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Chapter 14: Establishing pregnancy: from coitus to syngamy.

CHAPTER OBJECTIVES

* Provide an overview of pregnancy from coitus to parturition.

* Describe the maturation and transport of gametes and zygotes.

* Describe how a spermatozoon and oocyte encounter each other in the female reproductive tract.

* Describe the major steps of fertilization:

1) binding of the spermatozoon to the oocyte,

2) induction of the acrosome reaction, and 3) fusion of the spermatozoon and the oocyte.

OVERVIEW OF PREGNANCY

Prior to this point our consideration of mammalian reproduction has been limited to the production gametes and the physiologic and behavioral strategies that ensure mating occurs when a viable oocyte is available. Our major concern in the next three chapters will be with the mechanisms that are directly involved with the creation of new individuals. In other words, we will consider how the male and female gametes come to interact with each other to result in fertilization. In mammals, fertilization occurs within the oviduct. In all but the monotreme mammals, procreation requires development of offspring within the female's reproductive tract; that is, pregnancy. The pregnant state begins with fertilization and ends with parturition (Figure 14-1). For pedagogical reasons it is useful to divide discussions of pregnancy into three major areas of concern. The first area includes the transport of gametes and embryo as well as fertilization. The second includes events that precede implantation: the development of the embryo (embryogenesis), formation of the fetal membranes that will develop into the placenta, and maternal recognition of pregnancy. The third area of concern deals with how the pregnancy is maintained and finally terminated (parturition). This chapter will deal with the first area of concern.

TRANSPORT OF GAMETES AND ZYGOTES

Fertilization is predicated on the production of viable gametes as well as the successful delivery of spermatozoa and oocytes to the oviduct, the site of fertilization. The journey of spermatozoa to the oviduct is long and involves translocation from the testes, through the male genital ducts, and finally through most of the female's reproductive tract. The journey of the oocyte is much shorter; that is, from the ovary into the oviduct. Once fertilization occurs, the embryo is translocated from the oviduct into the uterus where it eventually implants and establishes pregnancy.

[FIGURE 14-1 OMITTED]

Transport of Spermatozoa Through the Male Reproductive System

As spermatids undergo morphogenesis to form spermatozoa, they remain in pockets of the Sertoli cells and move within the nurse cells toward the lumen of the seminferous tubule. Such movement is facilitated by tracks formed by microtubules. Once sperm detach from Sertoli cells (spermiation) they enter the lumen of the tubule and are passively transported (at this stage spermatozoa are nonmotile) toward the rete testis. The precise mechanism of tubular transport has not been elucidated, but it is unidirectional. The force that moves sperm through the tubules is the hydrostatic pressure arising from fluid produced by the Sertoli cells.

Once the spermatozoa enter the rete testis, they pass through the efferent ducts into the caput epididymis. This too is a passive process and is largely attributed to the positive pressure of the testes, which results from production of fluid by the seminferous tubules. Once inside the epididymis, the spermatozoa continue to move through the lumen, due in part to ciliary action of luminal epithelial cells as well as regular peristaltic contractions of the muscularis. Movement along the epididymis is slow, but constant. On average it takes 10 to 12 days for spermatozoa to move from the caput to the cauda epididymis. While spermatozoa move through the epididymis, they undergo maturational changes, including the acquisition of motility.

There is a steady trickle of spermatozoa from the cauda epididymis into the vas deferens. These cells enter the urethra and are usually excreted in urine. The release of spermatozoa into the urethra is much more dramatic during ejaculation. As noted in Chapter 13, ejaculation consists of emission and expulsion. During emission, pulsatile contractions of the epididymal muscularis move spermatozoa into the vas deferens and into the pelvic urethra. During ejaculation, movement of spermatozoa along the vas deferens is caused by rhythmic contractions of the ischiocavernosus and bulbospongiosus muscles. These contractions force seminal emissions into the pelvic urethra. As these fluids enter the urethra, secretions from the accessory sex glands are added to the ejaculate. These fluids make up 90 percent of the volume of semen. The total volume as well as the contribution of each gland to semen varies among species. In general, the vesicular glands contribute the most volume followed by the prostate and bulbourethral glands. Because these glands line the pelvic urethra in series, ejaculate consists of several fractions. For example, in humans the first fraction contains the highest concentration of sperm cells and primarily fluid secreted by the prostate gland. This portion of semen is rich in citric acid. The second fraction is primarily from the vesicular glands and contains high concentrations of fructose. Overall, the major difference between semen and other body fluids (e.g., blood plasma) is that semen contains high concentrations of citric acid, fructose, and numerous other compounds including some proteins unique to the prostate gland. The functions of these compounds are poorly understood. However, it is likely that they create an environment that supports sperm cells once they are released into the female's reproductive tract.

Transport of Spermatozoa Through the Female Reproductive System

During mating, the ejaculated semen is normally deposited into the anterior vagina (e.g., humans, cattle, sheep, and rabbits) or uterus (e.g., horses, pigs, and rodents), depending on the species.

In either case, spermatozoa face a series of barriers that must be successfully negotiated in order for fertilization to occur. Table 14-1 shows the number of spermatozoa in ejaculates and the site of semen deposition for several species of mammals. Several million sperm are normally deposited in the female reproductive tract. Most of these are lost at various locations in the female genital ducts, leaving only several hundred sperm at the site of fertilization in the oviduct (Figure 14-2). Only the most viable sperm complete the journey to the oviduct. Many die due to the hostile conditions of the female's reproductive tract. These nonviable sperm are removed from the female reproductive tract via retrograde (outward) flow as well absorption following degradation by leukocytes.

[FIGURE 14-2 OMITTED]

Sperm in the Vagina

The vaginal environment is extremely hostile to sperm. This is due to its low pH (less than 5.0) that is produced by the metabolic activity of a lactic acid-forming bacterium. Although seminal plasma may provide some buffering capacity, this effect is only temporary. Thus survival of sperm requires that they move rapidly into the cervix. A large percentage of ejaculated sperm are lost from the female reproductive tract via retrograde flow. Although much of this seems to be to outflow from the vagina, such loss can also occur following insemination into the uterus.

Sperm Transport through the Cervix

Contractions of the vagina and uterus appear to play important roles in transporting sperm through the female genital ducts. Spermatozoa have been found in the oviducts within 5 minutes after insemination; a response that can not be attributed to sperm motility alone. Such movement can be attributed to a negative vaginal pressure as well as an increase in vaginal and uterine contractions which occur following coitus. Together these responses propel sperm from the vagina into the cervix and through the uterus. This so-called rapid phase of sperm transport may not be too important with respect to fertility. The vast majority of sperm that reach the oviduct do so via a slow phase, which requires several hours. The cause of the postcoital contractions is unclear, but some evidence suggests that they might be induced by constituents of the seminal plasma (e.g., prostaglandins).

The cervix acts is a major barrier to sperm transport in cases of intra-vaginal insemination. In rabbits, less than 2 percent of the sperm deposited in the vagina during mating could be recovered in the uterus within 12 hours. With respect to sperm transport, this organ is best understood as a filter. Recall that the lumen of the cervix is highly convoluted and consists of many crypts. In addition, the cervical epithelium produces copious amounts of mucus which flows in a retrograde direction. Progressively motile sperm enter the cervix and become lodged in the crypts as they make their way through the lumen. In this way, viable sperm are physically protected from attack by marauding leukocytes. In contrast, less viable and dead sperm are carried out of the cervix via the mucus and are more prone to phagocytosis by leukocytes.

Whereas contractions of the vagina and uterus play a major role in the rapid transport of sperm through the cervix, progressive motility appears to be a major factor in the sustained or slow transport phase. The ability of spermatozoa to move through the cervix is directly dependent on the nature of the cervical mucus. This cervical gel consists of two major elements: a glycoprotein-rich fraction called mucin (40 percent) and an aqueous phase containing various soluble components consisting of inorganic salts, proteins, and low-molecular weight organic compounds (e.g., simple sugars, amino acids, and lipids). The consistency of the mucus gel changes with the stage of the ovarian cycle in response to changing concentrations of ovarian steroid hormones. During the peri-ovulatory period, when estradiol concentrations are high, the gel consists of 95 percent water and has a low viscosity. In this form, the mucin molecules line up in parallel chains (micelles). The aqueous spaces between these fibrils allow sperm cells to pass. However, during the luteal phase, when progesterone concentrations are high, the water content drops to 90 percent causing the fibrillar structure to disappear and raise viscosity of the gel. This latter structure is not compatible with sperm migration.

Spermatozoa are not evenly distributed within the lumen of the cervix. Most sperm are found near the mucosa. Those that migrate through the cervix toward the uterus occupy the crypts. Apparently the mucus produced in the base of the crypts is of lower viscosity than that produced in the apical regions. Thus sperm tend to migrate within these low-resistance pathways. The gradual emergence of sperm from the crypts into these "privileged pathways" is believed to account for the sustained release of spermatozoa from the cervix. The extent to which the cervix plays a role of a reservoir for sperm is unclear.

Sperm Transport through the Uterus

The morphology of the uterus doesn't impede migration of sperm in the way the cervix does. Nevertheless, the uterus is a barrier to sperm transport in the sense that these cells must cross the lumen of this organ in order to be transported to the oviducts. As noted earlier, uterine contractions play a much more important role in movement of spermatozoa through the uterus than sperm motility. This conclusion is based on the fact that 1) there is no known mechanism that ensures that sperm will move only toward the uterotubal junction, 2) uterine motility increases around the time of mating, and 3) inert particles are transported across the uterus when its motility is high (as it is around the time of mating). Once sperm enter the uterus, they are suspended in fluid secreted by the uterine mucosal cells. In addition to serving as a transport medium, this fluid provides support for the sperm as well as stimulates sperm activity.

Spermatozoa are found in the uterus up to 24 hours after insemination, but are not typically present thereafter. Most of the sperm move to the oviduct within a few hours after insemination (e.g., 6 to 8 hours in sheep and cattle). Those that remain in the uterus after this time are likely phagocytized by leukocytes that infiltrate the uterus within 4 hours after insemination. Interestingly, dead sperm and other debris are removed from the uterus via transport to the oviducts.

Sperm Transport Through the Uterotubal Junction

The final barrier to sperm transport is the uterotubal junction. This is the major barrier for sperm migration when insemination occurs within the uterus (e.g., pig, rodents). This structure is also a significant barrier for species in which intravaginal insemination occurs. The mechanism whereby the uterotubal junction impedes sperm migration is virtually unknown. Its ability to impair sperm transport does not appear to be related to its structure. Although the lumen of this region is tortuous, the junction does not contain a sphincter or valve. Research in some species supports the idea that sperm motility plays an important role in transport through this region.

Sperm Transport Through the Oviduct

Sperm migrate along the length of the oviduct and those that are not lost enter the peritoneal cavity via the infundibulum. Oviductal transport of sperm occurs in two ways. First, those that enter the oviduct shortly after mating move rapidly to the ampullary region. The second phase of transport is much slower. As spermatozoa pass through the uterotubal junction they accumulate in the proximal isthmus of the oviduct forming a reservoir. As sperm enter the isthmus, they become immotile and adhere to the epithelium, possibly due to the effects of mucus-rich fluid that is produced in this area. After a period of several hours, the sperm gradually regain their motility, break away from the epithelial cells, and resume their journey. As they leave the isthmus, spermatozoa become hyperactive and move quickly into the ampullary region. Whatever the cause of this delay in oviductal transport of sperm, it seems that this phenomenon is an important prerequisite for fertilization. For example, in sheep and cows, an accumulation period of 6 to 8 hours is necessary to achieve good fertilization rates.

Hyperactivity of sperm may be necessary for them to break away from the isthmus and resume their migration. However, this alone is not sufficient for transport into the ampulla. The oviduct itself appears to play an important role. In order to understand how the oviduct affects sperm transport it is necessary to review its morphology. The isthmus is a narrow, thick-walled segment containing a well-developed muscularis. The mucosal cells are not ciliated, but contain many secretory cells, which produce a thick, mucus-containing fluid. The anterior region of this segment opens into the larger ampullary region. This is the longest section of the oviduct and is thin walled with a thin muscularis. In addition, the mucosal epithelium consists of both secretory and ciliated cells. Secretions in the ampulla contain more water and are therefore less viscous than those produced in the isthmus. The most anterior segment is the infundibulum, which, like the ampulla, has a thin wall. The infundibulum is a funnel-shaped structure. The edges of the widest portion have a lacey appearance and the walls are densely folded (fimbriae). The mucosal cells are heavily ciliated throughout the infundibulum.

The oviduct effects anterior movement of sperm cells in the following way. Fluids produced by the anterior oviduct are pushed toward the uterus due to the beating of cilia. However, this fluid does not enter the uterus because the thicker oviductal fluid and the narrower lumen in the isthmus create resistance, which reverses the flow of fluids. Thus sperm transport is facilitated by a current, which moves fluid toward the infundibulum. The muscularis of the oviduct also plays a role in sperm transport. The oviduct exhibits peristaltic contractions at most times during the ovarian cycle. However, during the ovulatory period when estradiol concentrations are elevated, these contractions become more regular and are directed toward the ovary. The major consequence of this action is a mixing of oviductal contents, which might enhance the probability that sperm cells will encounter the oocyte.

Transport of the Oocyte

There are two main features of oocyte transport. The first deals with the "pick up" of the oocyte by the oviduct following ovulation. The second deals with migration of the oocyte to the site of fertilization.

The ovulated oocyte is embedded in a mass of granulosa cells (Figure 14-3). Cells of the corona radiata are attached to the zona pellucida. This layer of follicular cells is surrounded by a gelatinous matrix containing numerous cells of the cumulus oophorus. This "oocyte-cumulus complex" appears to be necessary for pick up and transport by the oviduct. Once ovulated, the complex of cells is translocated across the fimbriae and into the oviduct through the ostium (opening into the infundibular oviduct). This involves four mechanisms. First, negative pressure resulting from contractions of the muscularis of the oviduct may create a suction that draws the oocyte into the oviduct. Second, rhythmic contractions of smooth muscle of the ligament supporting the oviduct and ovary may cause the fimbriae to make contact with the ovary. Third, the cilia of the fimbriae make contact with the oocyte-cumulus mass and push it toward the lumen of the oviduct. Fourth, strands of oviductal mucus appear to make contact with the ovary during ovulation and may help guide the oocyte toward the oviduct.

[FIGURE 14-3 OMITTED]

Once the oocyte enters the oviduct it migrates toward the uterotubal junction. The time required to complete the journey from the fimbriae to the uterus averages 3 to 4 days in most species. Fertilization normally occurs in the ampullary-isthmic junction and does not appear to disrupt transport in most species. Transport of the oocyte or zygote through the oviduct is a complex process that is poorly understood. The extent to which oviductal cilia play a role in this process is unclear. It appears likely that contraction of the circular smooth muscle of the oviduct together with the flow of oviductal secretions interact to regulate movement of the oocyte and zygote through the duct towards the uterus. Movement of the oocyte or zygote appears to be almost random, but contractions of the oviduct seem to promote a directional flow towards the uterus. However, as the oocyte approaches the isthmus, its movement is impeded by the reverse flow of fluids described previously. The oocyte reaches this site within 24 hours after ovulation, but is retained there before resuming its migration through the isthmus. Resumption of transport may be related to the increase in diameter of the isthmus and uterotubal junction which occurs as estradiol levels fall and progesterone concentrations increase following ovulation.

The oocyte loses its complex of follicular cells by the time it enters the ampullary-isthmic junction. The timing of this process varies greatly among species. In most mammalian species the cumulus oophorus is present at the time of fertilization (e.g., primates). In others (e.g., cattle and sheep), the oocyte is encased only by the zona pellucida at the time of fertilization.

GAMETE MATURATION

The aforementioned mechanisms governing gamete transport permit sperm to make contact with an oocyte. However, this alone is not a sufficient condition for successful fertilization. It is also necessary for the gametes to be fully mature. This means that they are capable of interacting in a way that results in syngamy. Maturation of the oocyte typically refers to meiotic maturation; that is, the conversion of a full-grown primary oocyte into a secondary oocyte (unfertilized ovum). This occurs in response to the pre-ovulatory surge of LH and involves both the completion of meiosis 1 and metabolic changes that allow the oocyte to become fertilized and develop further. Unlike the oocyte, spermatozoa are not fully mature upon leaving the gonad. As noted earlier, sperm mature to the point where they are motile and have the potential to fuse with an oocyte. However, the capacity to fertilize isn't gained until the sperm resides in the female reproductive tract for several hours.

Capacitation of Spermatozoa

The physiologic changes that make spermatozoa capable of fertilizing an oocyte are referred to collectively as capacitation. This phenomenon is associated with two major changes in the physiology of spermatozoa (Figure 14-4). First, capacitation permits induction of the acrosome reaction, which is necessary for a sperm to penetrate the zona pellucida and ultimately fuse with the plasma membrane of the oocyte. Second, capacitation causes hyper-activation of the sperm. When this occurs the sperm becomes highly motile due to a frantic whipping motion of its tail. This may provide the thrust necessary to penetrate the zona pellucida.

[FIGURE 14-4 OMITTED]

[FIGURE 14-5 OMITTED]

The molecular basis for capacitation is poorly understood. Nevertheless, there is general agreement that capacitation involves changes in the plasma membrane of the sperm. One change that seems to be especially important is the removal or alteration of protein molecules that stabilize or mask membrane proteins that are necessary for the sperm cell to interact with an oocyte. Likewise, structural changes in the membrane in the tail region may be responsible for hyperactivation. Figure 14-5 illustrates the prevailing hypothesis of capacitation. Certain egg-binding proteins located on the surface of the plasma membrane of sperm have been shown to interact with proteins located on the zona pellucida. Prior to capacitation, these surface proteins are covered by so-called decapacitation factors, or protein constituents of seminal plasma. Solutes in fluids produced by the female reproductive tract strip the de-capacitation factors away from the sperm membrane, allowing them to interact with zona pellucida proteins, which in turn induce the acrosome reaction. Another effect of the capacitation-inducing compounds may be to alter the lipid composition of the plasma membrane, thereby altering the disposition of proteins involved with capacitation.

The identity(ies) of the chemical(s) that induce capacitation remain(s) unknown. However, they are presumed to be constituents of fluids secreted by the cervix and/or oviduct. In species where sperm are deposited in the uterus, the oviduct is clearly the site of capacitation. Recall that sperm accumulate in the caudal isthmus before moving on. In species where insemination occurs in the vagina, capacitation may occur in the cervix as well as in the oviduct. Due to variations in sperm characteristics as well as migration rate through the female tract, spermatozoa are capacitated at different times. This helps maintain a steady supply of capacitated sperm over several hours. Oviductal fluids are effective in inducing capacitation of sperm in vitro, as are artificial media designed to mimic the fluid produced by the oviduct. However, no single constituent appears to be the capacitation factor. Interestingly, production of capacitation factors doesn't appear to be organ specific.

FERTILIZATION

The site of fertilization is the ampullary-isthmic junction of the oviduct. Only a minute fraction of the spermatozoa ejaculated into a female's reproductive tract reach the oviduct, and only a small percentage of these find their way to the site of fertilization. According to some estimates as few as 100 spermatozoa occupy the ampullary-isthmic junction at the time of fertilization. It is unclear whether the meeting of the oocyte with sperm is due to chance, or if this is due to the oocyte producing a chemical agent that attracts spermatozoa (chemotaxis).

Fertilization can be divided into five major steps: 1) passage of the sperm through the cumulus oophorus (in species where this structure is present at the time of fertilization), 2) binding between the spermatozoon and oocyte, 3) induction of the spermatozoon's acrosome reaction, 4) fusion of the spermatozoon and oocyte, and 5) activation of the oocyte. Each of these steps will be considered in the following three sections.

Interaction of Spermatozoon with the Cumulus Oophorus

In most mammals, the oocyte is ovulated with its accompanying corona radiata and cumulus oophorus. However, in some ungulates such as the sheep and cattle, these cells are shed at the time of or shortly after ovulation. In the former cases, successful fertilization requires sperm to penetrate the cumulus before interacting with the zona pellucida. Penetrating the cumulus requires capacitation as well as an intact acrosome. Details regarding how sperm pass through this layer of cells is unclear, but the plasma membrane of sperm contain surface enzymes such as hyaluronidase, which lyses hyaluronic acid, a major structural component of the matrix supporting the cumulus cells.

Binding Between Spermatozoon and Oocyte

The next step in fertilization is the binding of the spermatozoon to the zona pellucida of the oocyte. Cell-to-cell adhesions are not uncommon in nature. Some familiar examples include the binding of bacteria and viruses to host cells. The mechanisms that mediate these types of interactions also mediate the interaction between a sperm and the zona pellucida. In all cases, it seems that binding is the result of interactions between complementary proteins located on the membranes of the adhering cells. As described in the previous section, capacitation appears to involve the unmasking of an egg-binding protein located on the plasma membrane of the sperm. This protein is a ligand for a sperm receptor located on the surface of the zona pellucida. The zona pellucida is composed entirely of three glycoproteins: ZP1, ZP2, and ZP3. ZP2 and ZP3 are bound by covalent bonds to form long filaments (Figure 14-6). These filaments are cross-linked by ZP1 molecules. It appears that ZP3 is the ligand for the sperm's egg binding protein, which is analogous to a receptor. This conclusion is based on the fact that disruption of expression of the ZP3 gene causes infertility in female mice.

[FIGURE 14-6 OMITTED]

The identity of the egg-binding protein on the sperm membrane remains elusive. However, the existence of such a protein is supported by the observation that purified ZP3 binds only to the plasma membrane covering the sperm head.

The Acrosome Reaction

You may recall that the acrosome is analogous to a large secretory vesicle that covers the nucleus in the apical region of the sperm's head (Figure 14-7). The outer acrosomal membrane lies just beneath the plasma membrane, whereas the inner acrosomal membrane overlies the nucleus. The acrosome reaction refers to structural changes in this arrangement that occur in response to the sperm binding the zona pellucida. During the reaction, the outer acrosomal membrane fuses with the plasma membrane in multiple locations along the head of the sperm, leading to the formation of numerous vesicles separated by channels through which acrosomal contents can escape. The vesicles are eventually dispersed, leaving only the inner acrosomal membrane overlying the nucleus. The contents of the acrosome include proteolytic enzymes such acrosin. These acrosomal lysins dissolve the zona pellucida creating a small hole through which the sperm can pass. At this point the hyperactivity of the sperm cell becomes important. The thrust created by the vigorous beating of the tail propels the sperm through the breach in the zona pellucida, leaving a small slit. The time required for a sperm to penetrate the zona averages between 7 and 30 minutes, depending on the species. Once the sperm enters, the membrane is repaired. In addition, once a sperm binds to the zona pellucida, ZP3 molecules are altered in a way that renders them unable to interact with egg-binding proteins.

[FIGURE 14-7 OMITTED]

At this point you might be wondering how binding of the sperm to the zona pellucida induces the morphologic changes associated with the acrosomal reaction. The best way to understand this process is to view the interaction between the egg-binding protein and ZP3 as analogous to the interaction between a receptor and its hormone. In other words, ZP3 binds to its receptor (egg binding protein) on the sperm membrane and induces intracellular responses that trigger vesicularization of the acrosome (Figure 14-8). Although the ZP3 receptor has not been identified, we know that its activation leads to an increase in intracellular calcium, which acts as a second messenger to trigger the acrosome reaction. In fact, an increase in calcium appears to be both a necessary and sufficient condition for this process.

[FIGURE 14-8 OMITTED]

Fusion of Sperm and Oocyte

Once the spermatozoon passes through the zona pellucida, it becomes lodged in the perivitelline space, makes contact with the membrane of the oocyte (vitelline membrane), and is eventually engulfed by the oocyte (Figure 14-9). The point of contact on the sperm is in the region of the equatorial segment. The inner acrosomal membrane and the plasma membrane of the sperm adhere to some of the numerous microvilli of the vitelline membrane. This is possible because the acrosome reaction renders the sperm capable of binding to the vitelline membrane. The exact mechanism is unknown, but some scientists hypothesize that dissolution of the acrosome induces changes in the remaining membrane that exposes proteins that allow it to dock with the membrane of the oocyte.

[FIGURE 14-9 OMITTED]

[FIGURE 14-10 OMITTED]

One of the hallmarks of fusion between sperm and egg is the cessation of movement by the sperm tail. Eventually the entire sperm, including the tail, is engulfed by the oocyte (Figure 14-10). The tail, including its mitochondria, degenerates quickly. While this is occurring the membranes in the head of the sperm degenerate and the nuclear contents decondense (disperse).

Activation of the Oocyte

As the sperm fuses with the oocyte, it induces a series of events known as activation. These include 1) the cortical reaction, 2) completion of meiosis 2 with extrusion of the second polar body, and 3) formation of pronucleus and syngamy. The timing of these events in the hamster is illustrated in Figure 14-11.

Soon after a sperm cell makes contact with the oocyte membrane, the socalled cortical granules develop along the outer edge (cortex) of the oocyte. These are actually secretory vesicles which release their contents via exocytosis soon after fusion begins (Figure 14-9). Exocytosis is preceded by an increase in intracellular [Ca.sup.2+], which occurs in response to the sperm binding to the oocyte. The compounds released by the cortical granules act on the zona pellucida and the vitelline membrane altering their structure in ways that prevent them from binding other sperm. These changes are known as the zona reaction and the vitelline block, respectively, and serve the function of preventing multiple fertilizations (i.e., polyspermy). The precise molecular mechanisms responsible for these responses remain unclear, but they appear to involve some sort of masking or chemical alteration of the proteins that interact with sperm proteins. For example, in the acrosome reaction, ZP3 loses its ability to bind to sperm cells.

[FIGURE 14-11 OMITTED]

Once the nucleus of the sperm cell enters the oocyte, it decondenses. In other words, its nuclear membrane disperses and the dense chromatin swells. As sperm cells mature in the epididymis, disulfide cross-links form among various nuclear proteins causing the nuclear material to become insoluble and condense. Once inside the oocyte, these disulfide bonds are chemically reduced, thus dispersing the proteins and liberating the chromosomes. As this occurs a new nuclear envelope organizes to form the male pronucleus. Little is known about the formation of the female pronucleus.

Once the two pronuclei have formed, they merge to form a single nucleus. This process is known as syngamy. Syngamy begins with the apposition of the two pronuclei. This is followed by the disintegration of the apposed membranes, reorganization of chromosomes, and formation of a single nuclear envelope encompassing the male and female chromosomes.

TIMING OF COPULATION, OVULATION, AND FERTILITY

We have encountered two major strategies whereby ovulation occurs harmoniously with delivery of spermatozoa. In the first case, sexual receptivity of females (estrus) coincides with ovulation (most mammals). In the second case, females are receptive continuously (e.g., most primates). However even with these adaptations there is a large chance that copulation will not result in fertile mating. Some of the variation in fertility can be attributed to the timing of copulation relative to ovulation. The key to successful fertilization is to have an oocyte arrive at the site of fertilization at a time that overlaps with the presence of viable spermatozoa. Sperm that arrive too early are likely to die before the oocyte arrives. On the other hand, an oocyte can lose its viability before the arrival of viable sperm cells. In general, sperm will live in the female tract for only a few days. The life span of oocytes is usually less than 24 hours. Therefore conception is possible only for a short period of time preceding and including the day of ovulation. Figure 14-12 illustrates this concept for humans. You may be surprised by the fact that the chance of pregnancy during this fertile period averages 20 percent. Because this period represents only 21 percent of the menstrual cycle, the average probability of conception for a particular copulation is only about 4 to 5 percent. This might seem very low in light of the exponential growth in global human population discussed in Chapter 1. However, when one considers the fact that the global population of humans includes billions of women of reproductive age, it becomes clear how such a low fertilization rate can result in tremendous increases in population. Consider the following example. There are approximately 80 million females of reproductive age (15 to 55 years of age) living in the United States. Assuming that this pool remains stable and that women engage in regular sexual activity without using birth control, a 5 percent conception rate would mean that 4 million new pregnancies are expected each year. This number is extremely close to the annual birth rate in the United States, which is surprising because an estimated 80 percent of women use some form of birth control in this country. It may be that the actual chance of pregnancy in the absence of birth control is higher than 5 percent because sexual intercourse is not evenly distributed across all days of the menstrual cycle. According to one recent study, women show the highest degree of sexual activity during the middle of the ovarian cycle, a time coinciding with the aforementioned window of fertility. Such a phenomenon might also account for the higher birth rates reported in many developing countries where contraception is not widely available to women.

[FIGURE 14-12 OMITTED]
BOX 14-1 Focus on Fertility: Postcopulatory Sexual Selection

One of the foundational principles of evolution theory
is sexual selection; that is, the idea that natural
selection promotes an increase in frequency of
genes that confer reproductive advantage. Darwin
understood this process to be confined to the precopulatory
period; that is, selection of a mate with
favorable features. This view is based on the
assumption that females are predominantly monogamous.
During the 1980s the notion of female monogamy
was challenged, and now there is wide
consensus that females of most animal species copulate
with multiple partners (polyandry). The idea
of female promiscuity raises the possibility that sexual
selection can occur following copulation. In
other words, it seems plausible that there is
"competition" among sperm from different males.
Evolutionary biologists have identified two types of
postcopulatory selection: 1) competition among
sperm of different males to fertilize the oocyte, and
2) the ability of the female to favor the sperm of
some males over others (cryptic female choice). The
former idea includes the notion that sperm from one
male can disrupt the fertilizing capacity of sperm
from another male ("kamikaze sperm"). The latter
idea appears to be the most plausible in mammals.

Evidence of postcopulatory selection can be
demonstrated by so-called heterospermic insemination
experiments. In these studies equal numbers of
sperm from two or more males are mixed and inseminated
in equal proportions. This approach eliminates
sire-related variations in fertilization rate that
can be attributed to differences in sperm number
and insemination time relative to ovulation. Results
of heterospermic insemination studies demonstrate
that the sperm of some sires display a fertilization
advantage over other sires. Recent research has
been devoted to understanding the physiologic basis
for such an advantage. The most widely accepted
idea is that the female is somehow selecting "desirable"
sperm based on certain phenotypic traits that
are the products of "fertilizing efficiency genes."
Traits that appear to be most important are motility
and the ability to undergo capacitation
(Figure 14-13). The idea of the female "selecting"
sperm requires elaboration. As noted earlier in this
chapter, the female reproductive tact can be portrayed
as hostile to sperm, meaning that it provides
various barriers to sperm. Sperm that overcome
these barriers are more likely to fertilize oocytes and
therefore pass on genes responsible for this fertilization
fitness. In addition to favoring genes that regulate
sperm physiology, the selection pressure
imposed by the female reproductive tract might also
favor anatomic, physiologic, and behavioral traits
that impact copulation. For example, large testicles
can be understood to be adaptations that ensure delivery
of large numbers of sperm cells. One of the
more unusual hypotheses is that the shape of the
penis of some animals is the result of selection
pressure that favors efficient delivery of sperm to
the female reproductive tract as well as to remove
seminal fluid from "competitors." Finally the deep
pelvic thrusting of males during copulation has been
portrayed as a behavioral adaptation that serves to
remove "rival" sperm. Whether or not such ideas
have scientific merit remains to be determined.

[FIGURE 14-13 OMITTED]

The theory of postcopulatory selection has been
used to explain why there is such tremendous variation
in the size, shape, and activity of sperm cells
within the animal kingdom. One of the more practical
implications of postcopulatory selection theory is the
possibility of developing new methods for evaluating
the fertility of males for livestock breeding programs.
If such tests were developed, the fertility of a sire
would be understood in terms of the fertilizing
ability of his sperm relative to those of other sires.


SUMMARY OF MAIN CONCEPTS

* The maturation and transport of gametes to the ampullary isthmic junction of the oviduct are necessary conditions for fertilization in mammals.

* Less than 1 percent of the sperm cells deposited in the female reproductive tract appear at the site of fertilization. The majority of sperm cells are lost due to retrograde flow and death.

* Transport of sperm in the oviduct involves movement of oviductal fluids by ciliary movement, peristaltic contractions of the muscularis, and motility of the sperm cell.

* Sperm gain the ability to fertilize an oocyte via the process of capacitation, which unmasks oocyte binding proteins and induces hypermotility of sperm.

* Fertilization is a multi-step process that includes binding of the sperm to the zona pellucida, induction of the acrosome reaction and penetration of the zona pellucida by the sperm, fusion of the sperm with the oocyte, and activation of the oocyte.

DISCUSSION

1. When spermatozoa collected from an ejaculate are added to a Petri dish containing an oocyte suspended in a common cell culture medium there is no fertilization. What is a reasonable explanation for this?

2. Injecting rabbits with certain drugs that induce contraction of smooth muscle, increases the number of sperm cells that are recovered from the oviduct following insemination. Explain how these drugs bring about this effect. Would you expect such treatments to increase fertilization rate (number of embryos per rabbit doe)? Why or why not?

3. Suppose a flock of 100 ewes is inseminated artificially with a mixture of semen from two different rams. The semen samples have equal concentrations of semen from each ram. You expect that the number of lambs from each sire would be equal (about 50 percent). However, you discover that 70 percent of the lambs are the offspring from one ram and 30 percent are from the other ram. Based on your knowledge of sperm transport and fertilization, develop a hypothesis to explain this response.

4. Some researchers have successfully decapacitated sperm cells. What does this mean? How might you go about accomplishing this?

REFERENCES

Bedford, J.M. 1982. Fertilization. In: C.R. Austin and R.V. Short, Reproduction in Mammals, Book 1: Germ Cells and Fertilization, Second Edition. Cambridge: Cambridge University Press, pp. 128-163.

Birkhead, T.R. and T. Pizzari. 2002. Postcopulatory sexual selection. Nature Reviews: Genetics 3:262-273.

Harper, M.J.K. 1982. Sperm and egg transport. In: C.R. Austin and R.V. Short, Reproduction in Mammals, Book 1: Germ Cells and Fertilization, Second Edition. Cambridge: Cambridge University Press:102-127.

Harper, M.J.K. 1994. Gamete and Zygote Transport. In: E. Knobil and J.D. Neill, The Physiology of Reproduction Vol. 2., Second Edition. New York: Raven Press, pp. 123-188.

Holt, W.V. and K.J.W. Van Look. 2004. Concepts in sperm heterogeneity, sperm selection and sperm competition as biological foundations for laboratory tests of semen quality. Reproduction 127:527-535.

Luke, M.C. and D.S. Coffey. 1994. The Male Accessory Sex Tissues: Structure, Androgen Action, and Physiology. In: E. Knobil and J.D. Neill, The Physiology of Reproduction Vol. 2., Second Edition. New York: Raven Press, pp. 1435-1488.

Wilcox, A.J., D.D. Baird, D.B. Dunson, D.R. McConnaughey, J.S. Kesner, and C.R. Weinberg. 2004. On the frequency of intercourse around ovulation: evidence for biological influences. Human Reproduction 19:1539-1543.

Wasserman, P.M. 1999. Mammalian fertilization: molecular aspects of gamete adhesion, exocytosis, and fusion. Cell 96:175-183.

Dean, J. 1992. Biology of mammalian fertilization: role of the zona pellucida. The Journal of Clinical Investigation, Inc. 89:1055-1059.

Wilcox, A.J., C.R. Weinberg and D.D. Baird. 1995. Timing of sexual intercourse in relation to ovulation: effects on the probability of conception, survival of the pregnancy and sex of the baby. New England Journal of Medicine 333:1517-1521.

Yanagimachi, R. 1994. Mammalian Fertilization. In: E. Knobil and J.D. Neill, The Physiology of Reproduction Vol. 2. Second Edition. New York: Raven Press:189-318.

Keith K. Schillo, PhD

Department of Animal and Food Sciences

University of Kentucky

Lexington, Kentucky
TABLE 14-1 Number of sperm ejaculated, site of sperm deposition, and
number of sperm reaching the oviduct in several species of mammals (1)

Species    Number of    Site of      Number of
           Sperm in     Deposition   Sperm in
           Ejaculate    (Natural     Oviduct
           (millions)   Mating)

Rat        58           Uterus       500
Rabbit     280          Vagina       250-500
Cattle     3000         Vagina       <100
Sheep      1000         Vagina       600-700
Swine      8000         Uterus       1000
Human      280          Vagina       200

(1) From Austin and Short (1982)
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Author:Schillo, Keith K.
Publication:Reproductive Physiology of Mammals, From Farm to Field and Beyond
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Date:Jan 1, 2009
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