Egg Transport

The first step in egg transport is capture of the ovulated egg by the uterine tube. Shortly before ovulation, the epithelial cells of the uterine tube become more highly ciliated, and smooth muscle activity in the tube and its suspensory ligament increases as the result of hormonal influences. By ovulation, the fimbriae of the uterine tube move closer to the ovary and seem to sweep rhythmically over its surface. This action, in addition to the currents set up by the cilia, efficiently captures the ovulated egg complex. Experimental studies on rabbits have shown that the bulk provided by the cellular coverings of the ovulated egg is important in facilitating the egg’s capture and transport by the uterine tube. Denuded ova or inert objects of that size are not so readily transported. Capture of the egg by the uterine tube also involves an adhesive interaction between the egg complex and the ciliary surface of the tube.

Even without these types of natural adaptations, the ability of the uterine tubes to capture eggs is remarkable. If the fimbriated end of the tube has been removed, egg capture occurs remarkably often, and pregnancies have even occurred in women who have had one ovary and the contralateral uterine tube removed. In such cases, the ovulated egg would have to travel free in the pelvic cavity for a considerable distance before entering the ostium of the uterine tube on the other side.

When inside the uterine tube, the egg is transported toward the uterus, mainly as the result of contractions of the smooth musculature of the tubal wall. Although the cilia lining the tubal mucosa may also play a role in egg transport, their action is not obligatory because women with immotile cilia syndrome are often fertile.

While in the uterine tube, the egg is bathed in tubal fluid, which is a combination of secretion by the tubal epithelial cells and transudate from capillaries just below the epithelium. In some mammals, exposure to oviductal secretions is important to the survival of the ovum and for modifying the composition of the zona pellucida, but the role of tubal fluid in humans is less clear.

Tubal transport of the egg usually takes 3 to 4 days, whether or not fertilization occurs (Fig. 2.2). Egg transport typically occurs in two phases: slow transport in the ampulla (approximately 72 hours) and a more rapid phase (8 hours) during which the egg or embryo passes through the isthmus and into the uterus (see p. 51). By a poorly understood mechanism, possibly local edema or reduced muscular activity, the egg is temporarily prevented from entering the isthmic portion of the tube, but under the influence of progesterone, the uterotubal junction relaxes and permits entry of the ovum.

By roughly 80 hours after ovulation, the ovulated egg or embryo has passed from the uterine tube into the uterus. If fertilization has not occurred, the egg degenerates and is phagocytized. (Implantation of the embryo is discussed in Chapter 3.)

Sperm Transport

Sperm transport occurs in both the male reproductive tract and the female reproductive tract. In the male reproductive tract, transport of spermatozoa is closely connected with their structural and functional maturation, whereas in the female reproductive tract, it is important for spermatozoa to pass to the upper uterine tube, where they can meet the ovulated egg.

After spermiogenesis in the seminiferous tubules, the spermatozoa are morphologically mature but are nonmotile and incapable of fertilizing an egg (Fig. 2.3). Spermatozoa are passively transported via testicular fluid from the seminiferous tubules to the caput (head) of the epididymis through the rete testis and the efferent ductules. They are propelled by fluid pressure generated in the seminiferous tubules and are assisted by smooth muscle contractions and ciliary currents in the efferent ductules. Spermatozoa spend about 12 days in the highly convoluted duct of the epididymis, which measures 6 m in the human, during which time they undergo biochemical maturation. This period of maturation is associated with changes in the glycoproteins in the plasma membrane of the sperm head. By the time the spermatozoa have reached the cauda (tail) of the epididymis, they are capable of fertilizing an egg.

On ejaculation, the spermatozoa rapidly pass through the ductus deferens and become mixed with fluid secretions from the seminal vesicles and prostate gland. Prostatic fluid is rich in citric acid, acid phosphatase, zinc, and magnesium ions, whereas fluid of the seminal vesicle is rich in fructose (the principal energy source of spermatozoa) and prostaglandins. The 2 to 6 mL of ejaculate (semen, or seminal fluid) typically consists of 40 to 250 million spermatozoa mixed with alkaline fluid from the seminal vesicles (60% of the total) and acid secretion (pH 6.5) from the prostate (30% of the total). The pH of normal semen ranges from 7.2 to 7.8. Despite the numerous spermatozoa (>100 million) normally present in an ejaculate, a number as small as 25 million spermatozoa per ejaculate may be compatible with fertility.

In the female reproductive tract, sperm transport begins in the upper vagina and ends in the ampulla of the uterine tube, where the spermatozoa make contact with the ovulated egg. During copulation, the seminal fluid is normally deposited in the upper vagina (see Fig. 2.3), where its composition and buffering capacity immediately protect the spermatozoa from the acid fluid found in the upper vaginal area. The acidic vaginal fluid normally serves a bactericidal function in protecting the cervical canal from pathogenic organisms. Within about 10 seconds, the pH of the upper vagina is increased from 4.3 to as much as 7.2. The buffering effect lasts only a few minutes in humans, but it provides enough time for the spermatozoa to approach the cervix in an environment (pH 6.0 to 6.5) optimal for sperm motility.

The next barriers that the sperm cells must overcome are the cervical canal and the cervical mucus that blocks it. Changes in intravaginal pressure may suck spermatozoa into the cervical os, but swimming movements also seem to be important for most spermatozoa in penetrating the cervical mucus.

The composition and viscosity of cervical mucus vary considerably throughout the menstrual cycle. Composed of cervical mucin (a glycoprotein with a high carbohydrate composition) and soluble components, cervical mucus is not readily penetrable. Between days 9 and 16 of the cycle, however, its water content increases, and this change facilitates the passage of sperm through the cervix around the time of ovulation; such mucus is sometimes called E mucus. After ovulation, under the influence of progesterone, the production of watery cervical mucus ceases, and a new type of sticky mucus, which has a much decreased water content, is produced. This progestational mucus, sometimes called G mucus, is almost completely resistant to sperm penetration. A highly effective method of natural family planning makes use of the properties of cervical mucus.

There are two main modes of sperm transport through the cervix. One is a phase of initial rapid transport, by which some spermatozoa can reach the uterine tubes within 5 to 20 minutes of ejaculation. Such rapid transport relies more on muscular movements of the female reproductive tract than on the motility of the spermatozoa themselves. These early-arriving sperm, however, appear not to be as capable of fertilizing an egg as do those that have spent more time in the female reproductive tract. The second, slow phase of sperm transport involves the swimming of spermatozoa through the cervical mucus (traveling at a rate of 2 to 3 mm/hour), their storage in cervical crypts, and their final passage through the cervical canal as much as 2 to 4 days later.

Relatively little is known about the passage of spermatozoa through the uterine cavity, but the contraction of uterine smooth muscle, rather than sperm motility, seems to be the main intrauterine transport mechanism. At this point, the spermatozoa enter one of the uterine tubes. According to some more recent estimates, only several hundred spermatozoa enter the uterine tubes, and most enter the tube containing the ovulated egg.

Once inside the uterine tube, the spermatozoa collect in the isthmus and bind to the epithelium for about 24 hours. During this time, they are influenced by secretions of the tube to undergo the capacitation reaction. One phase of capacitation is removal of cholesterol from the surface of the sperm. Cholesterol is a component of semen and acts to inhibit premature capacitation. The next phase of capacitation consists of removal of many of the glycoproteins that were deposited on the surface of the spermatozoa during their tenure in the epididymis. Capacitation is required for spermatozoa to be able to fertilize an egg (specifically, to undergo the acrosome reaction; see p. 29). After the capacitation reaction, the spermatozoa undergo a period of hyperactivity and detach from the tubal epithelium. Hyperactivation helps the spermatozoa to break free of the bonds that held them to the tubal epithelium. It also assists the sperm in penetrating isthmic mucus, as well as the corona radiata and the zona pellucida, which surround the ovum. Only small numbers of sperm are released at a given time. This may reduce the chances of polyspermy (see p. 31).

On their release from the isthmus, the spermatozoa make their way up the tube through a combination of muscular movements of the tube and some swimming movements. The simultaneous transport of an egg down and spermatozoa up the tube is currently explained on the basis of peristaltic contractions of the uterine tube muscles. These contractions subdivide the tube into compartments. Within a given compartment, the gametes are caught up in churning movements that over 1 or 2 days bring the egg and spermatozoa together. Fertilization of the egg normally occurs in the ampullary portion (upper third) of the uterine tube. Estimates suggest that spermatozoa retain their function in the female reproductive tract for about 80 hours.

After years of debate concerning the possibility that mammalian spermatozoa may be guided to the egg through attractants, more recent research suggests that this could be the case. Mammalian spermatozoa have been found to possess odorant receptors of the same family as olfactory receptors in the nose, and they can respond behaviorally to chemically defined odorants. Human spermatozoa also respond to cumulus-derived progesterone and to yet undefined chemoattractants emanating from follicular fluid and cumulus cells. Human spermatozoa are also known to respond to a temperature gradient, and studies on rabbits have shown that the site of sperm storage in the oviduct is cooler than that farther up the tube where fertilization occurs. It seems that only capacitated spermatozoa have the capability of responding to chemical or thermal stimuli. Because many of the sperm cells that enter the uterine tube fail to become capacitated, these spermatozoa are less likely to find their way to the egg.

Formation and Function of the Corpus Luteum of Ovulation and Pregnancy

While the ovulated egg is passing through the uterine tubes, the ruptured follicle from which it arose undergoes a series of striking changes that are essential for the progression of events leading to and supporting pregnancy (see Fig. 1.8). Soon after ovulation, the basement membrane that separates the granulosa cells from the theca interna breaks down, thus allowing thecal blood vessels to grow into the cavity of the ruptured follicle. The granulosa cells simultaneously undergo a series of major changes in form and function (luteinization). Within 30 to 40 hours of the LH surge, these cells, now called granulosa lutein cells, begin secreting increasing amounts of progesterone along with some estrogen. This pattern of secretion provides the hormonal basis for the changes in the female reproductive tissues during the last half of the menstrual cycle. During this period, the follicle continues to enlarge. Because of its yellow color, it is known as the corpus luteum. The granulosa lutein cells are terminally differentiated. They have stopped dividing, but they continue to secrete progesterone for 10 days.

In the absence of fertilization and a hormonal stimulus provided by the early embryo, the corpus luteum begins to deteriorate (luteolysis) late in the menstrual cycle. Luteolysis seems to involve both the preprogramming of the luteal cells to apoptosis (cell death) and uterine luteolytic factors, such as prostaglandin F₂. Regression of the corpus luteum and the accompanying reduction in progesterone production cause the hormonal withdrawal that results in the degenerative changes of the endometrial tissue during the last days of the menstrual cycle.

During the regression of the corpus luteum, the granulosa lutein cells degenerate and are replaced with collagenous scar tissue. Because of its white color, the former corpus luteum now becomes known as the corpus albicans (“white body”).

If fertilization occurs, the production of the protein hormone chorionic gonadotropin by the future placental tissues maintains the corpus luteum in a functional condition and causes an increase in its size and hormone production. Because the granulosa lutein cells are unable to divide and cease producing progesterone after 10 days, the large corpus luteum of pregnancy is composed principally of theca lutein cells. The corpus luteum of pregnancy remains functional for the first few months of pregnancy. After the second month, the placenta produces enough estrogens and progesterone to maintain pregnancy on its own. At this point, the ovaries can be removed, and pregnancy would continue.

Penetration of the Corona Radiata

When the spermatozoa first encounter the ovulated egg in the ampullary part of the uterine tube, they are confronted by the corona radiata and some remnants of the cumulus oophorus, which represents the outer layer of the egg complex (Fig. 2.4). The corona radiata is a highly cellular layer with an intercellular matrix consisting of proteins and a high concentration of carbohydrates, especially hyaluronic acid. It is widely believed that hyaluronidase emanating from the sperm head plays a major role in penetration of the corona radiata, but the active swimming movements of the spermatozoa are also important.

Attachment to and Penetration of the Zona Pellucida

The zona pellucida, which is 13 µm thick in humans, consists principally of four glycoproteins—ZP₁ to ZP₄. ZP₂ and ZP₃ combine to form basic units that polymerize into long filaments. These filaments are periodically linked by cross-bridges of ZP₁ and ZP₄ molecules (Fig. 2.5). The zona pellucida of an unfertilized mouse egg is estimated to contain more than 1 billion copies of the ZP₃ protein.

After they have penetrated the corona radiata, spermatozoa bind tightly to the zona pellucida by means of the plasma membrane of the sperm head (see Fig. 2.4). Spermatozoa bind specifically to a sialic acid molecule, which is the terminal part of a sequence of four sugars at the end of O-linked oligosaccharides that are attached to the polypeptide core of the ZP₃ molecule. Molecules on the surface of the sperm head are specific binding sites for the ZP₃ sperm receptors on the zona pellucida. More than 24 molecules have been proposed, but the identity of the zona-binding molecules remains unknown. Interspecies molecular differences in the sperm-binding regions of the ZP₃ molecule may serve as the basis for the inability of spermatozoa of one species to fertilize an egg of another species. In mammals, there is less species variation in the composition of ZP₃; this may explain why penetration of the zona pellucida by spermatozoa of closely related mammalian species is sometimes possible, whereas it is rare among lower animals.

On binding to the zona pellucida, mammalian spermatozoa undergo the acrosomal reaction. The essence of the acrosomal reaction is the fusion of parts of the outer acrosomal membrane with the overlying plasma membrane and the pinching off of fused parts as small vesicles. This results in the liberation of the multitude of enzymes that are stored in the acrosome (Box 2.1).

The acrosomal reaction in mammals is stimulated by the ZP₃ molecule acting through G proteins in the plasma membrane on the sperm head. In contrast to the sperm receptor function of ZP₃, a large segment of the polypeptide chain of the ZP₃ molecule must be present to induce the acrosomal reaction. An initiating event of the acrosomal reaction is a massive influx of calcium (Ca⁺⁺) through the plasma membrane of the sperm head. This process, accompanied by an influx of sodium (Na⁺) and an efflux of hydrogen (H⁺), increases the intracellular pH. Fusion of the outer acrosomal membrane with the overlying plasma membrane soon follows. As the vesicles of the fused membranes are shed, the enzymatic contents of the acrosome are freed and can assist the spermatozoa in making their way through the zona pellucida.

After the acrosomal reaction, the inner acrosomal membrane forms the outer surface covering of most of the sperm head (see Fig. 2.4D). Toward the base of the sperm head (in the equatorial region), the inner acrosomal membrane fuses with the remaining postacrosomal plasma membrane to maintain membrane continuity around the sperm head.

Only after completing the acrosomal reaction can the spermatozoon successfully begin to penetrate the zona pellucida. Penetration of the zona is accomplished by a combination of mechanical propulsion by movements of the sperm’s tail and digestion of a pathway through the action of acrosomal enzymes. The most important enzyme is acrosin, a serine proteinase that is bound to the inner acrosomal membrane. When the sperm has made its way through the zona and into the perivitelline space (the space between the egg’s plasma membrane and the zona pellucida), it can make direct contact with the plasma membrane of the egg.

Binding and Fusion of Spermatozoon and Egg

After a brief transit period through the perivitelline space, the spermatozoon makes contact with the egg. In two distinct steps, the spermatozoon first binds to and then fuses with the plasma membrane of the egg. Binding between the spermatozoon and egg occurs when the equatorial region of the sperm head contacts the microvilli surrounding the egg. Molecules on the plasma membrane of the sperm head, principally sperm proteins called fertilins and cyritestin, bind to α₆ integrin and CD9 protein molecules on the surface of the egg. The acrosomal reaction causes a change in the membrane properties of the spermatozoon because, if the acrosomal reaction has not occurred, the spermatozoon is unable to fuse with the egg. Actual fusion between spermatozoon and egg, mediated by integrin on the membrane of the oocyte, brings their plasma membranes into continuity.

After initial fusion, the contents of the spermatozoon (the head, the midpiece, and usually the tail) sink into the egg (Fig. 2.6), whereas the sperm’s plasma membrane, which is antigenically distinct from that of the egg, becomes incorporated into the egg’s plasma membrane and remains recognizable at least until the start of cleavage. Although mitochondria located in the sperm neck enter the egg, they do not contribute to the functional mitochondrial complement of the zygote. In humans, the sperm contributes the centrosome, which is required for cell cleavage.

Prevention of Polyspermy

When a spermatozoon has fused with an egg, the entry of other spermatozoa into the egg (polyspermy) must be prevented, or abnormal development is likely to result. Two blocks to polyspermy, fast and slow, are typically present in vertebrate fertilization.

The fast block to polyspermy, which has been best studied in sea urchins, consists of a rapid electrical depolarization of the plasma membrane of the egg. The resting membrane potential of the egg changes from about −70 to +10 mV within 2 to 3 seconds after fusion of the spermatozoon with the egg. This change in membrane potential prevents other spermatozoa from adhering to the egg’s plasma membrane. The fast block in mammals is short-lived, lasting only several minutes, and may not be as heavily based on membrane depolarization as that in sea urchins. This time is sufficient for the egg to mount a permanent slow block. The exact nature of the fast block in the human egg is still not well defined.

Very soon after sperm entry, successive waves of Ca⁺⁺ pass through the cytoplasm of the egg. The first set of waves, spreading from the site of sperm-egg fusion, is involved in stimulating completion of the second meiotic division of the egg. Later waves of Ca⁺⁺ initiate recruitment of maternal RNAs in the egg and act on the cortical granules as they pass by them. Exposure to Ca⁺⁺ causes the cortical granules to fuse with the plasma membrane and to release their contents (hydrolytic enzymes and polysaccharides) into the perivitelline space. The polysaccharides released into the perivitelline space become hydrated and swell, thus causing the zona pellucida to rise from the surface of the egg.

The secretory products of the cortical granules diffuse into the porous zona pellucida and hydrolyze the sperm receptor molecules (ZP₃ in the mouse) in the zona. This reaction, called the zona reaction, essentially eliminates the ability of spermatozoa to adhere to and penetrate the zona. The zona reaction has been observed in human eggs that have undergone in vitro fertilization. In addition to changes in the zona pellucida, alterations in sperm receptor molecules on the plasma membrane of the human egg cause the egg itself to become refractory to penetration by other spermatozoa.

Metabolic Activation of the Egg

The entry of the spermatozoon into the egg initiates several significant changes within the egg, including the aforementioned fast and slow blocks to polyspermy. In effect, the sperm introduces into the egg a soluble factor (currently thought to be a phospholipase [phospholipase C zeta]), which stimulates a pathway leading to the release of pulses of Ca⁺⁺ within the cytoplasm of the egg. In addition to initiating the blocks to polyspermy, the released Ca⁺⁺ stimulates a rapid intensification of the egg’s respiration and metabolism through an exchange of extracellular Na⁺ for intracellular H⁺. This exchange results in a rise in intracellular pH and an increase in oxidative metabolism.

Decondensation of the Sperm Nucleus

In the mature spermatozoon, the nuclear chromatin is very tightly packed, in large part because of the —SS— (disulfide) cross-linking that occurs among the protamine molecules complexed with the DNA during spermatogenesis. Shortly after the head of the sperm enters the cytoplasm of the egg, the permeability of its nuclear membrane begins to increase, thereby allowing cytoplasmic factors within the egg to affect the nuclear contents of the sperm. After reduction of the —SS— cross-links of the protamines to sulfhydryl (—SH) groups by reduced glutathione in the ooplasm, the protamines are rapidly lost from the chromatin of the spermatozoon, and the chromatin begins to spread out within the nucleus (now called a pronucleus) as it moves closer to the nuclear material of the egg.

Remodeling of the sperm head takes about 6 to 8 hours. After a short period during which the male chromosomes are naked, histones begin to associate with the chromosomes. During the period of pronuclear formation, the genetic material of the male pronucleus becomes demethylated, whereas methylation in the female genome is maintained.

Completion of Meiosis and the Development of Pronuclei in the Egg

After penetration of the egg by the spermatozoon, the nucleus of the egg, which had been arrested in metaphase of the second meiotic division, completes the last division and releases a second polar body into the perivitelline space (see Fig. 2.6).

The nucleus of the oocyte moves toward the cortex as the result of the action of myosin molecules acting on a network of actin filaments that connect one pole of the mitotic spindle to the cortex. The resulting contraction draws the entire mitotic apparatus toward the surface of the cell (Fig. 2.7). This determines the location at which both the first and second polar bodies are extruded.

A pronuclear membrane, derived largely from the endoplasmic reticulum of the egg, forms around the female chromosomal material. Cytoplasmic factors seem to control the growth of the female and the male pronuclei. Pronuclei appear 6 to 8 hours after sperm penetration, and they persist for about 10 to 12 hours. DNA replication occurs in the developing haploid pronuclei, and each chromosome forms two chromatids as the pronuclei approach each other. When the male and female pronuclei come into contact, their membranes break down, and the chromosomes intermingle. The maternal and paternal chromosomes quickly become organized around a mitotic spindle, derived from the centrosome of the sperm, in preparation for an ordinary mitotic division. At this point, the process of fertilization can be said to be complete, and the fertilized egg is called a zygote.

What is Accomplished by Fertilization?

The process of fertilization ties together many biological loose ends, as follows: