Medical Physiology, 3rd Edition

Transport of Gametes and Fertilization

Cilia and smooth muscle transport the egg and sperm within the female genital tract

Following ovulation, the fimbriae of the fallopian tube sweep over the ovarian surface and pick up the oocyte—surrounded by its complement of granulosa cells, the cumulus oophorus, and corona radiata (see p. 1122)—and deposit it in the fallopian tube. Shortly after ovulation, movements of the cilia and the smooth muscle of fallopian tube propel the oocyte-cumulus complex toward the uterus.

A man normally deposits 150 to 600 million sperm cells into the vagina at the time of ejaculation. Only 50 to 100 of these cells actually reach the ampullary portion of the fallopian tube, where fertilization normally occurs. However, the sperm get there very quickly, within ~5 minutes of ejaculation. The swimming motion of the sperm alone cannot account for such rapid transport. Forceful contractions of the uterus, cervix, and fallopian tubes propel the sperm into the upper reproductive tract during female orgasm. Prostaglandins in the seminal plasma may induce further contractile activity.

The “capacitation” of the spermatozoa that occurs in the female genital tract enhances the ability of the sperm cell to fertilize the ovum

Maturation of sperm continues while they are stored in the epididymis (see pp. 1102–1103). In most species, neither freshly ejaculated sperm cells nor sperm cells that are removed from the epididymis are capable of fertilizing the egg until these cells have undergone further maturation—capacitation—in the female reproductive tract or in the laboratory. Capacitation is a poorly understood physiological process by which spermatozoa acquire the ability to penetrate the zona pellucida of the ovum. The removal or modification of a protective protein coat from the sperm cell membrane appears to be an important molecular event in the process of capacitation. imageN56-0

N56-0

Role of a Bicarbonate-Activated Adenylyl Cyclase in Sperm Capacitation

Contributed by Emile Boulpaep, Walter Boron

It has long been recognized that the capacitation of mammalian sperm requires both image and adenylyl-cyclase activity, which led to the discovery of a image-stimulated soluble adenylyl cyclase (sAC). Recall that the adenylyl cyclase activated by heterotrimeric G proteins is a membrane-bound enzyme (p. 53). In contrast, sAC is present in the cytosol.

Reference

Wuttke MS, Buck J, Levin LR. Bicarbonate-regulated soluble adenylyl cyclase. J Pancreas. 2001;2:154–158.

Sperm cells do not need to pass through the cervix and uterus to achieve capacitation. Successful pregnancy can occur with gamete intrafallopian transfer (GIFT), in which spermatozoa and oocytes are placed directly into the ampulla of the fallopian tube, and also with direct ultrasound-guided intraperitoneal insemination, in which the sperm are deposited in the peritoneal cavity, near the fimbria. Thus, capacitation of sperm in the reproductive tract is not strictly organ specific. As evidenced by the success of in vitro fertilization (IVF) and embryo transfer (ET)—discussed in Box 56-1—capacitation is possible even if the sperm does not make contact with the female reproductive tract.

Box 56-1

In Vitro Fertilization and Embryo Transfer

IVF is a procedure in which an oocyte or oocytes are removed from a woman and then fertilized with sperm under laboratory conditions. Early development of the embryo also proceeds under laboratory conditions. Finally, the physician transfers one or more embryos to the uterine cavity, where the embryo will hopefully implant and develop.

Indications for IVF-ET

Indications for IVF-ET include disorders that impair the normal meeting of the sperm and the egg in the distal portion of the fallopian tube. In addition to ovulatory dysfunction, these disorders include tubal occlusion, tubal-peritoneal adhesions, endometriosis, and other disease processes of the female peritoneal cavity. In addition, IVF-ET is indicated in some cases of male-factor infertility (abnormalities in male reproductive function) or unexplained infertility.

Ovarian Stimulation

Because the success rates are <100% for each of the stages of IVF-ET, the physician needs several oocytes, all obtained in a single ovarian cycle. However, woman normally develops a single dominant follicle each cycle (see p. 1123). Thus, to obtain the multiple oocytes for IVF-ET, the physician must stimulate the development of multiple follicles in the woman by controlled ovarian hyperstimulation. Although this procedure qualitatively mimics the hormonal control of the normal cycle, the high dose of gonadotropins triggers the development of many follicles. The physician administers some combination of FSH and LH, or pure FSH, either intramuscularly or subcutaneously. Because exogenous gonadotropins stimulate the ovaries directly, GnRH analogs (see Box 55-2) are often used to downregulate the hypothalamic-pituitary axis during controlled ovarian stimulation. One usually administers these GnRH analogs before initiating gonadotropin therapy, primarily to prevent a premature LH surge and ovulation.

Cycle Monitoring

After administering the gonadotropins, the physician monitors the stimulated follicular growth in the ovaries with sonographic imaging. Size, number, and serial growth of ovarian follicles may be assessed daily or at other appropriate intervals. Serum estradiol levels provide an additional measure of follicular growth and function. When estradiol levels and follicular growth indicate—by established criteria—appropriate folliculogenesis, the physician simulates a natural LH surge by injecting hCG, which mimics the actions of LH (see p. 1111). However, in this case, the simulated LH surge completes the final maturation of multiple follicles and oocytes. Because ovulation usually occurs 36 to 39 hours following the beginning of the LH surge (see p. 1123), the physician plans oocyte retrieval in such a way that maximal follicular maturation can occur but the oocytes are still harvested prior to ovulation. Thus, retrievals are scheduled for 34 to 36 hours following the administration of hCG.

Oocyte Retrieval

The physician retrieves oocytes by aspirating them from individual follicles under sonographic guidance. With the patient under conscious or unconscious sedation, and after a local anesthetic is applied to the posterior vaginal wall, the physician inserts a probe equipped with a needle guide into the vagina. After inserting a 16- to 18-gauge needle through the vaginal wall, the physician aspirates the follicular fluid from each mature follicle and collects it in a test tube containing a small amount of culture medium. The eggs are identified in the follicular fluid, are separated from the fluid and other follicular cells, and are then washed and prepared for insemination. This procedure normally yields 8 to 15 oocytes.

Insemination

The sperm sample is subjected to numerous washes, followed by gradient centrifugation to separate the sperm cells from the other cells and from debris found in the ejaculate. Each egg is inseminated with 50,000 to 300,000 motile sperm cells in a drop of culture medium and is incubated overnight. Fertilization can usually be detected by the presence of two pronuclei in the egg cytoplasm after 16 to 20 hours. Fertilization rates generally range from 60% to 85%. Embryo development is allowed to continue in vitro for another 48 to 120 hours until embryos are transferred to the uterus.

Among couples whose male partner has very low numbers of motile sperm, high fertilization rates can be achieved using intracytoplasmic sperm injection (ICSI). Micromanipulation techniques are used to inject a sperm cell into the cytoplasm of each egg in vitro. Fertilization rates after ICSI are generally 60% to 70%, or approximately equivalent to conventional insemination in vitro.

Embryo Transfer

After culturing the cells for 48 to 120 hours, the physician transfers three or four embryos to the uterus at the four- to eight-cell stage (after 2 days) or fewer embryos at the blastocyst stage (after 5 days). Embryos are selected and are loaded into a thin, flexible catheter, which is inserted into the uterine cavity to the desired depth under ultrasonic guidance. The woman usually receives supplemental progesterone to support implantation and pregnancy. In certain cases, the embryos are transferred to the fallopian tube during laparoscopy. This procedure is referred to as tubal embryo transfer (TET). The rationale for this procedure is that the fallopian tube contributes to the early development of the embryo as it travels down the tube to the uterus.

Success Rates

Implantation rates usually range from 8% to 15% per embryo transferred. In the United States, the mean live birth rate per ET procedure is ~33%. Success rates in IVF-ET depend on numerous factors, including age as well as the type and severity of the disease causing infertility.

Fertilization begins as the sperm cell attaches to the zona pellucida and undergoes the acrosomal reaction, and it ends with the fusion of the male and female pronuclei

After ovulation, the egg in the fallopian tube is in a semidormant state. If it remains unfertilized, the ripe egg will remain quiescent for some time and eventually degenerate. When fertilization occurs, the sperm normally comes into contact with the oocyte in the ampullary portion of the tube, usually several hours after ovulation. Fertilization causes the egg to awaken (activation) and initiates a series of morphological and biochemical events that lead to cell division and differentiation. Fertilization occurs in eight steps:

Step 1: The sperm head weaves its way past the follicular cells and attaches to the zona pellucida that surrounds the oocyte (Fig. 56-1). The zona pellucida is composed of three glycoproteins; ZP1 cross-links the filamentous ZP2 and ZP3 into a latticework. Receptors on the plasma membrane of the sperm cell bind to ZP3, thereby initiating a signal-transduction cascade.

image

FIGURE 56-1 Fertilization. The illustration summarizes the eight steps of fertilization.

Step 2: As a result of the sperm-ZP3 interaction, the sperm cell undergoes the acrosomal reaction, a prelude to the migration of the sperm cell through the mucus-like zona pellucida. The acrosome (see p. 1103) is a unique sperm organelle containing hydrolyzing enzymes that are necessary for the sperm to penetrate the zona pellucida. During the acrosomal reaction, an increase in intracellular free Ca2+ concentration ([Ca2+]i) triggers fusion of the outer acrosomal membrane with the sperm cell's plasma membrane, resulting in the exocytosis of most of the acrosomal contents.

Step 3: The spermatozoon penetrates through the zona pellucida. One mechanism of this penetration is the action of the acrosomal enzymes. imageN56-2 Protease inhibitors can block the penetration of spermatozoa through the zona pellucida. The sperm cell also penetrates the zona pellucida by mechanical action. The sperm head rapidly oscillates about a fulcrum that is situated in the neck region. This rapid, vigorous, rocking action occurs at a frequency of ~6 to 8 per second. The sperm penetrates the zona pellucida at an angle, which creates a tangential cleavage slit and leaves the sperm head lying sideways against the oocyte membrane.

N56-2

Acrosomal Enzymes

Contributed by Ervin Jones

Among the many enzymes in the acrosome are acid hydrolases, the best characterized of which is proacrosin, the precursor to acrosin. Acrosin is a member of the serine protease superfamily; it is expressed only in spermatogenic cells. Two other enzymes released from the acrosome are neuraminidase and a special form of hyaluronidase. This particular hyaluronidase can be distinguished from the common lysosomal form of the enzyme, and it appears to be a spermatogenic cell–specific isozyme. Acrosin, hyaluronidase, and neuraminidase help the sperm penetrate the zona pellucida by hydrolyzing the sugar chains and the peptide chains of the glycoproteins of the zona pellucida.

Step 4: The cell membranes of the sperm and the oocyte fuse. Microvilli on the oocyte surface envelop the sperm cell, which probably binds to the oocyte membrane via specific proteins on the surfaces of the two cells. The posterior membrane of the acrosome—which remains part of the sperm cell after the acrosomal reaction—is the first portion of the sperm to fuse with the plasma membrane of the egg. The sperm cell per se does not enter the oocyte. Rather, the cytoplasmic portions of the head and tail enter the oocyte, leaving the sperm-cell plasma membrane behind, an action similar to a snake's crawling out of its skin.

Step 5: The oocyte undergoes the cortical reaction. As the spermatozoon penetrates the oocyte's plasma membrane, it initiates formation of inositol 1,4,5-trisphosphate (IP3); IP3 causes Ca2+ release from internal stores (see p. 60), which leads to an increase in [Ca2+]i and [Ca2+]i waves. This rise in [Ca2+]i, in turn, triggers the oocyte's second meiotic division—discussed below (see step 6)—and the cortical reaction. In the cortical reaction, small electron-dense granules that lie just beneath the plasma membrane fuse with the oocyte's plasma membrane. Exocytosis of these granules releases enzymes that act on glycoproteins in the zona pellucida, causing the zona pellucida to harden. This hardening involves the release of polysaccharides that impede the progression of the runners-up (i.e., sperm cells still in the zona pellucida). The cortical reaction also leads to the destruction of ZP receptors, which prevents further binding of sperm cells to the zona pellucida. From a teleological perspective, the cortical granule reaction prevents polyspermy.imageN56-3 Polyspermic embryos are abnormal because they are polyploid. They do not develop beyond the early cleavage stages.

N56-3

Block to Polyspermy

Contributed by Ervin Jones

Unlike the situation in some laboratory animals, in humans the block to polyspermy during fertilization does not involve receptors on the zona pellucida or on the cell membrane. It appears that the block to polyspermy in humans is due only to alterations of the inner aspect of the zona pellucida. In experiments on monospermic oocytes fertilized in vitro, other sperm cells added later can partially penetrate the zona pellucida but do not reach the inner half of the zona. When unfertilized oocytes, monospermic oocytes, and polyspermic oocytes are examined in vitro, similar numbers of sperm are found on and within the zona pellucida of each type of oocyte. Further evidence that the zona pellucida is the primary barrier to polyspermy in humans comes from experiments in which the zona pellucida is removed. In this case, the egg is usually penetrated by many sperm. In the in vitro fertilization of normal human oocytes, the rate of polyspermy is quite low, ~5% to 8%, even though 50,000 to 300,000 sperm are available to the oocyte. Thus, the zona pellucida block to polyspermy in humans is highly efficient.

Step 6: The oocyte completes its second meiotic division. The oocyte, which had been arrested in the prophase of its first meiotic division since fetal life (see p. 1073), completed its first meiotic division at the time of the surge of luteinizing hormone (LH), which occurred several hours before ovulation (see p. 1116). The result was the first polar body and a secondary oocyte with a haploid number of duplicated chromosomes (see Fig. 53-2C). Before fertilization, this secondary oocyte had begun a second meiotic division, which was arrested in metaphase. The rise in [Ca2+]i inside the oocyte—which the sperm cell triggers, as noted in step 5—causes not only the cortical reaction, but also the completion of the oocyte's second meiotic division. One result is the formation of the second polar body, which contains a haploid number of unduplicated maternal chromosomes. imageN56-4 The oocyte extrudes the chromosomes of the second polar body, together with a small amount of ooplasm, into a space immediately below the zona pellucida; the second polar body usually lies close to the first polar body. The nucleus of the oocyte also contains a haploid number of unduplicated chromosomes. As its chromosomes decondense, the nucleus of this mature ovum becomes the female pronucleus.

N56-4

Three Polar Bodies

Contributed by Sam Mesiano

Interestingly, although the small polar bodies are nonfunctional, they contain a full set of chromosomes and are responsive to cell cycle–regulatory mechanisms. Consequently, the first polar body also divides during the second meiotic division to produce a total of three polar bodies and a mature oocyte, each of which contain a haploid number of chromosomes. Thus, as with spermatogenesis, one diploid oogonia produces four haploid daughters; however, in oogenesis only one daughter becomes an functional gamete.

Step 7: The sperm nucleus decondenses and transforms into the male pronucleus, which, like the female pronucleus, contains a haploid number of unduplicated chromosomes (see Fig. 54-7). The cytoplasmic portion of the sperm's tail degenerates.

Step 8: The male and female pronuclei fuse, forming a new cell, the zygote. The mingling of chromosomes (syngamy) can be considered as the end of fertilization and the beginning of embryonic development. Thus, fertilization results in a conceptus that bears 46 chromosomes, 23 from the maternal gamete and 23 from the paternal gamete. Fertilization of the ovum by a sperm bearing an X chromosome produces a zygote with XX sex chromosomes; this develops into a female (see pp. 1073–1075). Fertilization with a Y-bearing sperm produces an XY zygote, which develops into a male. Therefore, chromosomal sex is established at fertilization.

LESION

REFLEXOGENIC ERECTION

PSYCHOGENIC ERECTION

EFFECT ON EJACULATION

Upper motor neuron

Present

Absent

Significantly impaired

Lower motor neuron

Absent

Present

Less impaired