Eggs of most species have thousands of adhesion sites on the vitelline envelope so that fertilization can occur wherever the single sperm may land on the egg. Yet, the enormous amounts of these adhesion sites can be problematic, since they could lead to the egg being fertilized by more than one sperm. This condition, known as polyspermy, is devastating. For example, fertilization of a sea urchin egg by two sperms results in a zygote with three haploid sets of chromosomes and the two centrosome introduced by the two sperms would set up a meiotic spindle with four poles. The next cleavage would produce cells with irregular numbers of chromosomes, which most often leads to early death (Kalthoff 91).
In many species polyspermy is prevented by two separate mechanisms. One of these mechanisms is known as the fast block to polyspermy, which occurs fast but is temporary. The other mechanism is called the slow block to polyspermy, since it takes more time to activate yet is permanent (Kalthoff 92).
As soon as one sperm makes contact with the egg, the fast block to polyspermy is initiated, and conditions change to prevent additional sperm from fusing to the egg. Inhibitory signals spread around the whole egg surface, starting from the site of where the sperm and egg plasma membrane first made contact. Even when sperm concentrations are high enough for many sperm-egg collisions per second, this mechanism is effective. A chemical signal would be too slow for a response time of less than one second so an electrical signal, which can travel fast enough, is the "activator". As the sperm and egg make contact, the membrane potential changes temporarily to a fertilization potential, which is more positive. The fertilization potential is caused by increased membrane permeability to certain ions, this potential remains positive for about one to two minutes and then slowly returns to its resting state (Kalthoff 92).
The second mechanism involves a cortical reaction that causes a slow block to polyspermy. The binding of gametes at fertilization produces a cytoplasmic Ca2+increase that triggers this cortical reaction. The cortical reaction is the exocytosis of cortical granules into the perivitelline space, between the plasma membrane and the vitelline envelope. Cortical granules are Golgi-derived organelles that rest beneath the egg plasma membrane, the cortex, of the oocyte (Hoodboy 2001). A mouse egg has about 4000 cortical granules, while a sea urchin egg has 15,000 or so (Kalthoff 93).
In sea urchin eggs, the cortical granules, which are in the cortex, undergo exocytosis, releasing several components into the perivitelline space. First, proteases cleave the proteins that attach the vitelline envelope to the plasma membrane. At the same time, sugars that were released from the cortical granules attract water into the perivitelline space and form a hyaline layer that lifts the vitelline envelope, (called the zona pellucida in mammals), off the egg plasma membrane. Next, peroxidases hardens the vitelline envelope, the hardened envelope is then called the fertilization envelope. Finally, enzymes released from the cortical granules modify the envelope's components, making the envelope impermeable to sperm (Kalthoff 94).
The cortical reaction begins soon after sperm and egg make contact, and is finished in one or two minutes, depending on egg size (Kalthoff 93). Binding of the gametes promotes a conformational change in a serpentine receptor in the intracellular domain, which affects its interaction with a hetertotrimeric GTP-binding stimulatory G protein, on the cytosolic side of the plasma membrane. When the G protein is bound to GDP it is inactive. When this G protein is affected by the conformational change of the receptor, it causes an exchange of GDP for GTP. The activated G protein thus activates a specific membrane bound Phospholipase C (PLC). PLC then cleaves phosphotidalinositol 4,5-bisphosphate to inosital trisphosphate (IP3) and diacyglycerol (DAG) in the plasma membrane. Inositol trisphosphate diffuses from the plasma membrane and heads to the endoplasmic reticulum, where it binds to specific IP3 receptors. This binding causes calcium channels in the endoplasmic reticulum to open and release calcium into the cytosol. The diacyglycerol and calcium activate protein kinase C (PKC) at the surface of the plasma membrane (Cox 456). The activation of these messengers triggers the cortical release.
Studies thus far indicate that unfertilized hamster oocyte (egg) cortical granules have diverse glycosylation components. Tests show that hamster cortical granules contain a least 12 heterogeneously glycosylated components. Nine of these glyconjugates associate with the blastomere (32 cell embryo) surface, cortical granule envelope, and/or zona pellucida after fertilization, and seven of them were still detected at the eight-cell stage of the embryo (Hoodbhoy et al. 2001). Cytochemical staining and lectin-binding studies show that cortical granules of mammalian eggs contain carbohydrates including a-D-mannose and a-D-GalNAc (Hernandez 2001).
In previous work, a 50 kDa protein (sp50) from guinea pig testicle was purified. The sp50 protein, which is linked to the sperm plasma and acrosomal membranes in a Ca2+dependent manner, also increases the acrosomal reaction (AR) brought out by Ca2+in permeabilized guinea pig sperm (Munoz-Gotera et al 2001). The acrosomal reaction allows the sperm to get through the egg's protective coat. Upon contact with the egg, the acrosome (at the tip of the sperm), releases acrosomal enzymes by exocytosis. These enzymes digest a hole through the egg's protective coat so that the sperm can release its nucleus into the egg (Kalthoff 78). So as to figure out what protein this was the first NH2-terminal amino acids of sp50 were sequenced and sp50 was identified as calreticulin (CRT) through a protein database (Munoz-gotera et al. 2001).
Realizing that both the cortical reaction (CR) and acrosomal reaction (AR) are exocytotic events, involving the calcium and calcium binding proteins, and that gametes arise from the same primordial germinal cell, they looked for CRT participation in egg exocytosis. CRT was detected in hamster eggs and observed in a granular pattern before egg activation, and outside the cell in the cortical granule envelope, when activated, (Munoz-gotera et al.2001).
The results indicate that calreticulin might be a cortical granule protein, which is released upon egg activation and stays in the perivitelline space, where it might act in the polyspermy blocking process (Munoz-gotera et al. 2001).
This purified sp50, which was found in guinea pig sperm, was put through 25 cycles of Edman degradation using an automated gas-phase protein sequencer to determine the NH2-terminal sequence. The first 21 amino acid residues of sp50 were individually identified, so as to make sure that the final product of the purification procedure was a homogeneous protein preparation and that it had no different subunits. The sequence EPAVY FKEQF LDG(DK)A WTNRW V was found. About the same amounts of D and K were recovered in position 14 indicating micro-heterogeneity of the protein, at least in those positions. A search of this sequence on a protein database showed a high degree of similarity, ranging from 90-100%, to corresponding sequences of CRT from different species. The first 10 amino acids of sp50 were found to be identical to CRT from murine cytolytic T lymphocytes and human lymphokine-activated killer (LAK) cells (Munoz-gotera et al. 2001). These results strongly showed that the sp50 protein of guinea pig testicles corresponds to CRT.
To further confirm that sp50 was CRT, immunoblot detection was done on Brij Sperm extract, using a polyclonal monospecific anti-CRTSC antibody (against to COOH-terminal sequence of CRT) and the polyclonal mono-specific anti-sp50/CRT antibody. Both anti-CRTSC (SC standing for sarcoplasmic) and anti-sp50/CRT antibodies immunolocalized a 50 kDA protein on the Brij sperm extract protein pattern. Sp50 was recognized by the anti-CRTSC antibody, as well as with the anti sp50/CRT. In addition, pure CRT was also recognized by both antibodies (Munoz-gotera et al. 2001). These results together, with the similarity between the amino acids sequence of both sp50 and CRT, support the idea that sp50 and CRT are the same protein.
With the knowledge that sp50 is CRT, it was time to locate its position on the egg. First the anti-sp50/CRT antibody was used to detect CRT. Immunofluorescence confocal microscopy of non-activated eggs showed that CRT, was localized in a pattern of granular fluorescence, mainly at the egg cortex. The same pattern was also observed with anti-CRTSC antibody. The localization and observation of CRT pattern brought on suspicion that the protein might be linked to the egg's cortical granules. So, CRT localization was determined in activated eggs, using anti-sp50 antibody. Pale fluorescence was observed in activated eggs fixed at 5 or 30 minutes postactivation (Munoz-gotera et al. 2001). These results, suggest that CRT is inside the cortical granule and that the protein is released during cortical granule exocytosis.
The data indicated that CRT may be a cortical granule constituent, and it is also known that alpha-D-mannose is a major carbohydrate constituent of hamster egg cortical granules (Hoodbhoy et al. 2001). Therefore the lectin, lens culynaris agglutinin (LCA), was used to reveal mannose as control. To gather more information on egg CRT location, eggs were double stained with anti-sp50/CRT antibody and rhodamine labeled second antibody (red fluorescence) for CRT, and with lectin LCA coupled to Fluorescence (green fluorescence) for mannose. The double staining result was the same. A granular pattern of yellow fluorescence was observed, indicating colocalization of CRT with the internal cortical granule carbohydrate mannose (Munoz-gotera et al. 2001).
CRT was then revealed in zona pellucida intact eggs, activated for 5 minutes with A23187 (calcium inophores), and fixed 45 minutes later. By indirect IIF, the sp50 antibody stained a bright fluorescent halo in the perivitelline space, seen in optical sections in the middle of the egg, using CLSM. Fluorescence was more intense nearer the egg plasma membrane and in the polar body of the eggs than in the remaining perivitelline space. The controls, non-activated stained eggs, did not show fluorescence (Munoz-gotera et al 2001).
The secretion of the intragranular components occur during the cortical reaction, induced with A23187 in hamster eggs. Once the cortical reaction happens, the cortical granule constituents can be detected in the egg's incubation medium. As mentioned earlier, CRT-related fluorescence decreased in activated eggs, therefore it was determined that CRT was present in the egg's incubation medium after egg activation. CRT was detected in the egg's incubation medium obtained at 2 and 25 minutes post activation. No protein band was recognized in the egg's incubation medium obtained before egg activation. The highest CRT was observed in all of the 300 non-activated eggs extracted, and in the 300 activated eggs, less CRT content was observed (Munoz-gotera et al. 2001). In each of the analyzed samples, anti-sp50/CRT antibody identified only one protein band, Mr of 60 kDa (Munoz-gotera et al. 2001).
To evaluate the egg's plasma membrane integrity, calmodulin (CaM) was used for reference, since it is not an exocytotic protein. If CaM was found in the egg's incubation medium it would indicate that the membrane was ruptured. Results were that CaM was not detected in the incubation medium samples obtained before 2, 37, and 137 minutes after the eggs were activated (Munoz-gotera et al. 2001).
In this major study it was reported that: the sp50 protein which was isolated from the guinea pig testicle has an NH2-terminal sequence identical to CRT and is recognized by CRT antibodies; in hamster egg protein extracts, anti-sp50/CRT as well as anti-CRTSC antibodies, specifically recognized a band with a molecular weight of 60 kDa, as is Xenopus egg's CRT 61 kDa. It also concluded that the egg's CRT amount decreases after cell activation, as could be followed by inmunofluorescence and immunobloting; decreased levels of CRT protein in activated eggs correlate with the presence of CRT in the egg's activation medium; the egg's CRT is found in the cortical granules; and CaM was not detected in activated egg's incubation medium but only present inside the eggs (indicating that the plasma membrane's integrity was conserved) (Munoz-gotera et al. 2001).
Sp50's complete identity was located between the NH2-terminal sequence and that of cytotoxic T-lymphocyte CRT and LAK CRT. Additionally, anti-sp50/CRT and anti-CRTSC antibodies recognized pure sp50, as well as pure CRT. They also cross-react with a single 50 kDa protein band from Brij guinea pig spermatozoa extract and with a 60 kDa protein band from hamster egg extracts (Munoz-gotera et al. 2001). Together, these results prove that sp50 and CRT are the same.
CRT is a highly conserved (Johnson et al. 2001) and a protein that is found throughout the body. The molecular mass of CRT, based on amino acid sequence deduced from murine cDNA, was estimated to be about 46 kDa (Michalak et al 1999). But, when analyzed by SDS-PAGE, CRT migrates with a molecular mass of 60-63 kDa. It was also found that hamster egg CRT migrated to a mass of 60 kDa (Munoz-gotera et al 2001). The appearance of CRT as about 60 kDa is likely due to its highly negative charge (pI =4.7) and/or other structural features (Coppolino et al. 1997).
It has been known that CRT is a major calcium binding protein of the endoplasmic reticulum (Johnson et al. 2001). It has a function in calcium binding, maintaining Ca2+in the ER, and a molecular chaperone function to fold proteins (Coppolino et al. 1997). CRT interacts in a Ca2+dependant manner. However, CRT continues to be found outside of the endoplasmic reticulum, places like in the acrosomal area of spermatids and mature spermatozoa, in the nuclear envelope and within the nucleus, in the cell cytoplasm, as well as on the surface and within natural killer cells (NK), neutrophils, LSK, and cytotoxic T-lymphocyte granules (Andrin et al. 1998). This recent discovery that CRT is inside cortical granules of unfertilized hamster eggs, correlates well with past findings, which have found CRT in neutrophils and lymphocytes granules.
It has also long been recognized that CRT, as mentioned above, is a chaperone protein of the endoplasmic reticulum. It acts as granzymes and perforinas, chaperone proteins found in granules of natural killer cells and cytotoxic T-lymphocyte (Andrin et al. 1998). Cortical granules of vertebrates contain a number of components, which during egg activation are exocytosed into the perivitelline space (Kaltoff 93), which modify egg vestments. As in the endoplasmic reticulum, T-lymphocyte, and natural killer cells, cortical granule CRT may perform as a chaperone for some of these lytic exocytosed components, and allow their enzymatic function on the egg vestments (Munoz-gotera et al. 2001).
Calreticulin participates in many cellular functions, but one of the major functions it has is chaperoning. Molecular chaperones prevent the accumulation of partially folded proteins, increase the yield of correctly folded proteins, and assembly, and also increase the rate of correctly folded intermediates by recruiting other folding enzymes. Chaperones are highly versatile being involved in the "quality-control" process during the synthesis of a variety of molecules (Michalak et al. 1999).
There is an interesting hypothesis, by John et al, suggesting the lectin-like region of CRT may play a dual role in the ER lumen: chaperoning of newly synthesized integral and secreted proteins and modulation of "functional" conformation of the mature, fully functional integral ER glycoproteins (Michalak et al. 1999).
Ca2+ is taken up into the ER lumen by SERCA. Camacho's' group carried out a study to see if calreticluin affects the function of SERCA. They concluded that co-expression of calreticulin with SERCA 2b results in a sustained elevation in Ca2+release without concomitant oscillations. SERCA 2b has a transmembrane segment and a C-terminal 12 residue tail localized to the ER lumen and having a putative N-glycoslation site. Studies have shown that this residue, N1036 is critical for calreticluin-dependent effects on SERCA 2b function and for its isoform-specific functional differences. Effects of CRT on SERCA 2b involve the P-domain of the protein, suggesting involvement of the chaperone function of CRT (Michalak et al. 1999).
With these observations in mind, John et al, proposed that the C-terminal tail of SERCA 2b may be glycosylated in vivo and that CRT modulates SERCA 2b Ca2+-transport activity by a direct interaction with glycosylated C-terminal tail of the pump. CRT binding to SERCA 2b might be regulated by changes in the ER calcium reminiscent of the role of Ca2+in CRT-carbohydrate interactions. Under conditions of Ca2+stores, CRT would not interact with SERCA2b and the ATPase would exhibit full enzymatic activity for efficient refilling of the stores.
CRT also acts as a lectin, a protein that binds a carbohydrate, usually a oligosaccharide, with very high affinity and specificity, mediating cell-to-cell interactions, involved in chaperoning glycoproteins (Spiro et al. 1996). Since CRT was seen within the perivitelline space after the cortical reaction and it was observed that the sperm binds to a glycoprotein, this suggests that the exocytosed CRT may link to and interfere with some components of the egg surface, resulting in a direct block to polyspermy. CRT acting as a chaperone may also be indirectly involved in the polyspermy block (Munoz-gotera et al. 2001), because CRT would chaperone (fold) proteins that were in cortical granules, which would later be involved in the blocking process.
CRT participated in the polyspermy blocking process in several ways. If CRT functions as a chaperone, it might also modulate enzymatic activity either inside the cortical granule or, at exocytosis, in the pervitelline space and/or on the zona pellucida. As a lectin, CRT might be able to block carbohydrates on the egg plasma membrane, and most definitely, CRT is a component of the new cortical granule envelope, the structure that forms in the perivitelline space together with other components, which are exocytosed during the cortical reaction ( Munoz-gotera et al. 2001).
Copyright © 2002 Jose Bosque and Koni Stone
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