The process of fertilization is complex, and knowledge of that process in the human has been limited in a large part because of ethical constraints. However, with the advent of assisted reproductive technologies (ARTs), understanding of the complexities involved in successful fertilization has been greatly enhanced. Although there were a number of major driving forces (e.g., anovulation) behind the development and implementation of in vitro fertilization techniques, one of the principal motivators was to address those situations for which tubal pathology (e.g., hydrosalphinx) was the primary cause for infertility. As ART has developed, so has understanding of the essentials for human reproductive success. Still, a great deal more is known about reproductive processes in nonhuman mammals. This review focuses on what is currently known about egg transport and fertilization in the human.

Historically “egg transport” has referred to the movement of the oocyte, over time, from the point of rupture out of the ovarian follicle to entry and deposition into the uterine cavity. However, to apply this phrase to the sequence of events previously described is inaccurate. First, the word “egg” (more correctly, oocyte) refers only to the haploid female gamete, but after fertilization the oocyte, now termed a zygote, is diploid. Second, once the zygote has cleaved it is called a preimplantation embryo, and it is at this stage of development that entry into the uterine cavity occurs for subsequent implantation. Therefore, for purposes of clarity and accuracy, the term egg transport covers postovulation and prefertilization stages (i.e. the haploid life span of the ovulated oocyte). A subsequent section provides details concerning transport of the fertilized (diploid) oocyte (i.e. zygote) and preimplantation embryo.

Before discussing the actual passage of the oocyte into and through the fallopian tube (oviduct), it is essential to briefly describe the anatomy and physiology of that structure.¹ The oviduct is attached distally to the ovary and proximally to the uterus. It is a muscular tube with an overall length on average of about 11 to 12 cm and is composed of four regions. The most distal portion is referred to as the infundibulum; it is approximately 1 cm in length and contains the finger-like fimbria. The epithelial lining of the fimbria is densely ciliated and highly convoluted. The former quality, along with the muscle-controlled movements of the fimbria, is thought to be important for capture of the cumulus-oocyte complex. The next portion of the oviduct is called the ampulla. This segment averages 5 to 8 cm in length. It is within this highly ciliated portion of the oviduct that fertilization and early embryo development occur. The ampulla is most often the site for ectopic implantation. The next region, approximately 2 to 3 cm in length, is the isthmus. Like the ampulla, it too is ciliated yet less densely so. The isthmus is thought to regulate sperm and embryo transport. The last segment of the oviduct is called the intramural segment; it is the link between the isthmus of the oviduct and the uterine cavity.

Each portion of the oviduct appears to be preferentially regulated by hormones that cause a distinct regionalization of activities, depending on the day in the female reproductive cycle.² For example, in the follicular phase (day 4), propulsive forces operating throughout the length of the oviduct are pro-uterus. However, as the menstrual cycle continues to unfold, so do differences in regional activity of the oviduct. At day 8, the ampulla has alternating pro-uterus and pro-ovary propulsive forces. At the time of ovulation, ipsilateral transport to the ovary increases with increasing follicular diameter.³ It has been observed that pregnancy rates after intercourse are higher in those women who demonstrate ipsilateral transport, as opposed to those who fail to show lateralization.³ Therefore, it would appear that oviductal function is critical for early stages of fertilization.

At the time of ovulation, the oocyte is surrounded by a mass of granulosa cells termed the cumulus oophorus, and they are collectively called the cumulus-oocyte complex (COC). The several innermost cell layers of the cumulus (i.e. those immediately overlying the zona pellucida of the oocyte) are called the coronal cells. (After cumulus maturation the same cells are called the corona radiata because of their “sunburst” appearance.) These cells have processes that extend through the acellular glycoprotein matrix (i.e. the zona) to contact the oocyte plasma membrane for metabolic exchange (e.g. nutrients). The cumulus of the mature COC is rather sticky, and it is thought that this attribute facilitates the adherence of the COC to the surface of the ovary.

The mechanism by which the COC is picked up and gains entry into the oviductal lumen is uncertain. One possibility is that the fimbriated end of the ipsilateral oviduct sweeps over the ovary, picks up the COC, and draws it into the tubular lumen by muscular control. Paradoxically, women have become pregnant who were missing the oviduct on the side where ovulation occurred. This evidence implies that other forces are in place to facilitate oocyte pickup. Another possibility is that the rhythmic and unidirectional beating of cilia in the ampullary and isthmic regions of the oviduct draw the COC into the tube. However, this cannot be the sole mechanism by which the COC is picked up and transported through the oviduct, because women with immotile cilia syndrome (Kartagener's syndrome) are often fertile. Another possibility is that negative pressure results from muscular contractions of the oviduct, and the COC is sucked from the surface of the ovary and into the lumen. However, capping and suturing of the fimbriated end in women has failed to prevent pregnancy.⁴ More recently, researchers using sophisticated measuring techniques reported that the uterus and oviduct appear to act as a peristaltic pump. The pumping frequency increases on the ipsilateral side, in the direction where ovulation will occur, and as the follicular diameter increases.³ A novel alternative to the aforementioned mechanisms for COC pickup is one involving mucus strand connections between fimbria and ovary that act as a tether between the two structures to facilitate fimbrial capture of the COC.⁵ The entire process of pickup and deposition of the COC into the lumen takes between 2 and 3 minutes after ovulation. Therefore, it would seem that at least several mechanisms are involved with COC pickup, the most important of which are ciliary beating, sweeping of the ovarian surface by the fimbria, and peristaltic pumping of the female tract.

After ovulation, the fertilizable life span of the mature human oocyte is estimated to be 12 to 24 hours. In contrast, the fertilizable life span of the human spermatozoon is probably 48 to 72 hours. Sperm motility can persist for much longer (and has been documented in vivo for up to 5 days), but fertilizing ability is lost before motility is.

In addition to the female factors described herein and in other chapters, male-related conditions must be satisfied for successful fertilization. The first condition is that a sufficient number of mature, viable spermatozoa must be present in the ejaculate. Second, the morphology of the sperm must be such that the cervical mucus will allow passage into the uterus. Cervical mucus serves as an effective filter to prevent passage into the uterus of not only seminal plasma (seminal prostaglandins cause uterine contractions) but also those spermatozoa with abnormal shape and poor motility. Sperm with abnormal morphology are known to have decreased fertilizing ability. Third, it is essential that a good percentage of these sperm have forwardly progressive motion to propel them from the seminal plasma, through the cervical mucus, and into the uterine cavity and the fallopian tube for ultimate encounter with the COC. Fourth, at some time during sperm transport, and presumably close to the time of acrosome reaction and zona penetration, sperm motion should change to a hyperactivated state. Fifth, it is critical that the fluids contributed by the accessory glands and the components contained within those fluids function to facilitate viability, prevent premature activation by stabilizing the sperm membranes, coat the sperm membranes with elements essential for protection from the hostile environment of the vagina, and have reversibility in the binding of coating proteins to allow for membrane modifications (capacitation) so that the integral proteins (receptors) necessary for initiation of events required for fertilization (acrosome reaction) can be expressed.^6,7

It is not until ejaculated human spermatozoa are removed from seminal plasma and the process of capacitation has been initiated that sperm gain the ability to fertilize a fully invested human oocyte. At the moment sperm enter into cervical mucus, they become isolated from the seminal plasma and the process of capacitation is initiated. One reason sperm must be removed from seminal plasma is that proteins present in the seminal plasma act to biochemically restrict the sperm plasma membrane, at least as it relates to fertilizing ability. As sperm swim through the cervical mucus, proteins adsorbed to the plasma membrane begin to be removed. It is at the point of removal from exposure to the aforementioned proteins that changes begin to occur, in crescendo-like fashion, to prepare the spermatozoon for fertilization. These unique changes have collectively been termed capacitation, and they were first described by Chang and Austin.^8,9

Some events that occur during the time course to induce capacitation help to characterize this process, and they are (1) an increase in membrane fluidity⁶; (2) a decrease in net surface charge⁶; (3) an increase in oxidative processes and cyclic adenosine monophosphate (cAMP) production^6,10,11; (4) a decrease in the ratio of plasma membrane cholesterol to phospholipid^6,12,13; (5) expression of mannose binding sites as a consequence of cholesterol removal¹³; (6) an increase in tyrosine phosphorylation^10,11; (7) an increase in reactive oxygen species¹⁰; and (8) changes in sperm swimming patterns, termed hyperactivation.^6,14 The only clear indicator as to the progress of capacitation is susceptibility to acrosome reaction induction, but occurrence of the reaction merely signifies that a sperm cell has become fully capacitated. The processes of capacitation and acrosomal exocytosis have been shown experimentally to be distinct and separable in human spermatozoa.^15,16

The acrosome reaction is an exocytotic process occurring in the sperm head that is essential for successful penetration of the oocyte zona pellucida. Sperm that have not undergone an acrosome reaction on or in extremely close proximity to the zona pellucida cannot fertilize the oocyte without assistance.e.g.^6,7 Contained within the anterior portion of the sperm head and immediately underlying the plasma membrane is a membrane-bound, cap-like structure called the acrosome. The acrosome is a unique organelle that is analogous to both a lysosome and a regulated secretory vesicle.^17,18 Contained within the acrosomal vesicle are several lytic enzymes that are released as a consequence of the acrosome reaction. One of the principal enzymes is a serine glycoproteinase called acrosin, and it exists in a proenzyme form called proacrosin.¹⁷ The precise in vivo mechanism by which proacrosin (inactive form) is converted to acrosin (active form) is ill defined, but a change in acrosomal pH is thought to be involved.

Because changes in intracellular calcium play an important role in somatic cell exocytosis, it has been reasoned that this may also be true for exocytosis in spermatozoa.⁶ Although a large amount of data support a role for calcium in certain important aspects of human sperm function (e.g., capacitation, motility), its absolute requirement for the acrosome reaction is a matter of debate. For example, the addition of periovulatory follicular fluid or progesterone to capacitated spermatozoa stimulates an influx of calcium ions that is coincident with the acrosome reaction.^{19,20,21,22,23} Because periovulatory follicular fluid contains progesterone, it is reasoned that this is the mechanism by which follicular fluid stimulates calcium influx and the acrosome reaction. However, other acrosome reaction-stimulating factors (e.g., atrial natriuretic peptide) have been detected in this complex fluid, and their role in fertilization cannot be discounted.²⁴

Other data suggest a less important role for calcium in the acrosome reaction. Studies using solubilized human zona pellucida have demonstrated that the acrosome reaction can occur in the absence of extracellular calcium. However, the response was roughly half that obtained when calcium was included in the medium.²⁵ In final evaluation, it seems reasonable to conclude that calcium may contribute to the overall acrosome reaction response but may not be absolutely required for the reaction to proceed to completion.

If one considers the temporal and spatial aspects of fertilization, then it would seem critical that sperm receptors for zona ligands be properly situated and primed as a result of capacitation so that the subsequent ligand-receptor-induced signaling events that culminate in the acrosome reaction (e.g., localized activation and release of acrosomal enzymes) occur in immediate proximity to the oocyte. In fact, an increasing amount of evidence supports the idea that human spermatozoa must be acrosome intact when they contact, bind, and are induced to acrosome react by the zona pellucida glycoproteins.²⁶ Therefore, prematurity or disorganization in the aforementioned sequence of events is likely to lead to reduced fertilization potential.

A species-selective barrier to fertilization surrounds the mammalian oocyte and is called the zona pellucida. The zona pellucida is an acellular matrix consisting of three glycoproteins: ZP1, ZP2, and ZP3.²⁷ Experiments have shown that the zona pellucida is responsible for the initiation of sperm-zona binding and the acrosome reaction.^25,26,27,28 Using recombinant technology, the role of ZP3 in the fertilization process has been clarified. Specifically, ZP3 is now characterized as the primary ligand for sperm-zona binding and acrosome reaction induction.^29,30

Although ZP3 has been fairly well characterized as a ligand for sperm, such is not the case for ZP3 receptors on the sperm plasma membrane. The majority of current data concerning sperm receptors for zona glycoproteins is restricted to nonhuman mammalian and nonmammalian species. In the human, one of the best described ZP3 receptor candidates is a lectin that binds mannose-containing ligands.¹³ Another ZP3 receptor candidate on human sperm is a 95-kd receptor tyrosine kinase (RTK).³¹ This receptor is thought to initiate intracellular pH changes that culminate in the acrosome reaction. Interestingly, not only does intact zona pellucida stimulate tyrosine phosphorylation but so also does progesterone.³² Whether these two agonists act via the same RTK is questionable.

Based on a variety of data, the possibility exists that one or more signaling or second-messenger pathways interact or communicate in the sequence of events that terminates in the acrosome reaction, and subsequent penetration of the oocyte vestments by the spermatozoon.^16,25,33 Indeed, this might endow the spermatozoon with sensitive control mechanisms for regulating cellular responses as it swims through the varied and changing environment of the female reproductive tract. In fact, this arrangement could potentially provide these cells with the ability to sense and respond to molecules or ligands present in the female reproductive tract that have been shown to initiate the acrosome reaction, such as follicular and oviductal fluids and cumulus oophorus.

After a spermatozoon has undergone the acrosome reaction and its zona-penetrating enzymes have become activated, it is then able to pass through the zona. Enzymatic degradation of zona proteins and vigorous flagellar motion (hyperactivated motility) facilitate entry and passage of the sperm through the zona, leaving a penetration slit in its wake. In order to gain access into the oocyte interior the spermatozoon must contact, bind to, and fuse with the oocyte plasma membrane. The acrosome reaction not only culminates in the release of enzymes but also brings about a remodeling of the sperm plasma membrane. As a result, new sperm membrane proteins become exposed that are likely to prove integral for sperm-oocyte fusion. Although the process of sperm-oocyte fusion is only beginning to be understood, several features can be identified.

Data indicate that sperm-oocyte fusion is initiated by signal transduction processes that involve adhesion molecules in the form of ligands and receptors on both sperm and oocyte plasma membranes. These molecules are beginning to be characterized, and it can be stated with some confidence that adhesion of the spermatozoon to the oolemma is mediated by integrins.^34,35,36 Integrins are a class of heterodimeric adhesion receptor molecules that participate in cell-to-cell and cell-to-substratum interactions, and they are present on essentially all human cells. Further, all mammalian oocytes express integrins on their plasma membrane surface.

Integrins that recognize the Arg-Gly-Asp sequence (RGD) have been detected on the plasma membrane of human oocytes. Fibronectin and vitronectin are glycoproteins that contain functional RGD sequences, and they are present on human spermatozoa.^34,35,36,37 When oligopeptides specifically designed to block fibronectin or vitronectin receptors were tested on human spermatozoa in a zona-free hamster oocyte assay, it was found that the peptide for blocking cell attachment to fibronectin was without effect but the other peptide, which blocks both fibronectin and vitronectin receptors, inhibited sperm-oocyte binding. These data suggest that a possible mechanism for sperm-oocyte adhesion and fusion involves an integrin-vitronectin receptor-ligand interaction.³⁸

Another potential ligand for oolemmal integrin is human fertilin.^39,40 Fertilin, formerly PH30, is a heterodimeric sperm surface protein with binding and fusion domains compatible for interaction with integrin receptors on the oocyte. Because of its domains, human fertilin β can be identified as a member of the ADAM family (membrane-anchored proteins having A Disintegrin And Metalloprotease domain).^39,40 Although fertilin has been relatively well described in nonhuman mammalian systems, more investigation is required to determine its precise role in human sperm-oocyte adhesion and fusion. The possibility exists that fertilin and vitronectin act together or in a parallel fashion during gamete interaction.

At some point during or after the fusion process, the oocyte is induced by the spermatozoon to become activated.⁴¹ Activation involves the resumption of meiosis, as evidenced by extrusion of the second polar body, and the release of cortical granules into the perivitelline space. The cortical granules modify zona glycoproteins 2 and 3 on the inner aspect of the zona pellucida, resulting in a loss of their ability to stimulate the acrosome reaction and tight binding, so as to prevent penetration into the oocyte by supernumerary spermatozoa. This latter event apparently occurs before or simultaneously with the resumption of meiosis. Failure of the oocyte to synthesize or exocytose the cortical granules in a timely fashion results in polyspermic fertilization and embryodysgenesis.

The first and most notable event after incorporation of the spermatozoon into the oocyte is the production of sperm-induced calcium (Ca²⁺ ) transients. Calcium is the main intracellular signal responsible for the initiation of oocyte activation. These calcium fluxes occur in series and over time (termed “calcium oscillations”); when only a single transient is induced, either by chemical or mechanical stimulation, the oocyte fails to activate. The mechanism by which sperm induce calcium transients is unknown, but there are data that support essentially two models for sperm-induced oocyte activation.^41,42

One proposed mechanism for sperm-induced oocyte activation is borrowed from classic ligandreceptor interaction that culminates in effector activation.⁴¹ In this model, initiation for the signal cascade occurs as a result of binding of a ligand, in this case the spermatozoon, to a sperm receptor on the oolemma, which results in G-protein activation, activation of an amplifying enzyme, and generation of an intracellular second messenger within the oocyte.

A second possible mechanism for sperm-induced oocyte activation can loosely be termed the “fusion hypothesis.”⁴¹ A prerequisite for this model is that at the time of sperm and oocyte membrane fusion a “latent” period ensues. It is proposed that during the latent period a soluble sperm-derived factor diffuses from the sperm into the oocyte's cytoplasm and results in oocyte activation.^43,44,45,46 One problem in assigning a role for such a “sperm factor” in oocyte activation is that to date there are no published reports in which it was shown that the extract from a single spermatozoon was able to activate an oocyte.

Regardless of which mechanism or collection of mechanisms is ultimately responsible for oocyte activation, the issue remains that at the time of sperm-oocyte fusion the initiating factor must be appropriately primed and situated to effect the desired response. Abnormalities in transcription, translation, or any other significant molecular process responsible for producing the oocyte-activating ligand/effector molecule during spermatogenesis or spermiogenesis will ultimately render the fertilization event moot.

As the sperm nucleus is undergoing oocytemediated decondensation, the sperm centrosome is orchestrating pronuclear mobilization, syngamy, and, ultimately, early cleavage. The paternally inherited human sperm centrosome, with the assistance of maternal γ-tubulin, nucleates sperm astral microtubules, unites paternal and maternal genomes, and forms the mitotic spindle. At the time of fertilization, the human sperm centrosome restores the zygotic centrosome, which is the organizing center for microtubules. In doing so, is establishes the polarity and three-dimensional structure of the embryo.^47,48

As mentioned previously, fertilization occurs in the ampulla. Transit time of the newly fertilized oocyte (zygote) from the ampulla to the ampulla-isthmic junction is approximately 30 hours, after which the zygote remains in the isthmus another 30 hours before resuming transit through the isthmus. It is not until the fifth or sixth day after fertilization that the preimplantation embryo is deposited into the uterine cavity. During the time frame from fertilization to deposition of the embryo in the uterus, the propulsive forces in the oviduct are pro-uterus.²

There is general consensus that, for embryo development to proceed to a point adequate for implantation, residence in the oviduct is required. However, a 1998 report demonstrated that the transfer to the uterus of zygotes resulting from in vitro fertilization results in pregnancy rates that are on a par with rates achieved from multicell embryos.⁴⁹ In contrast, when human embryos are cocultured on human oviductal epithelial cells, higher implantation and lower spontaneous abortion rates are achieved.⁵⁰ Therefore, it would appear that complex interactions take place between the oviductal epithelium and the embryo, and much more knowledge is necessary before we can understand the contributions of the tubal environment to embryo development. However, it can be clearly stated that synchrony between uterine endometrium and embryo development must be in place for successful implantation to be achieved.

There is still much more knowledge that needs to be gained regarding human fertilization. The ARTs have provided us with numerous tools to better understand this complex process, although it can be argued that the in vitro environment can never completely replicate the in vivo one. Perhaps new and improved tissue culture conditions will facilitate advances in elucidating the complexities of sperm-oocyte interaction. Much activity is now being directed toward the refinement of oviductal cell culture in an effort to better understand oviductal physiology, function, and sperm-epithelial cell interaction. Many fascinating results are being reported from these pioneering investigations.^50,51,52,53

In conclusion, a wealth of information has been obtained in the last decade concerning the processes of human fertilization and implantation. A further expansion of this knowledge will take place rapidly over the next decade as better, smaller, more accurate invasive and noninvasive microprobes are evolved for revealing the subtle and complex nature of oocyte transport, gamete interaction, and early embryonic development and transport.

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