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Critically evaluate evidence for plc zeta as the oocyte activation factor.
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CRITICALLY EVALUATE EVIDENCE FOR PLC ZETA AS THE OOCYTE ACTIVATION FACTOR.
Fertilization is the process where in a haploid sperm fuses with a haploid egg to form a zygote which undergoes repeated division to for a blastula. Proper fertilization required the presence of several recognition molecules where the egg and sper recognize each other and begin the fusion process. Recombination occurs during this stage and the genetic material is exchanged. Fertilization is immediately followed by the formation of a fertilization envelope and the influx of calcium ions. Calcium ions are sequesterd in the endoplasmic reticulum which are released during fertilization.
Infertility is one of the major problems in the growing population.It not only is a social problem but also a psychological and physiological factor involved in family medicine. Several government are investing a large amount of money on treating infertility in various health areas. The aim is to understand the nasic methods of reproduction after copulation which leads to various stages of embryo development. The most important method is in vitro fertilization. Females having abnormal ovulation problems can be treated by a method called in vitro maturation (IVM). Here a mature oocyte is prepared for invitro fertilization and is implanted into the body for development. An important role is played by the oocyte to activate itself. The capacity to produce calcium oscillations is one step in the activation process. The details of these are not properly understood and is required for further studies. Proper research in this are and targeting the right molecule can actually cause a drastic rise in the development of artificial fertilization techniques and treating several cases of infertility. Currently the best understood mechanism is the sequence of events after fertilization.
Oocyte activation commences when a sperm fuses with an oocyte leading an intricate cascade of reactions in the form of a signaling pathway leading to the development of early embryonic stage. The most important events occurring during this stage are the exocytosis of the cortical granule, formation of pronucleus, the recruitment of maternal mRNAs release of G1 arrest phase and beginning of embryonic gene expression. Ca2+ plays and important role in the activation of oocyte. Ca2+ is stored in the ER and is released during oocyte activation. This is predicted to be achieved by the involvement of a sperm- oocyte activation factor (SOAF). When the sperm fuses with the oocyte SOAF is released. Egg activation is triggered by a cross talk in between the sperm and egg whichlater leads to a cascade of signalling pathways eventually causing the release of phospholipase C (PLC). Even though this mechanism is accepted to a certain extent, the exact way in which this is done is a topic of debate and discussion among researchers. Injection of sperm into the egg known by the term cytoplasmic sperm injection paves way to strong dilemma over the receptor ligand mechanism in sperm and egg binding process. (Kline at al, 1992).
In order for any substance to be considered as a SOAF, any specif criteria must be attached to its function. This particular sperm factor should be capable of eliciting a Ca2+ response inseparable from that occurs during mammalian fertilization. One of the criteria high production of IP3 through phosphor inositol signaling pathway. Phospholipases are a group of enzymes present within the cytosol that are involved in the catalysis of phosphatidylinositol 4,5-biphosphate (PIP2) to produce inositol triphosphate and di acyl glycerol. IP3 results in the release of Ca2+ ions sequestered within the endoplasmic reticulum. The Ca2+ ions are involved in mediating cellular responses. Release of calciyum causes increase in the cytosolic calcium content by calcium induced calcium release mechanism. Currently about 384 phospholipases have been discovered which are isozymes that differ in their structure and regulatory mechanisms. PLCs may act as SOAFs since they play an important role in the activation of oocyte.
Embryological studies are based mainly on the analysis of a specific soluble sperm derived substance called Phospholipase c (PLC ZETA) (Strickeret al, 1999) in similarly to the events occurring during development of an embryo and fertilization. When a recombinant PLC Zeta protein molecule in mouse was injected, it immediately showed the beginning of calcium oscillations. PLC Zeta was discovered in 2002. Since then research has started in various labs around the world to understand its existence and to check whether it is the ultimate reason for beginning of Ca2+ oscillation. Experiments by Swann et al concluded the wide discussion and debate among researchers by discovering that Ca2+ oscillation assisted the activation of oocyte in non mammals like frog (Strickeret al, 1999).
Initially the molecules identified to be SOAFs were glucosamine-6-phosphate isomerase (GPI) andcitrate synthase but were later discarded. GPI is a molecule derived from the sperm and was known to induce the activation of the egg. After seveal failed experiments the molecule was discareded. Citrate synthase was also found to be a good SOAF but however the experiments were successful only in newt and were not promising in humans. Hence it was also removed from the limelight.
Initially it was believed that a sperm derived PLC protein was the most probable SOAF, due to its production of IP3 and the interaction with other PLC molecules. However there was a conclusion that no available moolecules were capable of inducing fertilization as that occurs in the normal fertilization in the body. Mouse expressed sequence tag database was uded to identify a novel form of PLC called PLC zeta, which is the smallest member among this family.
The structure of every biomolecule is well suited to its function, PLC zeta is a typical example for this. The structure is composed of two catalytic domains (X and Y),
EF hands, and a C2 domain. Any mutation in these domains lead to a falure in calcium evocation and can cause abnormal release. The activity of PLC zeta is high when it is injected into the ooplasm. Hence it is supposed that the oocyte plays an important role in inducing the enzymatic role of PLC zeta upon fertilization. The interaction of PLCC Zeta with vecicles with receptors in the oocyte which contains an important role for the egg
Along with the increasing evidence of biological and functional roles in oocyte activation, a large number of clinical experiments has helped to elucidate the role of PLZ zeta as the prominent SOAF candidate. The exact spatial location of the PLC zeta was to be identified to analyse the importance in human infertility. Inside the sperm head there are three locations where PLC zeta expression is concentrated. They are the acrosomal region, theequatorial segment, and the postacrosomal region. This suggests that the different populations are involved in plying multiple functional roles in the fertilization process and are not only limited to the activation of sperm. In males, sperms devoid of PLC zeta were unsuccessful in causing a Calcium oscillation. They also failed in ICSI when they were administered into the oocytes. In such cases the capability to cause oscillations were retained
Genetic associations found between infertility and PLC zeta were able to provide a support for the clinical relations and proved highly important in livestock breeding in a agriculture. An infertile nonglobozoospermic male sperm was taken and two PLC zeta mutant forms were identified. Detailed study of the structure revealed the
The first genetic link involved the identification of dysfunctional PLC zeta was the mutational substitution by histidine for proline at 398th position and at 233dr position by histidine instead of proline. These two locations were found in the catalytic domains, X and Y. The secondary structure of PLC zeta were disrupted due to these changes leading to an alteration in the function of the molecule. The altered structure failed to release the calcium or lead to the abnormal production of calcium oscillations.
ICSI is the most important milestone in the treatment modality for curing infertility in males and is currently considered as one of the most reliable techniques in this field. There is less tghat 5% chance of failure of ICSI and is considered to be due to Oocyte
activation deficiency. In such cases the common practice is to make use of artificial oocyte activators. This technique is used in several clinics but ther are sevelar controversies surrounding the harmful effects this technique has on the embryo. It is now opined that instead of using artificial oocyte activators, the use of a PLC zeta molecue would be much more effective and safer method for triggering calcium oscillation upon fertilization. The major challenge in this case is to extract the purified protein which is to be done using mammalian lysates of from cellines in bacteria. Even though several possibilities are present there are still concerns regarding the same when it comes to cinical application.
When a Nus-A-tagged protein was injected into the oocyte of a mouse it showed the presence of calcium oscillation similar to that found in normal fertilization. This is promising study carried out in a bacterial cell line. If mutations are present in the catalytic domains of the molecule also the newly administered PLC ZETA will play an important role in the activation of oocyte. There are several concerns regarding the length of the NUS-A tag and its necessity in the activation and stabilization of the sperm, there are enough evidences which prove that they play an important role as a SOAF candidate. Recent experiments also question the availability of proteins other that PLC zeta to complement its action so as to develop better drug candidates with similar effects.
About 20% of zinc in the cell were lost by the activation of mature eggs arrested at metaphase 2 by the exocytosis of zinc present in the cortex of the egg. This phenomenon, called zinc sparks is also found to be present in two other primate species other than humans. In order to identify the loss of zinc in human egg during activation process, the egg was treated with Caionomycin. This treatment causes the exogenous calcium from entering the egg even without the sperm induction and causes a rise in the intracellular calcium. (Saunders A et al., 2007). The presence of calcium within the cell and zinc outside the cell were observed with the help of live cell imaging using the dyes Fluo-4-AM and FluoZin-3. On exposing to 20 µM Ca-ionmycin, it was observed that a parallel rise in the Zinc was seen accordingly with that of interacellular calcium levels. Hence zinc exocytocis was coupled with an increase in calcium levels in the intracellular side. Hence a correlation between calcium transient and zinc spark can be observed in association with exocytosis of zinc ions. There are two different pathways for confirming that zinc spark was induced by the activation of egg. 1. Release of stored calcium while using ionomycin without calcium induction 2. microinjection of human PLC ZETA cRNA. In a male specific PLC Zeta, calcium osillations induced by sperm can be assisted by producing 1,4,5 inositol triphosphate from phosphatidyl inositol. This reaction is involved in mammalian fertilization through Calcium release mediated by IP3. (Kashir J. et al.,2014) (Carroll et all.,2002). According to this theory the first calcium transient is apparently linked to the production of Zinc sparks. On observing zinc exocytosis with FluoZin-3 during activation of the egg, it was noted that the amplitude of zinc sparks were not changed between individual eggs or gametes which inturn suggest a difference in the quality of gametes. These evidences prove that egg activation is coupled with the manifestation of zinc sparks
Even 10 years after the discovery of PLC Zeta no other candidate was discovered to play an equally satisfying role in producing calcium oscillations after the egg has been fertilized. Molecular an clinical experiments have been conducted to examine the role of another SOAF in a number of laboratories. After years of research a compound has evolved with success which plays almost a similar role to that of PLC ZETA. This molecule is postacrosomal WW-binding domain protein, or PAWP. PAWP is specific for sperm and is known to increase arrest at meiosis and begin the formation of a pro nucleus after fertilization thus suggesting a prospective role in the activation of egg. There is a striking similarity between the structure of PAWP and PLC zeta. The N terminal region of PLC Zeta is similar to the WW domain-binding protein 2. The exact mechanism of the signalling of PAWP is currently unknown however WW domain-binding is utilized by PAWP to bind and express in the egg. The egg PLC gamma consist of an SH3 domain where the yes associated protein binds and causes phosphor inositide signalling pathway. The pathway strill is hypothetical and elucidating it is highly important in understanding the signalling mechanism.
PAWP has been suggested in various papers to be the most important factor in membrane fusion of the egg and sperm. Activation of the oocyte is preceeded by changes in calcium levels which is again preceeded by membrane fusion of the egg and sperm. phospholipase C zeta (PLC ZETA) is a protein present in serum. It is responsible for the activation of egg and increases the level of calcium. This inturn causes a rise in the production of Ip3. The high levels of IP3 in turn causes a release of the calcium ions from the endoplasmic reticulum.
Research indicates that the involvement of Postacrosomal WW Domain-binding protein (PAWP) in triggering ca2+ oscillation along with the development of the zygote. The first step in fertilization is the fusion of the sperm and egg. Immediately after fusion, a fertilization envelope will be formed around the zygote. Increase in calcuim level is the next event. The discussions give an impression that PPXY/WWI domain is an important component in the beginning of mammalian oocyte activation.
After a substantial amount of research and clinical proofs it has been concluded that PLC-zeta is the major factor which is responsible for prodiuction of oscillations in calcium levels. Initial research in Phospholipase C Zeta was initiated in 2002 after which several research has been performed in several laboratories all around the world and was experimentally substantiated that it is the sole factor involved in fertilization and oocyte activation. (TABLE 1) The different institutions where research on PLC zeta is flourisihing are given in the following table. The following figure gives a brief idea about the mechanism of PLC ZETA. PLC ZETA is located in the intracellular location of the oocyte and is hydrolyzed to phosphatidylinositol 4,5- bisphosphate from the sperm head. IP3 release causes the release of intracellular calcium from the endoplasmic reticulum. The increase in Calcium also increases the development of the egga and its activation.
In contradiction to the above mentioned data recently an article authored by Arabi et al.,2014phosphatidylinositol 4,5- bisphosphate that calcium oscillations can be produced from a pronucleus in mammalian and mouse eggs in the presence of PAWP. PAWP. PAWp protein is a sperm derived protein present in the afteracrosomal sheath of the theca of perinucleus. PAWP consist of an alkali labile protein with sequence similarity that of the N terminal half of WW domain-binding protein 2. Proline, imino acid is present at the C terminal halif of the protein. Combined injection of an inhibitor peptide extracted from WWI domain of PAWP is responsible of the inhibition of calcium oscillation induced by PAWP. In order for a successful fertilization to take place PAWP binding molecule (fig 1) righthand side.
The figure gives the mode of action of PLC (to the lefthand side) and PAWP (to the righthand side side) in egg of mammals.
Zn chelation leads to the induction of MII egg activation
Zinc spark is a clear indicator of the early activation of oocytes. There are three routes for activation of zinc sparks1. Ca-ionomycin 2. ionomycin 3. PLC ZETA cRNA. To elucidate the mechanisms loss of zinc, a zinc exactchelator N,N,N`,N`-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) was applied formimicing the loss of Zinc. .(Fig)( Ducibella et al, 2007). MII eggs were treated with 50 µM TPEN cytoplasmic labile zinc which down regulated the levels of zinc. This was counter stained using zinc-exactfluorophore Fluozin-3-AM. (Figure A). No alteration in the levels of labile Zinc in MII eggs cured with DMSO. Zinc insufficiency was induced by only TPEN which caused MII egg activation. Mesh like microtubules were present which is a proof of the interphase phase of mitosis which implicates TPEN has entered into mitosis. This also differentiates MII meiosis and undivided Meiosis II. DMSO treated cells were arrested in Meiosis II. This is an indication of the presence of mitotic spindle. Hence it can be concluded that the transition from meiosis to mitosis is achieved by decreasing the calcium concentration. This is one of the few important steps in the transfer of embryo from mitosis to meiosis.
Through all the above experiments we can comprehend the importance of PLC ZETA, PAWP and Zinc spark during the developmental concept. The Sperm quality parameters and sperm production is not affected by the presence of PLC ZETA. The ability of an egg to bind with the sperm does not block the loss of PLC ZETA. It may be also noted that PLC zeta might be the only physiological factor acting as a trigger for calcium oscillations. A number of laboratories are studying about the role of PLC ZETA. Whether PLC ZETA is the only activator of is yet to be confirmed and and is a topic of debate.
There is also an extensive reach discussion on zinc spark being the major motive behind egg commencement. More investigation has to be done in this area to prevent bias in the exact mechanisms. If the exact mechanism is determined it is possible to alter in vitro fertilization mechanisms and several ther mechanisms for arrteficial conceptions and other embryological studies. We further await for advances clinical and experimental support for confirming the mechanisms. It is highly risky and costly to discover a new medicine from the scratch with very little knowledge about the molecule and this knowledge would be a new arena in medical sciences.
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