Abstract
Mature human gametes ready for fertilization differ in their state of nuclear maturation: the spermatozoon has completed meiosis and the oocyte is arrested at metaphase II. However, both gametes must also undergo a process of cytoplasmic maturation before they are capable of fertilization. This involves a complex series of biochemical, physiological and structural events that occur in a carefully orchestrated temporal and spatial pattern in parallel with, but independent from, nuclear maturation. Cytoplasmic and nuclear maturation are often asynchronous (Dale, 2018a): therefore, a cohort of human metaphase II oocytes collected after controlled ovarian hyperstimulation in an IVF program may appear to be similar with regards to the nuclear apparatus, but they are in fact at various stages of cytoplasmic maturation. This may partly explain the different developmental capabilities of embryos generated from a single cohort of oocytes.
Mature human gametes ready for fertilization differ in their state of nuclear maturation: the spermatozoon has completed meiosis and the oocyte is arrested at metaphase II. However, both gametes must also undergo a process of cytoplasmic maturation before they are capable of fertilization. This involves a complex series of biochemical, physiological and structural events that occur in a carefully orchestrated temporal and spatial pattern in parallel with, but independent from, nuclear maturation. Cytoplasmic and nuclear maturation are often asynchronous (Dale, 2018a): therefore, a cohort of human metaphase II oocytes collected after controlled ovarian hyperstimulation in an IVF program may appear to be similar with regards to the nuclear apparatus, but they are in fact at various stages of cytoplasmic maturation. This may partly explain the different developmental capabilities of embryos generated from a single cohort of oocytes.
Sperm–oocyte interaction is a complex process of cell–cell interaction that requires species-specific recognition and binding of the two cells. While interacting, each gamete triggers a cascade of events in its partner which changes them from arrested to developmentally competent cells. Controlled, synchronous gamete activation is essential for embryonic development; however, the biochemical and physiological processes that ultimately lead to the fusion of the male and female pronuclei are still poorly understood. In human assisted reproductive technology, although the technique of intracytoplasmic injection of spermatozoa (ICSI) essentially bypasses the initial stages of fertilization, including sperm capacitation and the interaction of the gametes, successful fertilization still requires the controlled and correct activation of both the spermatozoon and oocyte.
Mammalian fertilization is internal, and the male gametes must be introduced into the female tract at coitus. Coitus itself ranges from minutes in humans to hours in camels but is accompanied by many physiological changes. In the human, tactile and psychogenic stimuli can initiate penile erection, caused by decrease in resistance and consequently dilatation in the arteries supplying the penis, with closure of the arterio-venous shunts and venous blood valves. Vasocongestion can increase the volume of the testes by as much as 50%. Sequential contraction of the smooth muscles of the urethra and the striated muscles in the penis results in ejaculation of semen, with mixing of three different components: prostatic liquid rich in acid phosphatase, the vas deferens fraction containing spermatozoa and the seminal vesicle fraction containing fructose.
In the woman, tactile stimulation of the glans clitoris and vaginal wall leads to engorgement of the vagina and labia majora, and the vagina expands. Orgasm is accompanied by frequent vaginal contractions, with uterine contractions beginning in the fundus and spreading to the lower uterine segment. In man, rabbit, sheep, cow and cat, the semen is ejaculated into the vagina. In the pig, dog and horse, it is deposited directly into the cervix and uterus. In many species, the semen coagulates rapidly after deposition in the female tract, as a result of interaction with an enzyme of prostatic origin. The coagulation may serve to retain spermatozoa in the vagina or to protect them from the acid environment.
In the human, this coagulum forms a loose gel which is dissolved within 1 hour by progressive action of a second proenzyme, also of prostatic origin. Within minutes of coitus, spermatozoa may be detected in the cervix or uterus; 99% of the spermatozoa are lost from the vagina, but the few that enter the tract may survive for many hours in the cervical crypts of mucus. Abnormal sperm with poor motility are less able to penetrate the cervical mucus, and this may represent one means of sperm selection in natural conception. In the absence of progesterone, cervical mucus permits sperm penetration into the upper female tract, and contractions of the myometrium during the periovulatory period may assist sperm movement toward the utero-tubal junction. Kinz et al. (1996) suggested that there may be a mechanism that preferentially draws sperm toward the isthmus on the same side as the ovarian dominant follicle. A few thousand sperm swim through the utero-tubal junctions to reach the oviducts, where they interact with oviductal epithelium: the oviductal environment and its secretions play a critical role in the transport and interaction of both gametes. Sperm are trapped and stored in the initial part of the oviduct, which may contribute to preventing polyspermic fertilization by allowing only a few sperm to reach the ampulla at a time.
Sperm–Oocyte Ratios: Reduction by Elimination
In the course of evolution, spermatozoal wastage has apparently been retained as a requirement for the union of one spermatozoon with one oocyte. In most animals, spermatozoa are produced in huge excess, irrespective of whether fertilization occurs externally or internally: in humans and other mammals the sperm:oocyte ratio at origin can be as high as 109:1. If we examine animal sperm size and number in relation to body mass, it appears that evolutionary responses have favored sperm number rather than sperm size with increasing body size: larger animals such as elephants produce more sperm per ejaculate (corrected for body mass) than a small mammal such as a mouse. Despite large sperm numbers in mammals, behavioral adaptations are required to ensure fertilization. Mating must be synchronized, and the sperm need to be deposited in the female tract. The vast majority of spermatozoa are rapidly eliminated from the tract, and only a minute fraction successfully migrate to the site of fertilization.
In mice, the major barrier for sperm ascent is the utero-tubal junction, with spermatozoa being progressively released from the lower part of the oviductal isthmus at ovulation. In humans, the first barrier to sperm ascent is the highly folded mucus-filled cervix, where sperm are retained and released over a period of several days. In-vivo studies that counted spermatozoa in situ revealed a few hundred spermatozoa in sheep ampullae and only five in a human female ampulla (Dale & Monroy, 1981). An in-vivo study of fertilization in the mouse showed a 1:1 sperm–oocyte ratio in the ampullae – supernumerary spermatozoa were never observed.
Human in-vitro fertilization practice routinely involves inseminating cumulus-intact oocytes with a concentration of at least 100 000 spermatozoa/mL. The oocytes remain in this sperm bath for 18 hours before transfer to fresh medium to check for fertilization. However, removing oocytes from the sperm bath at various times prematurely followed by culture in sperm-free medium showed that an initial exposure of 5 minutes was sufficient for fertilization to progress (Gianaroli et al., 1996). In these experiments, approximately 10 spermatozoa entered the cumulus complex, with one successfully reaching and fertilizing the oocyte. The remainder were blocked at various levels in the cumulus complex. This confirms the concept that large numbers of spermatozoa are not required for fertilization in mammals and reinforces the idea that not more than a handful of spermatozoa approach the oocyte in natural fertilization. There is evidence to show that sperm respond to chemotactic stimuli from mature oocytes and surrounding cumulus cells: progesterone acts as the principal chemoattractant, and calcium entry is an important second messenger (see Tosti & Ménézo, 2016). An olfactory receptor gene expressed in the testis may be involved in sperm chemotaxis in humans (Spehr et al., 2004). Chemical modulation of the zona pellucida (ZP) by oviductal-specific glycoproteins before the oocyte encounters the spermatozoon has also been described (Coy & Aviles, 2010), and this may also be involved in fine tuning sperm–oocyte interactions in mammals.
In summary, although great quantities of spermatozoa are produced, few reach the oocyte under natural conditions. Those that do must then traverse and interact with the cumulus and coronal cells, which reduce the number of spermatozoa that can reach the oocyte to bind to and penetrate the ZP. The ZP is composed of several glycoproteins that differ between species, and this layer impedes sperm progression even further. The ZP modulates sperm binding and protects the embryo during early development, but we know little about its topographical constitution and whether sperm entry is piloted to a specific site. In many animals, including mammals, the extracellular coats of the oocyte may be removed without inhibiting fertilization, and initial gamete interactions may be bypassed by microinjecting the spermatozoon directly into the oocyte. This is a laboratory artifact, and it is often erroneously interpreted as showing redundancy of the extracellular coats. In nature, passage through these coats is a prerequisite for normal fertilization, oocyte activation and subsequent paternal nuclear decondensation.
Polyspermy
The entry of more than one spermatozoon into a mammalian oocyte results in abnormal cleavage, and the embryo will block in development: this is known as pathological polyspermy. Images of oocytes in the laboratory with hundreds of spermatozoa attached to their surface have led to the notion that oocytes have evolved mechanisms that allow the penetration of a single spermatozoon, while repelling supernumerary spermatozoa. However, these images are a laboratory artifact. As discussed above, under natural conditions, the number of spermatozoa at the site of fertilization is extremely low compared with the numbers originally deposited in the female tract. This is regulated initially by the female reproductive tract, and then by a bottleneck created by extracellular coats of the oocyte. In order to reach the oocyte plasma membrane, the fertilizing spermatozoon must encounter and respond to a correct sequence of signals from the oocytes’ extracellular coats. Those that fail to respond are halted in their progression by defective signaling and fall by the wayside. In nature, final sperm:oocyte ratios approach unity, and it would therefore appear that the achievement of monospermy has evolved via selective pressures rather than mechanisms that prevent polyspermy.
The initial stages of fertilization depend principally on two structures: the acrosome of the spermatozoon and the ZP of the oocyte. Three major events are involved in sperm–oocyte interactions:
1. The spermatozoon attaches to the ZP.
2. The spermatozoon undergoes the acrosome reaction, releasing digestive enzymes, and exposing the inner acrosomal membrane.
3. This highly fusogenic sperm membrane makes contact with the oocyte plasma membrane and the two membranes fuse together.
Gamete Activation
Both spermatozoa and oocytes are in a quiescent state that is maintained by numerous carefully orchestrated cellular and molecular mechanisms, and their transition from arrested to developmentally competent cells is known as gamete activation. The cascades of events involved in mutual gamete activation are interconnected, with shared molecules and signaling pathways: calcium represents a key molecule for both gametes in each step of the process. Successful fertilization is dependent upon both gametes achieving full competence after the complex series of events necessary for their activation.
Sperm Activation
Before the male gamete can initiate the steps required for successfully fertilizing an oocyte, the spermatozoon must itself be activated, a process that involves several behavioral, physiological and structural changes. Some of these changes are induced by exposure to environmental signals, and others are induced whilst the spermatozoon is interacting with the oocyte and its extracellular investments. The steps include changes in motility, capacitation, acrosome reaction, penetration of the ZP, binding to the oolemma and membrane fusion.
Motility
Spermatozoa are maintained in the testis in a quiescent state. Metabolic suppression is regulated by physical restraint, low pH and low oxygen tension in the seminal fluid. They acquire motility during the process of epididymal maturation, but only become fully motile after ejaculation and capacitation. Sperm motility is regulated by intracellular ions and is associated with changes in the membrane potential, in particular a potassium-induced hyperpolarization (Miller et al., 2015).
Capacitation and Hyperactivation
Spermatozoa are not capable of fertilization immediately after ejaculation. They develop the capacity to fertilize (capacitate) after a period of time in the female genital tract; since epididymal maturation and capacitation are unique to mammals, this may represent an evolutionary adaptation to internal fertilization. During capacitation, the spermatozoa undergo a series of changes that give them the ‘capacity’ for binding to and penetrating the oocyte. These changes include an increase in membrane fluidity, cholesterol efflux, ion fluxes that alter sperm membrane potential, increased tyrosine phosphorylation of proteins, induction of hyperactive motility and the acrosome reaction.
Hyperactivation involves a change in flagellar beating, with an increase in the amplitude of flagellar bend; this may provide a force that helps in the release of spermatozoa from the oviductal epithelium and enhances the sperm’s ability to navigate toward the oocyte. Hyperactivation may also help the sperm to penetrate the ZP.
Capacitation is a transitory state: the time required for capacitation varies from species to species and ranges from less than 1 hour in the mouse to 1–4 hours in the human. Only 10% of the available population are capacitated at any one time: capacitated spermatozoa are continuously replaced from the stored pool, ensuring that fertile spermatozoa are available over the period of several hours when ovulation may occur (Forman & Fissore, 2015).
Two changes take place: the epididymal and seminal plasma proteins coating the spermatozoa are removed, followed by an alteration in the glycoproteins of the sperm plasma membranes (an antigen on the plasma membrane of the mouse spermatozoon, laid down during epididymal maturation, cannot be removed by repeated washing, but disappears, or is masked during capacitation). The events are regulated by the activation of intracellular signaling pathways, involving cAMP, protein kinase A, receptor tyrosine kinases and non-receptor tyrosine kinases. A number of different molecules regulate these pathways, including calcium, bicarbonate, reactive oxygen species, GABA, progesterone, angiotensin and cytokines. Phosphorylation of sperm proteins is an important part of capacitation, and this has been shown to be associated with the change in the pattern of sperm motility described above, hyperactive motility, recognizable by an increase in lateral head displacement. There is also some evidence that spermatozoa can translate some mRNA species during capacitation (Gur and Breitbart, 2006).
In the human, capacitation in vivo probably starts while the spermatozoa are passing through the cervix. Many enzymes and factors from the female tract have been implicated in causing capacitation, such as arylsulfatase, fucosidase and taurine. The factors involved are not species specific, and capacitation may be induced in vitro in the absence of any signals from the female tract. Follicular fluid can also promote capacitation in vitro. A low molecular weight motility factor found in follicular fluid, ovary, uterus and oviduct may increase sperm metabolism (and hence motility) by lowering ATP and increasing cyclic AMP levels within the sperm. Table 4.1 demonstrates the duration of fertility and motility of mammalian spermatozoa within the oviduct, together with the fertilizable life of oocytes.
Table 4.1 Survival parameters of mammalian gametes in vitro
| Time required for capacitation (h) | Duration of sperm motility (h) | Duration of sperm fertility (h) | Fertilizable life of oocytes (h) | |
|---|---|---|---|---|
| Mouse | <1 | 13 | 6 | 15 |
| Sheep | 1–5 | 48 | 30–48 | 12–15 |
| Rat | 2–3 | 17 | 14 | 12 |
| Hamster | 2–4 | – | – | 9–12 |
| Pig | 3–6 | 50 | 24–48 | 10 |
| Rabbit | 5 | 43–50 | 28–36 | 6–8 |
| Rhesus monkey | 5–6 | – | – | 23 |
| Man | 5–6 | 48–60 | 24–48 | 6–24 |
| Dog | – | 268 | 134 | 24 |
Chemically defined media with appropriate concentrations of electrolytes, metabolic energy sources and a protein source (serum albumin) will also induce the acrosome reaction in a population of washed sperm. The removal or redistribution of glycoproteins on the sperm cell surface during capacitation exposes receptor sites that can respond to oocyte signals, leading to the acrosome reaction.
Acrosome Reaction (AR)
The acrosome is a membrane-bound cap that covers the anterior portion of the sperm head; it contains a large array of hydrolytic enzymes including hyaluronidase, acrosin, proacrosin, phosphatase, arylsulfatase, collagenase, phospholipase C and β-galactosidase. The acrosome originates in the Golgi system of the early spermatid, in a series of concentrically arranged membranes around an aggregation of small vesicles. One of the vesicles increases in size and fills with particulate material, and the vesicle grows by fusion of several smaller vesicles. When the future acrosomal vesicle reaches a certain size, it migrates toward the nucleus. The nucleus then starts to elongate, the vesicle loses much of its fluid content and its membrane wraps around the front of the nucleus to form the typical acrosome.
When the capacitated spermatozoon attaches to the ZP, the permeability of the sperm plasma membrane is altered, causing a transient change in the concentration of several intracellular ions. This triggers the acrosome reaction, which is the final prerequisite step in the activation of the spermatozoon before gamete fusion is possible. The reaction consists of three stages:
1. The outer acrosomal membrane fuses with the overlying sperm head plasma membrane, allowing the contents to be released; this can be monitored in vitro using a fluorescent tag in the acrosome reaction to ionophore challenge (ARIC) test.
2. The acrosomal granule breaks down, releasing lysins. These enzymes ‘dissolve’ a pathway through the ZP.
3. When the sperm head plasma membrane contacts the oocyte plasma membrane, the two membranes fuse.
It appears that some of the sperm that may reach the zona are not triggered into the acrosome reaction, and are not able to penetrate the egg. In order for sperm to attach, a specific molecular fit may be required to induce the acrosome reaction. In the human, the membranes start to fuse near the border between the acrosomal cap and its equatorial segment. Once the correct trigger signals have been received, the acrosome reaction is relatively rapid, taking 2–15 minutes in vitro. Gametes collected from the ampullae of mammals after mating show that free-swimming spermatozoa have unreacted acrosomes, and those within the cumulus mass have either reacted acrosomes or are in the process of reacting. The majority of spermatozoa attached to the surface of the ZP surface have reacted acrosomes.
The acrosome reaction only occurs in the presence of Ca2+: it may be induced artificially by adding Ca ionophore A23187, a chemical that carries Ca2+ across cell membranes to the sperm cytoplasm (Figure 4.1), or simply by increasing the external concentration of Ca2+. An artificially high pH of about 9–9.5 will also induce the AR. It appears that the physiological events leading to the AR parallel those leading to activation of the oocyte, including changes in the ion permeability of the plasma membrane, alterations in the intracellular level of free Ca2+ and an alkalinization of the cytoplasm. The influx of calcium triggers the fusion of the acrosomal membranes and the exocytosis of the acrosomal contents. The sequence of events leading to exocytosis may involve several second messenger pathways, including:
Changes in intracellular calcium
Activation of cAMP and phosphokinase A pathways
Phospholipase C generating InsP3 and diacylglycerol (DAG)
Phospholipase D generating phosphatidic acid
Activation of phospholipase A2 generating arachidonic acid.
Completion of the acrosome reaction does not necessarily ensure successful fertilization in vitro; enormous variability can be found in a population of spermatozoa surrounding the cumulus mass. Some will acrosome react too soon, others too late: in some the trigger stimulus will be inadequate, perhaps in others the transduction mechanism will fail at some point. The cumulus mass is composed of both cellular and acellular components, and the acellular matrix is made up of proteins and carbohydrates, including hyaluronic acid. As described earlier, in vivo, very few spermatozoa reach the site of fertilization: therefore, the idea that large populations of spermatozoa surrounding the oocyte mass dissolve the cumulus matrix, as observed during IVF, is probably incorrect in vivo. Fertilization occurs before the dispersion of the cumulus mass, and in vivo the sperm:oocyte ratio is probably close to 1:1.
Figure 4.1 Transmission electron microscope (TEM) section through a human spermatozoon showing the plasma membrane (PM) and outer (OAM) and inner (IAM) acrosomal membranes. To the right is a TEM of a human spermatozoon after exposure to the calcium ionophore A23187 has triggered the acrosome reaction.
Oocyte Activation
The Zona Pellucida
The ZP is secreted by the growing oocyte, a glycoprotein sheet several micrometers thick that provides a protective coat for the oocyte and developing embryo. If we accept the concept that polyspermy prevention is a laboratory artifact, it probably serves mainly as a protective coat for the developing embryo. Electron microscopy shows the outer surface to have a latticed appearance, consisting of 70% protein, 20% hexose, 3% sialic acid and 2% sulfate. In the mouse oocyte, the zona contains three glycoproteins, ZP1, ZP2 and ZP3, with apparent molecular weights of 200 000, 120 000 and 83 000, respectively; each is heavily glycosylated. Filaments of ZP2 and ZP3 polymers are crosslinked noncovalently, and ZP1 dimers create bridges between them to form a matrix (Wassarman et al., 1996). In the mouse, ZP2 is distributed throughout the thickness of the zona, and ZP3 binds to primary receptors on capacitated spermatozoa, inducing a cascade of events that lead to the acrosome reaction. Sperm receptors for ZP2, in contrast, are located on the inner acrosomal membrane and therefore are unmasked only after the AR has taken place. Following the AR, ZP2 binds the sperm to the zona, and the sperm penetrate the ZP to fuse with the oocyte plasma membrane. The zona in many species, including humans, contains a fourth zona protein, ZP4, which is absent in mice.
The ZP gene family has an ancient phylogeny, and the coding sequences of the murine and human ZP genes are 74% identical. All ZP proteins contain a structural element, the ZP domain (ZPD), composed of 260 amino acids, and this structural element is also found in many other proteins with different functions, including receptors and intracellular signaling related to differentiation and morphogenesis. There are 10 ZPD proteins in nematodes, more than 20 in flies and over 100 in birds.
Synthesis of these glycoproteins is regulated in a temporal sequence during oogenesis. In mammals and amphibians, ZP genes are transcribed exclusively by oocytes and/or follicle cells. ZP2 is expressed at low levels in resting oocytes, but ZP1 and ZP3 are expressed only in growing oocytes. In fish and birds, the ZP proteins are synthesized in the ovary and/or the liver. ZP3, an 83-kDa glycoprotein, appears to be the primary adhesion molecule. The bioactive component within ZP3 is thought to be related to its carbohydrate composition, with the terminal sugar residue being either a terminal alpha-linked galactose or a terminal N-acetylglucosamine. Other studies have suggested that ZP1 may also be involved in primary adhesion events in the rabbit and the human. ZP2 and ZP3 are the two major subunits of the ZP; the N-terminal region of ZP2 may regulate sperm recognition in mouse and human oocytes. When sperm bind to ZP3, they undergo the acrosome reaction.
Studies carried out using human ZP have revealed that ZP2 is modified by cleavage, similar to the mouse model. Sperm penetration through the zona is inhibited after fertilization. After sperm penetration, the zona undergoes modifications consistent with its role as a protective device. However, unlike the mouse, the ZP can still bind spermatozoa and induce the AR. Thus, fertilization does not abolish ZP bioactivity. This difference may be linked to the presence of ZP4 – the precise events that describe the role of the zona proteins in gamete interaction require further study in humans (Lefièvre et al., 2004; Conner et al., 2005; Patrat et al., 2006; Tanphaichitr et al., 2015; Tosti & Ménézo, 2016).
Cytoplasmic Maturation
During stages of oogenesis, the oocyte has accumulated reserves of proteins and mRNAs that allow it to remain quiescent, in a state of developmental arrest that is characterized by blocks at both nuclear and cytoplasmic levels. Arrest during the first meiotic prophase is characterized by the germinal vesicle; following germinal vesicle breakdown, meiosis is again arrested, and this block is removed by the fertilizing spermatozoon (Table 4.2). Two types of protein kinase in the cytoplasm maintain the second meiotic arrest: maturation promoting factor (MPF) and cytostatic factor (CSF). Oocytes acquire competence for successful fertilization and the ability to sustain early development via cytoplasmic maturation, a process that may be considered a parallel to sperm capacitation. Several milestones in development must be reached before an oocyte is capable of being fertilized correctly (see Tosti & Ménézo, 2016 for review and Figure 4.2 for overview):
1. MPF is expressed at a high level.
Core components of MPF = CSF and cyclin-dependent kinase1(Cdk1/Cdk2).
CSF maintains the anaphase-promoting complex/cyclosome (APC/C) inactive, via a signaling cascade involving early mitotic inhibitors, Emi2/Erp1.
2. High levels of other factors are present within the oocyte, including c-mos, mitogen-activated protein kinase (MAPK) and active p34cdc2.
3. Progression to the MII stage of meiosis. The first polar body must be extruded into the perivitelline space, between the oolemma and ZP.
4. Virtually all transcription ceases by the time of germinal vesicle breakdown (GBVD). The expression of genes beyond this stage switches to translation of stored mRNA.
These points are summarized in Table 4.2 and Figure 4.2.
Table 4.2 Expression of factors during late stages of oogenesis in preparation for fertilization
| G2 → M phase | ||||||
|---|---|---|---|---|---|---|
| First meiotic block | Metaphase I | Second meiotic block | Post fertilization | |||
| Germinal vesicle | Germinal vesicle breakdown | First polar body | Metaphase II | |||
| Phos p34cdc2 | ++ | − | − | − | − | ++ |
| Active p34cdc2 | − | ++ | ++ | ++ | ++ | − |
| MPF | − | ++ | ++ | − | ++ | − |
| c-mos | + | + | ++ | ++ | ++ | − |
| MAPK | − | ++ | ++ | ++ | ++ | − |
| cAMP | ++ | − | − | − | − | − |
Figure 4.2 Sperm-induced oocyte activation. Left panel: image representing events occurring during sperm-induced oocyte activation: upon release of the sperm factor (SF), electrical modification of oocyte plasma membrane properties generates an outward (in mammals) or inward ion current (non-mammals). Release of calcium from the intracellular stores generates calcium oscillations. Physical changes of the oocyte occur by release of CG contents, and finally meiosis is resumed, allowing completion of the cell cycle, extrusion of the polar body and triggering of zygote formation. Right panel: table reporting the event and the components involved. PLCζ = phospholipase C; PAWP = postacrosomal sheath WW domain-binding protein; PIP2 = phosphatidylinositol (4,5)-bisphosphate; IP3 = inositol 1,4,5-trisphosphate; cADPr = cyclic adenosine diphosphoribose; NAADP = nicotinic acid adenine dinucleotide phosphate; TPCs = two-pore channels; SFE1, SFE9 = structural matrix proteins; PKC = protein kinase C; SNAP = N-ethylmaleimide-sensitive factor attachment protein alpha; NSF = N-ethylmaleimide sensitive factor; MPF = maturation-promoting factor; MAPK = mitogen-activated protein kinase; CAMKII = calcium calmodulin-dependent protein kinase; EMI/ERP = early mitotic inhibitors; Mos = serine/threonine kinase; Cdk1 = cyclin-dependent kinase; CG = cortical granule; SF = sperm factor.
The first event of activation in oocytes of most species is an increase in ionic permeability of the plasma membrane. In the human, the spermatozoon induces an outward current in the oocyte plasma membrane by activating calcium-gated potassium channels. In vitro, the activation competence of oocytes is continually changing, and is not a stable, prolonged feature of ovulated eggs; therefore, timing is critical in the handling of in-vitro manipulations.
Sperm–oocyte Fusion
The process of membrane fusion between gametes is temperature, pH and Ca2+ dependent, and the two membranes must be in close approximation. Fusion appears to be mediated or facilitated by membrane-associated proteins, but terminal saccharides of glycoproteins are not directly involved in the process. During penetration of the zona, the spermatozoon loses its acrosomal contents, and only the inner acrosomal membrane is in direct contact with the zona. In eutherian mammals, the post-acrosomal region of the sperm head plasma membrane only attains fusibility after the acrosome reaction; this area apparently fuses with the oocyte plasma membrane, and the two membranes become continuous (Figure 4.3).
The surface of the oocyte membrane is organized into evenly spaced short microvilli that seem to facilitate gamete fusion; these microvilli have a low radius of curvature which may help to overcome opposing electrostatic charges. In mouse and hamster, the area overlying the metaphase spindle is microvillus-free, and spermatozoa are not able to, or are less likely to, fuse with this area. The human oocyte, however, has microvilli present over the entire surface, with no obvious polarity at this stage. There may, however, be ‘hotspots’ for sperm entry into the oocyte; this is an area that requires further research.
Figure 4.3 (a) Transmission electron micrograph showing the point of sperm–oocyte fusion in the sea urchin. Sperm factor must flow through this cytoplasmic bridge of 0.1 mm diameter. The large granule (1 mm) below the spermatozoon is a cortical granule. (b) Stages in sperm–oocyte fusion in the mammal.
The Oocyte Plasma Membrane
Lipids in the plasma membrane are organized asymmetrically into ‘rafts,’ domains of 10–200 nm, which make up to 20% of the surface area of somatic cells. They are thought to be platforms for membrane trafficking, signal transduction and viral entry, containing regions of high cholesterol, sphingomyelin and gangliosides, and are enriched in phospholipids with saturated fatty acyl chains. Caveolin, a major raft component, serves as a scaffolding to embed and inactivate many proteins and enzymes. Lipid rafts are less fluid than the rest of the plasma membrane and display lateral movement in response to physiological stimuli. In mouse, sea urchin and amphibians, fertilization may be inhibited by methyl-beta-cyclodextrin (MBCD), which disrupts rafts by dispersing important raft proteins, such as CD9, and inhibits Src kinase activation and the completion of meiosis. In mouse oocytes, MBCD disrupts both planar and caveolar rafts, which are thought to be the sites of mammalian sperm–egg binding and fusion. Phosphatidic acid (PA) may be responsible for stabilizing rafts. Other lipids may also affect rafts and membrane fusion, for example production of ceramide during fertilization may lead to clustered rafts and an increase in raft diameter. Cortical microfilaments are important in raft biology, and PA is a major regulator of cytoskeletal fibers.
Several lipids seem to have roles in membrane fusion events at fertilization, including the acrosome reaction, gamete fusion and cortical exocytosis, regulating receptors and releasing intracellular calcium. In Xenopus oocytes, phosphatidic acid can activate Src tyrosine kinase or phospholipase C during fertilization, leading to an increase in intracellular calcium. Lipases such as phospholipase D, C and A2, sphingomyelinase, lipin 1 and autotaxin are involved in generating lipid second messengers at fertilization.
In the human oocyte, lipid raft microdomains are enriched in the ganglioside GM1 and the tetraspanin protein CD9. GM1 is involved in a variety of processes such as virus docking, signal transduction and protein binding, while CD9 seems to be the most important membrane component involved in sperm penetration in mammals. Sperm penetration into the human oocyte appears to be dependent on the density and organization of GM1 microdomains at the site where the sperm arrives – these can be considered docking sites. Sperm bind at these GM1 microdomains, but do not penetrate. The lipid rafts with CD9, distinct to those with GM1, may be sites for stable binding. GM1 organization and the stability of these plasma membrane rafts depend on underlying mitochondrial activity and efficiency.
