acceptors is a protein such as serum albumin. Supplementing culture media with serum albumin and using it to culture sperm cells for a few hours would lead to an increase in capacitation in the cells.
- This change in membrane fluidity due to cholesterol loss affects the availability of soluble membrane proteins, such as SACY.
- Looking at the CatSper channel, it is known there is a natural inhibitor that exists endogenously in the sperm cell. This is 2AG. In the absence of any activation, this is responsible for keeping the CatSper channel inactive.
- A recent study in 2016 showed when progesterone binds to ABHD2 (endocannabinoid receptor, known as Alpha/Beta hydrolase domain containing protein 2) in the sperm membrane, this complex breaks down 2AG (2-arachidonoylglycerol). Since it breaks down 2AG, it releases the CatSper channel and allows calcium influx from the extracellular environment. Breakthrough study that led to the discovery of the progesterone receptor. It was known that progesterone stimulated calcium influx and a lot of other physiological changes, but it was not known what the progesterone receptor was prior to that study in 2016.
- It is known that the progesterone/ABHD2 complex breaks down 2AG into arachidonic acid and glycerol. This frees up the CatSper channel for calcium influx. SACY (Soluble adenylyl cyclase) is a membrane protein; in order for it to be activated, calcium influx and bicarbonate influx are both required. Bicarbonate enters through the sodium-coupled bicarbonate transporter (NBC) Once activated, SACY converts ATP into cAMP. CAMP then goes on to activate PKA. PKA then facilitates the tyrosine phosphorylation of specific proteins that are involved in the changes associated with sperm capacitation, e.g. hyperactivated motility.
- While the diagram suggests progesterone is the main extracellular ligand, this is just for illustrative purposes. In real time, in vivo, the follicular fluid itself contains a vast array of proteins and steroids that would be in the extracellular space. When it comes into contact with the COC, there are lots of other things found in the extracellular space that interact with the sperm membrane.
- Losing cholesterol from the cell membrane affects membrane fluidity and the availability of SACY, because it is usually located very close to the membrane in its natural state. Once progesterone (or whatever the ligand is) has freed up the CatSper channel and allowed calcium and bicarbonate influx, SACY will be activated. It is this protein that then converts ATP into cAMP and results in the changes associated with sperm capacitation.
What happens in the sperm-oocyte interaction?
- Once a few capacitated sperm make it to the site of fertilisation, they come in contact with the cumulus-oocyte complex (COC) in the oviducts and a much higher concentration of follicular fluid (FF).
- FF in addition to secretions from the COC, e.g. progesterone, further modulate the spermatozoa to initiate the process of sperm-oocyte interaction and subsequent fertilisation.
1) Penetration of the cumulus oophorus (cumulus cells that surround the oocyte)
2) Sperm-zona binding (sperm binds to the ZP surrounding the oocyte)
3) Acrosome reaction
4) Sperm-oocyte fusion and oocyte activation
The first step in the sperm-oocyte interaction is penetration of the cumulus-oophorus. How does this occur?
- A visible feature of sperm capacitation is the switch from progressive motility to hyperactivated motility, characterised by whiplash tail movements. This is necessary for penetrating the oocyte vestments. Starting with penetration of cumulus cells; at this point, the sperm cell is capacitated, whiplash tail movements provide the momentum required to get through the cumulous cell mass. Looking at the oocyte, the inner circle shows oocyte cell membrane, the next circle shows the ZP and the surrounding cells with nuclei are cumulus cells.
- Once sperm come in contact with the cumulus-oophorus cell mass surrounding the oocyte, the enzyme hyaluronidase present on the surface of the sperm head dissolves hyaluronic acid (hyaluronan) – the major cementing factor between cells that constitute the cumulus-oophorus cell mass.
- Hyaluronidase, present in the sperm head, dissolves the binding factor (hyaluronic acid). Hyaluronic acid is the factor that holds the cumulus cells together. Hyaluronidase in the sperm cells breaks down hyaluronic acid to make its way through to the oocyte.
The second step in the sperm-oocyte interaction is sperm-zona binding (sperm binds to the ZP surrounding the oocyte). How does this occur?
- Following penetration of the cumulus-oophorus, the sperm cell can be seen at the zona. In real time, there are usually a bunch of sperm that surround the egg and a lot of them are working away at the cumulus, but only one makes it in.
- Four zona pellucida proteins have been characterised to be present in the ZP, the protective glycoprotein coat that surrounds the oocyte. The four zona pellucida (ZP) glycoproteins expressed = ZP1, ZP2, ZP3, ZP4.
- Studies across mammalian species have shown that sperm bind to ZP2 and ZP3 via receptors present on the sperm’s plasma membrane.
- Recent studies by Yauger et al. (2011) and Baibakov et al. (2012) show that human sperm bind to ZP2.
- These studies used transgenic mice containing oocytes from mice with human ZP. They mixed these with human sperm and found that human sperm only bound to ZP2 on these transgenic oocytes.
The third step in the sperm-oocyte interaction is the acrosome reaction. What is this?
- This is, by definition, the release or the endocytosis of enzymes that are present in the sperm, in the head (the acrosome region). This is triggered usually in response to sperm-oocyte interaction or in response to signals within the female reproductive tract.
- Triggered in response to sperm-oocyte interaction
- Looking at the intact sperm, just underneath the plasma membrane, the outer acrosomal membrane can be seen. This outer acrosomal membrane fuses with the plasma membrane just over it and this results in perforations in the membrane that result in the release of acrosomal enzymes.
- Fusion of the sperm’s plasma membrane with it’s outer acrosomal membrane results in the release of hydrolytic and proteolytic enzymes e.g. hyaluronidase and acrosin.
- Some literature says acrosome reaction occurs when sperm comes in contact with the COC, while others say acrosome reaction occurs during sperm-zona binding. However, in vitro studies that culture sperm cells with progesterone can trigger acrosome reaction, culturing sperm cells with calcium ionophores will also trigger acrosome reaction. This shows that sperm acrosome reaction can be triggered in vitro and, in vivo, it means sperm acrosome reaction can occur outside of the vicinity of the oocyte. Important to note = when it comes to acrosome reaction, there are different subpopulations of sperm undergoing acrosome reaction at different points in their journey in the female reproductive tract; the ones that make it to the oocyte are the ones that undergo acrosome reaction at the right place and time.
- When a couple of eggs are inseminated during IVF, the sperm will make it to different stages.
- When a couple of eggs are inseminated with thousands of sperm, many sperm will be seen having done the heavy lifting (undergoing acrosome reaction to break down this COC), some cells will also be seen in the perivitelline space that didn’t quite make it through (underwent acrosome reaction in contact with the zona), but only one sperm cell will have made it in (underwent acrosome reaction at the right place and time).
- There are some schools of thought that believe that there are certain things in the female tract that keep all of the sperm from undergoing acrosome reaction too early before it gets to the site of fertilisation. One thing is for sure, it is possible for spontaneous acrosome reaction to occur before it gets to the oocyte, but the ones that actually make it to fertilise the oocyte undergo the acrosome reaction at the right place and time.
The fourth step in the sperm-oocyte interaction is sperm-oocyte fusion and oocyte activation. How does this occur?
- When looking at sperm-oocyte fusion and oocyte activation, recent studies have shown there is a protein binding that occurs in order for sperm-oocyte fusion to be successful (Juno-Izumo binding = Juno on the surface on the egg binds with Izumo on the sperm). This is something that has to occur in the case of sperm-oocyte fusion. When sperm successfully fuse with the oocyte, there is intracellular calcium release from intracellular stores within the oocyte (usually the endoplasmic reticulum). This triggers a cortical reaction, where there is a release of cortical granules) that prevents further sperm entering.
- This intracellular calcium spike in turn triggers oocyte activation. This comprises of a series of events, starting with the release from meiotic arrest and the completion of meiosis II.
- At the point of ovulation, the oocyte is arrested in metaphase II (meiosis II). From the LH surge and ovulation, there is maturation that releases the oocytes from arrest in meiosis I and takes them to meiosis II. Oocyte activation releases the oocyte from metaphase II arrest and completes meiosis II.
- Another event in oocyte activation is the incorporation of sperm DNA and pronuclear formation. Embryo cleavage also begins. The completion of that second meiotic division is characterised by the extrusion of the second polar body.
- Intracellular calcium spike triggers a cortical reaction → Release from meiotic arrest and completion of meiosis II,
Incorporation of sperm DNA and pronuclear formation, Embryo cleavage.
How does the sperm trigger oocyte activation?
- Phospholipase C Zeta (PLC-ζ) is a soluble sperm protein that is introduced in the oocyte to trigger the events of oocyte activation.
- This was recently discovered in 2002 by Saunders et al. and is now widely accepted as the oocyte activation factor introduced by sperm. There has been some data that has been published and some coverage on it by a group in Canada where they challenge the role of PLC-Z as the soluble sperm activator of the oocyte. They proposed an alternative protein (post-acrosomalWW-domainbinding protein(PAWP)). They have been able to show some evidence of activation as well, but PLC-zeta is still widely accepted as the oocyte activation factor introduced by sperm.
- It acts by activating inositol trisphosphate (IP3) production, leading to repetitive calcium release (oscillations) from the oocyte’s calcium stores.
- PLC-Z breaks down phosphatidylinositol 4,5-bisphosphate (PIP2) into IP3 and diacylglycerol (DAG). IP3 then binds to the IP3 receptors on the endoplasmic reticulum and there is calcium release.
- A lot of clinical studies have shown that PLC-Z deficiency has been linked with male infertility (including poor sperm quality).
- Has been identified in all species studied so far, including human.
What are the two key features that are assessed in fertilisation via ART?
- When the embryologist assesses fertilisation (under the microscope or in real time during assisted reproduction), there are two key features that are assessed in the oocyte = pronuclei and polar bodies.
- In a normally fertilised oocyte, the second polar body should be extruded due to the completion of meiosis II and there should also be two pronuclei (one from the sperm’s genetic material and the other from the oocyte’s genetic material. Should also have well-positioned nucleoli as well.
- Clinically, a normally fertilised oocyte is characterised as 2PN and 2PB
1) Pronuclei (PN)
- 2PN = Normal fertilisation. Normally fertilised oocytes should contain two juxtaposed and centrally located PN, with distinctly clear membranes and nucleolar precursor bodies (NPBs).
- 0PN = No fertilisation
- 1PN = Abnormal fertilisation. Contains a haploid set of chromosomes (nuclear material of only one gamete was incorporated). There are cases where a 1PN zygote contains a diploid set of chromosomes, resulting from errors in the synchrony of PN formation/fusion. Zygotes in this category often have 2 polar bodies.
- ≥3PN = Abnormal fertilisation. Contains additional set(s) of chromosomes, which could be either digynic or diandric (can come from the egg or sperm). Significantly linked to the formation of aneuploid embryos.
2) Polar body (PB)
- 0PB = Oocyte at the Metaphase I stage of meiosis.
- 1PB = Mature oocyte at the Metaphase II stage of meiosis with one polar body extruded.
No fertilisation.
- 2PB = Oocyte has completed second meiotic division and extruded second polar body in response to fertilisation.
- There are some cases where there is 1PN and 2PB. This means that it has completed meiosis II in response to the sperm and it is likely that fertilisation has occurred. This may be because there is a fault in the process that results in the formation of pronuclei, so it occurs out of sync. Normally, when looking at the formation of pronuclei in a normal case (in a time-lapse), they usually appear in sync, but there are some cases where one will appear a while before the other. At the time it is being observed in the embryology lab, it may either have been missed or one hasn’t appeared yet. In these cases, they can be closely observed as they develop into embryos.
- The classical case where fertilisation did not occur properly = 1PN and 1PB.
- Digynic is when the nuclear material is internalised (instead of the oocyte releasing the polar body and completing meiosis II). The set of chromosomes present in the polar body appears as an extra pronucleus. The diandric case is when multiple sperm manage to enter the egg (incorporated as additional pronuclei).
- Often, during oocyte retrieval in IVF, there are metaphase I oocytes as well, especially in cases where they have been scheduled for ICSI (where sperm is manually injected into the oocytes,. The surrounding cumulus cells have to be removed to see the maturity of the oocyte, as only mature oocytes can be injected. The desired metaphase II oocytes are separated but, in some cases, metaphase I can be left in culture and observed again later. If they manage to cross over to metaphase II then they are added to the rest of the cohort of injection. If they do not crossover, they are discarded.
Which of these cases are fertilised
1) 0PN, 1PB
2) 2PN, 2PB
3) 1PN, 1PB
4) 2PN, 2PB
5) 1PN, 1PB
6) 3PN, 2PB
1) Unfertilised – 0PN, 1PB
2) Normal Fert – 2PN, 2PB
3) Unfertilised/abnormal fertilisation – 1PN, 1PB
4) Normal Fert – 2PN, 2PB
5) Unfertilised – 1PN, 1PB
6) Abnormal Fert – 3PN, 2PB
- It is important to notes that the oocyte is not a 2D structure; may have to roll it within culture to see all polar bodies clearly under the microscope.
- In a case of abnormal fertilisation when there are multiple pronuclei, there can be cases where there is one polar body or cases where there are two. One polar body would be a digenic situation; the oocyte internalised the nuclear material from the polar body instead of extruding it. The nuclear material from that polar body is manifesting as the third pronucleus. In cases where there are two polar bodies, it is likely to be diandric (additional sperm entered). It is also possible to have a combination of sperm (additional sperm and internalisation of the genetic material from the polar body as well).
Summarise the stages of embryo development from fertilised egg to hatched blastocyst.
- Once pronuclei formation and fertilisation is complete, oocyte activation continues by initiation of cleavage into the 2-cell embryo (made up of two blastomeres). These series of divisions continue.
- The morula is a ball of compacted blastomeres (would expect to have 32+ cells on average; the junctions and the spaces between each blastomere become tighter before they compact into what is termed the morula (appears mulberry-shaped)).
- On day 5, the formation of an early blastocyst can be seen; there is clear differentiation between the two blastocyst cell lines (the inner cell mass and the trophoblast with the blastocoele fluid).
- Going into day 6, the blastocyst starts to expand which is crucial for blastocyst hatching. There are proteolytic enzymes at the abembryonic pole (area of a blastocyst opposite the embryo or embryoblast); they digest the zona (still surrounds the embryo; quite thick until the embryo expands). Digestion at the abembryonic pole facilitates the hatching. With further expansions and contractions, through the bit that has been digested, it can make its way and herniate out.
- Once successful hatching has occurred, the blastocyst is now able to interact with the uterus.
