Phase 1: Origin and Migration of Germ Cells

Primordial germ cells, the earliest recognizable precursors of gametes, arise outside the gonads and migrate into the gonads during early embryonic development. Human primordial germ cells first become readily recognizable at 24 days after fertilization in the endodermal layer of the yolk sac (Fig. 1.1A) by their large size and high content of the enzyme alkaline phosphatase. In the mouse, their origin has been traced even earlier in development (see p. 390). Germ cells exit from the yolk sac into the hindgut epithelium and then migrate* through the dorsal mesentery until they reach the primordia of the gonads (Fig. 1.1B). In the mouse, an estimated 100 cells leave the yolk sac, and through mitotic multiplication (6 to 7 rounds of cell division), about 4000 primordial germ cells enter the primitive gonads.

Misdirected primordial germ cells that lodge in extragonadal sites usually die, but if such cells survive, they may develop into teratomas. Teratomas are bizarre growths that contain scrambled mixtures of highly differentiated tissues, such as skin, hair, cartilage, and even teeth (Fig. 1.2). They are found in the mediastinum, the sacrococcygeal region, and the oral region.

Phase 2: Increase in the Number of Germ Cells by Mitosis

After they arrive in the gonads, the primordial germ cells begin a phase of rapid mitotic proliferation. In a mitotic division, each germ cell produces two diploid progeny that are genetically equal. Through several series of mitotic divisions, the number of primordial germ cells increases exponentially from hundreds to millions. The pattern of mitotic proliferation differs markedly between male and female germ cells. Oogonia, as mitotically active germ cells in the female are called, go through a period of intense mitotic activity in the embryonic ovary from the second through the fifth month of pregnancy in the human. During this period, the population of germ cells increases from only a few thousand to nearly 7 million (Fig. 1.3). This number represents the maximum number of germ cells that is ever found in the ovaries. Shortly thereafter, numerous oogonia undergo a natural degeneration called atresia. Atresia of germ cells is a continuing feature of the histological landscape of the human ovary until menopause.

Spermatogonia, which are the male counterparts of oogonia, follow a pattern of mitotic proliferation that differs greatly from that in the female. Mitosis also begins early in the embryonic testes, but in contrast to female germ cells, male germ cells maintain the ability to divide throughout postnatal life. The seminiferous tubules of the testes are lined with a germinative population of spermatogonia. Beginning at puberty, subpopulations of spermatogonia undergo periodic waves of mitosis. The progeny of these divisions enter meiosis as synchronous groups. This pattern of spermatogonial mitosis continues throughout life.

The biological significance of meiosis in humans is similar to that in other species. Of primary importance are (1) reduction of the number of chromosomes from the diploid (2n) to the haploid (1n) number so that the species number of chromosomes can be maintained from generation to generation, (2) independent reassortment of maternal and paternal chromosomes for better mixing of genetic characteristics, and (3) further redistribution of maternal and paternal genetic information through the process of crossing-over during the first meiotic division.

Meiosis involves two sets of divisions (Fig. 1.4). Before the first meiotic division, deoxyribonucleic acid (DNA) replication has already occurred, so at the beginning of meiosis, the cell is 2n, 4c. (In this designation, n is the species number of chromosomes, and c is the amount of DNA in a single set [n] of chromosomes.) The cell contains the normal number (2n) of chromosomes, but as a result of replication, its DNA content (4c) is double the normal amount (2c).

In the first meiotic division, often called the reductional division, a prolonged prophase (see Fig. 1.4) leads to the pairing of homologous chromosomes and frequent crossing-over, resulting in the exchange of segments between members of the paired chromosomes. Crossing-over even occurs in the sex chromosomes. This takes place in a small region of homology between the X and Y chromosomes. Crossing-over is not a purely random process. Rather, it occurs at sites along the chromosomes known as hot spots. Their location is based on configurations of proteins that organize the chromosomes early in meiosis. One such protein is cohesin, which helps to hold sister chromatids together during division. Hypermethylation of histone proteins in the chromatin indicates specific sites where the DNA strands break and are later repaired after crossing-over is completed. Another protein, condensin, is important in compaction of the chromosomes, which is necessary for both mitotic and meiotic divisions to occur.

During metaphase of the first meiotic division, the chromosome pairs (tetrads) line up at the metaphase (equatorial) plate so that at anaphase I, one chromosome of a homologous pair moves toward one pole of the spindle, and the other chromosome moves toward the opposite pole. This represents one of the principal differences between a meiotic and a mitotic division. In a mitotic anaphase, the centromere between the sister chromatids of each chromosome splits after the chromosomes have lined up at the metaphase plate, and one chromatid from each chromosome migrates to each pole of the mitotic spindle. This activity results in genetically equal daughter cells after a mitotic division, whereas the daughter cells are genetically unequal after the first meiotic division. Each daughter cell of the first meiotic division contains the haploid (1n) number of chromosomes, but each chromosome still consists of two chromatids (2c) connected by a centromere. No new duplication of chromosomal DNA is required between the first and second meiotic divisions because each haploid daughter cell resulting from the first meiotic division already contains chromosomes in the replicated state.

The second meiotic division, called the equational division, is similar to an ordinary mitotic division except that before division the cell is haploid (1n, 2c). When the chromosomes line up along the equatorial plate at metaphase II, the centromeres between sister chromatids divide, allowing the sister chromatids of each chromosome to migrate to opposite poles of the spindle apparatus during anaphase II. Each daughter cell of the second meiotic division is truly haploid (1n, 1c).

Meiosis in Females

The period of meiosis involves other cellular activities in addition to the redistribution of chromosomal material. As the oogonia enter the first meiotic division late in the fetal period, they are called primary oocytes.

Meiosis in the human female is a very leisurely process. As the primary oocytes enter the diplotene stage of the first meiotic division in the early months after birth, the first of two blocks in the meiotic process occurs (Fig. 1.5). The suspended diplotene phase of meiosis is the period when the primary oocyte prepares for the needs of the embryo. In oocytes of amphibians and other lower vertebrates, which must develop outside the mother’s body and often in a hostile environment, it is highly advantageous for the early stages of development to occur very rapidly so that the stage of independent locomotion and feeding is attained as soon as possible. These conditions necessitate a strategy of storing up the materials needed for early development well in advance of ovulation and fertilization because normal synthetic processes would not be rapid enough to produce the materials required for the rapidly cleaving embryo. In such species, yolk is accumulated, the genes for producing ribosomal ribonucleic acid (rRNA) are amplified, and many types of RNA molecules are synthesized and stored in an inactive form for later use.

RNA synthesis in the amphibian oocyte occurs on the lampbrush chromosomes, which are characterized by many prominent loops of spread-out DNA on which messenger RNA (mRNA) molecules are synthesized. The amplified genes for producing rRNA are manifested by the presence of 600 to 1000 nucleoli within the nucleus. Primary oocytes also prepare for fertilization by producing several thousand cortical granules, which are of great importance during the fertilization process (see Chapter 2).

The mammalian oocyte prepares for an early embryonic period that is more prolonged than that of amphibians and that occurs in the nutritive environment of the maternal reproductive tract. Therefore, it is not faced with the need to store as great a quantity of materials as are the eggs of lower vertebrates. As a consequence, the buildup of yolk is negligible. Evidence indicates, however, a low level of ribosomal DNA (rDNA) amplification (two to three times) in diplotene human oocytes, a finding suggesting that some degree of molecular advance planning is also required to support early cleavage in the human. The presence of 2 to 40 small (2-µm) RNA-containing micronuclei (miniature nucleoli) per oocyte nucleus correlates with the molecular data.

Human diplotene chromosomes do not appear to be arranged in a true lampbrush configuration, and massive amounts of RNA synthesis seem unlikely. The developing mammalian (mouse) oocyte produces 10,000 times less rRNA and 1000 times less mRNA than its amphibian counterpart. Nevertheless, there is a steady accumulation of mRNA and a proportional accumulation of rRNA. These amounts of maternally derived RNA seem to be enough to take the fertilized egg through the first couple of cleavage divisions, after which the embryonic genome takes control of macromolecular synthetic processes.

Because cortical granules play an important role in preventing the entry of excess spermatozoa during fertilization in human eggs (see p. 31), the formation of cortical granules (mainly from the Golgi apparatus) continues to be one of the functions of the diplotene stage that is preserved in humans. Roughly 4500 cortical granules are produced in the mouse oocyte. A higher number is likely in the human oocyte.

Unless they degenerate, all primary oocytes remain arrested in the diplotene stage of meiosis until puberty. During the reproductive years, small numbers (10 to 30) of primary oocytes complete the first meiotic division with each menstrual cycle and begin to develop further. The other primary oocytes remain arrested in the diplotene stage, some for 50 years.

With the completion of the first meiotic division shortly before ovulation, two unequal cellular progeny result. One is a large cell, called the secondary oocyte. The other is a small cell called the first polar body (see Fig. 1.5). The secondary oocytes begin the second meiotic division, but again the meiotic process is arrested, this time at metaphase. The stimulus for the release from this meiotic block is fertilization by a spermatozoon. Unfertilized secondary oocytes fail to complete the second meiotic division. The second meiotic division is also unequal; one of the daughter cells is relegated to becoming a second polar body. The first polar body may also divide during the second meiotic division. Formation of both the first and second polar bodies involves highly asymmetric cell divisions. To a large extent, this is accomplished by displacement of the mitotic spindle apparatus toward the periphery of the oocyte through the actions of the cytoskeletal protein actin (see Fig. 2.7).

Meiosis in Males

Meiosis in the male does not begin until after puberty. In contrast to the primary oocytes in the female, not all spermatogonia enter meiosis at the same time. Large numbers of spermatogonia remain in the mitotic cycle throughout much of the reproductive lifetime of males. When the progeny of a spermatogonium have entered the meiotic cycle as primary spermatocytes, they spend several weeks passing through the first meiotic division (Fig. 1.6). The result of the first meiotic division is the formation of two secondary spermatocytes, which immediately enter the second meiotic division. About 8 hours later, the second meiotic division is completed, and four haploid (1n, 1c) spermatids remain as progeny of the single primary spermatocyte. The total length of human spermatogenesis is 64 days.

Disturbances that can occur during meiosis and result in chromosomal aberrations are discussed in Clinical Correlation 1.1 and Figure 1.7.

Clinical Correlation 1.1   Meiotic Disturbances Resulting in Chromosomal Aberrations

Chromosomes sometimes fail to separate during meiosis, a phenomenon known as nondisjunction. As a result, one haploid daughter gamete contains both members of a chromosomal pair for a total of 24 chromosomes, whereas the other haploid gamete contains only 22 chromosomes (Fig. 1.7). When such gametes combine with normal gametes of the opposite sex (with 23 chromosomes), the resulting embryos contain 47 chromosomes (with a trisomy of 1 chromosome) or 45 chromosomes (monosomy of 1 chromosome). (Specific syndromes associated with the nondisjunction of chromosomes are summarized in Chapter 8.) The generic term given to a condition characterized by an abnormal number of chromosomes is aneuploidy.

In other cases, part of a chromosome can be translocated to another chromosome during meiosis, or part of a chromosome can be deleted. Similarly, duplications or inversions of parts of chromosomes occasionally occur during meiosis. These conditions may result in syndromes similar to those seen after the nondisjunction of entire chromosomes. Under some circumstances (e.g., simultaneous fertilization by two spermatozoa, failure of the second polar body to separate from the oocyte during the second meiotic division), the cells of the embryo contain more than two multiples of the haploid number of chromosomes (polyploidy).

Chromosomal abnormalities are the underlying cause of a high percentage of spontaneous abortions during the early weeks of pregnancy. More than 75% of spontaneous abortions occurring before the second week and more than 60% of those occurring during the first half of pregnancy contain chromosomal abnormalities ranging from trisomies of individual chromosomes to overall polyploidy. Although the incidence of chromosomal anomalies declines with stillbirths occurring after the fifth month of pregnancy, it is close to 6%, a 10-fold higher incidence over the 0.5% of living infants who are born with chromosomal anomalies. In counseling patients who have had a stillbirth or a spontaneous abortion, it can be useful to mention that this is often nature’s way of handling an embryo destined to be highly abnormal.

Of the roughly 2 million primary oocytes present in the ovaries at birth, only about 40,000—all of which are arrested in the diplotene stage of the first meiotic division—survive until puberty. From this number, approximately 400 (1 per menstrual cycle) are actually ovulated. The rest of the primary oocytes degenerate without leaving the ovary, but many of them undergo some further development before becoming atretic. Although some studies suggested that adult mammalian ovaries contain primitive cells that can give rise to new oocytes, such reports remain controversial.

The egg, along with its surrounding cells, is called a follicle. Maturation of the egg is intimately bound with the development of its cellular covering. Because of this, considering the development of the egg and its surrounding follicular cells as an integrated unit is a useful approach in the study of oogenesis.

In the embryo, oogonia are naked, but after meiosis begins, cells from the ovary partially surround the primary oocytes to form primordial follicles (see Fig. 1.5). By birth, the primary oocytes are invested with a complete layer of follicular cells, and the complex of primary oocyte and the follicular (granulosa) cells is called a primary follicle (Fig. 1.8). Both the oocyte and the surrounding follicular cells develop prominent microvilli and gap junctions that connect the two cell types.

The meiotic arrest at the diplotene stage of the first meiotic division is the result of a complex set of interactions between the oocyte and its surrounding layer of follicular (granulosa) cells. The principal factor in maintaining meiotic arrest is a high concentration of cyclic adenosine monophosphate (cAMP) in the cytoplasm of the oocyte (Fig. 1.9). This is accomplished by both the intrinsic production of cAMP by the oocyte and the production of cAMP by the follicular cells and its transport into the oocyte through gap junctions connecting the follicular cells to the oocyte. In addition, the follicular cells produce and transport into the oocyte cyclic guanosine monophosphate (cGMP), which inactivates phosphodiesterase 3A (PDE3A), an enzyme that converts cAMP to 5′AMP. The high cAMP within the oocyte inactivates maturation promoting factor (MPF), which at a later time functions to lead the oocyte out of meiotic arrest and to complete the first meiotic division.

As the primary follicle takes shape, a prominent, translucent, noncellular membrane called the zona pellucida forms between the primary oocyte and its enveloping follicular cells (Fig. 1.10). The microvillous connections between the oocyte and follicular cells are maintained through the zona pellucida. In rodents, the components of the zona pellucida (four glycoproteins and glycosaminoglycans) are synthesized almost entirely by the egg, but in other mammals, follicular cells also contribute materials to the zona. The zona pellucida contains sperm receptors and other components that are important in fertilization and early postfertilization development. (The functions of these molecules are discussed more fully in Chapter 2.)

In the prepubertal years, many of the primary follicles enlarge, mainly because of an increase in the size of the oocyte (up to 300-fold) and the number of follicular cells. An oocyte with more than one layer of surrounding granulosa cells is called a secondary follicle. A basement membrane called the membrana granulosa surrounds the epithelial granulosa cells of the secondary follicle. The membrana granulosa forms a barrier to capillaries, and as a result, the oocyte and the granulosa cells depend on the diffusion of oxygen and nutrients for their survival.

An additional set of cellular coverings, derived from the ovarian connective tissue (stroma), begins to form around the developing follicle after it has become two to three cell layers thick. Known initially as the theca folliculi, this covering ultimately differentiates into two layers: a highly vascularized and glandular theca interna and a more connective tissue–like outer capsule called the theca externa. The early thecal cells secrete an angiogenesis factor, which stimulates the growth of blood vessels in the thecal layer. This nutritive support facilitates growth of the follicle.

Early development of the follicle occurs without the significant influence of hormones, but as puberty approaches, continued follicular maturation requires the action of the pituitary gonadotropic hormone follicle-stimulating hormone (FSH) on the granulosa cells, which have by this time developed FSH receptors on their surfaces (see Fig. 1.10). In addition, the oocyte itself exerts a significant influence on follicular growth. After blood-borne FSH is bound to the FSH receptors, the stimulated granulosa cells produce small amounts of estrogens. The most obvious indication of the further development of some of the follicles is the formation of an antrum, a cavity filled with a fluid called liquor folliculi. Initially formed by secretions of the follicular cells, the antral fluid is later formed mostly as a transudate from the capillaries on the outer side of the membrana granulosa.

Formation of the antrum divides the follicular cells into two groups. The cells immediately surrounding the oocyte are called cumulus cells, and the cells between the antrum and the membrana granulosa become the mural granulosa cells. Factors secreted by the oocyte confer different properties on the cumulus cells from the mural granulosa cells. In the absence of a direct stimulus from the oocyte, the granulosa cells follow a default pathway and begin to assemble hormone receptors on their surface (see Fig. 1.10). In contrast, the cumulus cells do not express hormone receptors, but under the influence of the oocyte, they undergo changes that facilitate the release of the ovum at the time of ovulation.

Enlargement of the follicle results largely from the proliferation of granulosa cells. The direct stimulus for granulosa cell proliferation is a locally produced signaling protein, activin, a member of the transforming growth factor-β family of signaling molecules (see Table 4.1). The local action of activin is enhanced by the actions of FSH.

Responding to the stimulus of pituitary hormones, secondary follicles produce significant amounts of steroid hormones. The cells of the theca interna possess receptors for luteinizing hormone (LH), also secreted by the anterior pituitary (see Fig. 1.15). The theca interna cells produce androgens (e.g., testosterone), which pass through the membrana granulosa to the granulosa cells. The influence of FSH induces the granulosa cells to synthesize the enzyme (aromatase), which converts the theca-derived androgens into estrogens (mainly 17β-estradiol). Not only does the estradiol leave the follicle to exert important effects on other parts of the body, but also it stimulates the formation of LH receptors on the granulosa cells. Through this mechanism, the follicular cells are able to respond to the large LH surge that immediately precedes ovulation (see Fig. 1.16).

Under multiple hormonal influences, the follicle enlarges rapidly (Fig. 1.11; see Fig. 1.10) and presses against the surface of the ovary. At this point, it is called a tertiary (graafian) follicle. About 10 to 12 hours before ovulation, meiosis resumes.

The resumption of meiosis in response to the LH surge is initiated by the cumulus (granulosa) cells, because the oocyte itself does not possess LH receptors. Responding to LH, the cumulus cells shut down their gap junctions (see Fig. 1.9B). This reduces the transfer of both cAMP and cGMP from the cumulus cells into the oocyte. The resulting reduction of cGMP in the oocyte allows the activation of PDE3A. The activated PDE3A then breaks down the intra-oocytic cAMP into 5′AMP. The decline in the concentration of cAMP sets off a signaling pathway leading to the activation of MPF and the subsequent resumption of meiosis.

The egg, now a secondary oocyte, is located in a small mound of cells known as the cumulus oophorus, which lies on one side of the greatly enlarged antrum. In response to the preovulatory surge of gonadotropic hormones, factors secreted by the oocyte pass through gap junctions into the surrounding cumulus cells and stimulate the cumulus cells to secrete hyaluronic acid into the intercellular spaces. The hyaluronic acid binds water molecules and enlarges the intercellular spaces, thus expanding the cumulus oophorus. In keeping with the hormonally induced internal changes, the diameter of the follicle increases from about 6 mm early in the second week to almost 2 cm at ovulation.

The tertiary follicle protrudes from the surface of the ovary like a blister. The granulosa cells contain numerous FSH and LH receptors, and LH receptors are abundant in the cells of the theca interna. The follicular cells secrete large amounts of estradiol (see Fig. 1.16), which prepares many other components of the female reproductive tract for gamete transport. Within the antrum, the follicular fluid contains the following: (1) a complement of proteins similar to that seen in serum, but in a lower concentration; (2) 20 enzymes; (3) dissolved hormones, including FSH, LH, and steroids; and (4) proteoglycans. The strong negative charge of the proteoglycans attracts water molecules, and with greater amounts of secreted proteoglycans, the volume of antral fluid increases correspondingly. The follicle is now poised for ovulation and awaits the stimulus of the preovulatory surge of FSH and LH released by the anterior pituitary gland.

The reason only one follicle normally matures to the point of ovulation is still not completely understood. Early in the cycle, as many as 50 follicles begin to develop, but only about 3 attain a diameter of as great as 8 mm. Initial follicular growth is gonadotropin independent, but continued growth depends on a minimum “tonic” level of gonadotropins, principally FSH. During the phase of gonadotropin-induced growth, a dominant enlarging follicle becomes independent of FSH and secretes large amounts of inhibin (see p. 19). Inhibin suppresses the secretion of FSH by the pituitary, and when the FSH levels fall below the tonic threshold, the other developing follicles, which are still dependent on FSH for maintenance, become atretic. The dominant follicle acquires its status about 7 days before ovulation. It may also secrete an inhibiting substance that acts directly on the other growing follicles.

Spermatogenesis

Spermatogenesis begins in the seminiferous tubules of the testes after the onset of puberty. In the broadest sense, the process begins with mitotic proliferation of the spermatogonia. At the base of the seminiferous epithelium are several populations of spermatogonia. Type A spermatogonia represent the stem cell population that mitotically maintains proper numbers of spermatogonia throughout life. Type A spermatogonia give rise to type B spermatogonia, which are destined to leave the mitotic cycle and enter meiosis. Entry into meiosis is stimulated by retinoic acid (a derivative of vitamin A). Many spermatogonia and their cellular descendants are connected by intercellular cytoplasmic bridges, which may be instrumental in maintaining the synchronous development of large clusters of sperm cells.

All spermatogonia are sequestered at the base of the seminiferous epithelium by interlocking processes of Sertoli cells, which are complex cells that are regularly distributed throughout the periphery of the seminiferous epithelium and that occupy about 30% of its volume (see Fig. 1.6). As the progeny of the type B spermatogonia (called primary spermatocytes) complete the leptotene stage of the first meiotic division, they pass through the Sertoli cell barrier to the interior of the seminiferous tubule. This translocation is accomplished by the formation of a new layer of Sertoli cell processes beneath these cells and, slightly later, the dissolution of the original layer that was between them and the interior of the seminiferous tubule. The Sertoli cell processes are very tightly joined and form an immunological barrier (blood-testis barrier [see Fig. 1.6]) between the forming sperm cells and the rest of the body, including the spermatogonia. When they have begun meiosis, developing sperm cells are immunologically different from the rest of the body. Autoimmune infertility can arise if the blood-testis barrier is broken down.

The progeny of the type B spermatogonia, which have entered the first meiotic division, are the primary spermatocytes (see Fig. 1.6). Located in a characteristic position just inside the layer of spermatogonia and still deeply embedded in Sertoli cell cytoplasm, primary spermatocytes spend 24 days passing through the first meiotic division. During this time, the developing sperm cells use a strategy similar to that of the egg—producing in advance molecules that are needed at later periods when changes occur very rapidly. Such preparation involves the production of mRNA molecules and their storage in an inactive form until they are needed to produce the necessary proteins.

A well-known example of preparatory mRNA synthesis involves the formation of protamines, which are small, arginine-rich, and cysteine-rich proteins that displace the lysine-rich nuclear histones and allow the high degree of compaction of nuclear chromatin required during the final stages of sperm formation. Protamine mRNAs are first synthesized in primary spermatocytes but are not translated into proteins until the spermatid stage. In the meantime, the protamine mRNAs are complexed with proteins and are inaccessible to the translational machinery. If protamine mRNAs are translated before the spermatid stage, the chromosomes condense prematurely, and sterility results.

After completion of the first meiotic division, the primary spermatocyte gives rise to 2 secondary spermatocytes, which remain connected by a cytoplasmic bridge. The secondary spermatocytes enter the second meiotic division without delay. This phase of meiosis is very rapid, typically completed in approximately 8 hours. Each secondary spermatocyte produces 2 immature haploid gametes, the spermatids. The 4 spermatids produced from a primary spermatocyte progenitor are still connected to one another and typically to as many as 100 other spermatids as well. In mice, some genes are transcribed as late as the spermatid stage.

Spermatids do not divide further, but they undergo a series of profound changes that transform them from ordinary-looking cells to highly specialized spermatozoa (singular, spermatozoon). The process of transformation from spermatids to spermatozoa is called spermiogenesis or spermatid metamorphosis.

Several major categories of change occur during spermiogenesis (Fig. 1.12). One is the progressive reduction in the size of the nucleus and tremendous condensation of the chromosomal material, which is associated with the replacement of histones by protamines. Along with the changes in the nucleus, a profound reorganization of the cytoplasm occurs. Cytoplasm streams away from the nucleus, but a condensation of the Golgi apparatus at the apical end of the nucleus ultimately gives rise to the acrosome. The acrosome is an enzyme-filled structure that plays a crucial role in the fertilization process. At the other end of the nucleus, a prominent flagellum grows out of the centriolar region. Mitochondria are arranged in a spiral around the proximal part of the flagellum. During spermiogenesis, the plasma membrane of the head of the sperm is partitioned into several antigenically distinct molecular domains. These domains undergo numerous changes as the sperm cells mature in the male and at a later point when the spermatozoa are traveling through the female reproductive tract. As spermiogenesis continues, the remainder of the cytoplasm (residual body [see G in Fig. 1.12]) moves away from the nucleus and is shed along the developing tail of the sperm cell. The residual bodies are phagocytized by Sertoli cells (Box 1.1 and Fig. 1.13).

Box 1.1   Passage of Sperm Precursors through the Blood-Testis Barrier

During spermatogenesis, developing sperm cells are closely linked with Sertoli cells, and the topography of maturation occurs in regular but complex patterns. A striking example involves the coordinated detachment of mature spermatids from the apical surface of Sertoli cells and the remodeling of the inter-Sertoli cell tight-junction complex that constitutes the blood-testis barrier (see Fig. 1.13). Type B spermatogonia, which are just entering the preleptotene stage of the first meiotic division and becoming primary spermatocytes, are located outside (basal to) the blood-testis barrier. Late-stage spermatids are attached to the apical surface of Sertoli cells by aggregates of tight-junction proteins, called surface adhesion complexes.

At a specific stage in spermatid development, the surface adhesion complexes break down, and the mature spermatids are released into the lumen of the seminiferous tubule. Biologically active laminin fragments, originating from the degenerating surface adhesion complexes, make their way to the tight-junction complex that constitutes the blood-testis barrier. These fragments, along with certain cytokines and proteinases, degrade the tight-junctional proteins of the blood-testis barrier, and the blood-testis barrier, located apically to the preleptotene primary spermatocyte, breaks down. Then testosterone, which is 50 to 100 times more concentrated in the seminiferous tubule than in the general circulation, stimulates the synthesis of new tight-junction proteins on the basal side of that preleptotene spermatocyte, thus reestablishing the integrity of the blood-testis barrier. In parallel, a new set of spermatids becomes adherent to the apical surface of the Sertoli cells through the formation of new surface adhesion complexes.

For many years, gene expression in postmeiotic (haploid) spermatids was considered to be impossible. Molecular biological research on mice has shown, however, that gene expression in postmeiotic spermatids is not only possible but also common. Nearly 100 proteins are produced only after the completion of the second meiotic division, and many additional proteins are synthesized during and after meiosis.

On completion of spermiogenesis (approximately 64 days after the start of spermatogenesis), the spermatozoon is a highly specialized cell well adapted for motion and the delivery of its packet of DNA to the egg. The sperm cell consists of the following: a head (2 to 3 µm wide and 4 to 5 µm long) containing the nucleus and acrosome; a midpiece containing the centrioles, the proximal part of the flagellum, and the mitochondrial helix; and the tail (about 50 µm long), which consists of a highly specialized flagellum (see Fig. 1.12). (Specific functional properties of these components of the sperm cell are discussed in Chapter 2.)

Abnormal Spermatozoa

Substantial numbers (up to 10%) of mature spermatozoa are grossly abnormal. The spectrum of anomalies ranges from double heads or tails to defective flagella or variability in head size. Such defective sperm cells are highly unlikely to fertilize an egg. If the percentage of defective spermatozoa increases to greater than 20% of the total, reduced fertility may result.

Ovaries and Uterine Tubes

The ovaries and uterine (or fallopian) tubes form a functional complex devoted to the production and transport of eggs. In addition, the uterine tubes play an important role as a conduit for spermatozoa and in preparing them to be fully functional during the fertilization process. The uterine tube consists of three anatomically and functionally recognizable segments: the ampulla, the isthmus, and the intramural segments.

The almond-shaped ovaries, located on either side of the uterus, are positioned very near the open, funnel-shaped ends of the ampullary segments of the uterine tubes. Numerous fingerlike projections, called fimbriae (Fig. 1.14), project toward the ovary from the open infundibulum of the uterine tube and are involved in directing the ovulated egg into the tube. The uterine tube is characterized by a complex internal lining, with a high density of prominent longitudinal folds in the upper ampulla. These folds become progressively simpler in parts of the tube closer to the uterus. The lining epithelium of the uterine tubes contains a mixture of ciliated cells that assist in gamete transport and secretory cells that produce a fluid supporting the early development of the embryo. Layers of smooth muscle cells throughout the uterine tubes provide the basis for peristaltic contractions. The amount and function of many of these components are under cyclic hormonal control, and the overall effect of these changes is to facilitate the transport of gametes and the fertilized egg.

The two segments of the uterine tubes closest to the uterus play particularly important roles as pathways for sperm transport toward the ovulated egg. The intramural segment, which is embedded in the uterine wall, has a very thin lumen containing mucus, the composition of which varies with phases in the menstrual cycle. This segment serves as a gateway regulating the passage of spermatozoa into the uterine tube, but it also restricts the entry of bacteria into the tube. The middle isthmus segment serves as an important site of temporary sperm storage and participates in the final stages of functional maturation of sperm cells (see Chapter 2).

Uterus

The principal functions of the uterus are to receive and maintain the embryo during pregnancy and to expel the fetus at the termination of pregnancy. The first function is carried out by the uterine mucosa (endometrium) and the second by the muscular wall (myometrium). Under the cyclic effect of hormones, the uterus undergoes a series of prominent changes throughout the course of each menstrual cycle.

The uterus is a pear-shaped organ with thick walls of smooth muscle (myometrium) and a complex mucosal lining (see Fig. 1.14). The mucosal lining, called the endometrium, has a structure that changes daily throughout the menstrual cycle. The endometrium can be subdivided into two layers: a functional layer, which is shed with each menstrual period or after parturition, and a basal layer, which remains intact. The general structure of the endometrium consists of (1) a columnar surface epithelium, (2) uterine glands, (3) a specialized connective tissue stroma, and (4) spiral arteries that coil from the basal layer toward the surface of the endometrium. All these structures participate in the implantation and nourishment of the embryo.

The lower outlet of the uterus is the cervix. The mucosal surface of the cervix is not typical uterine endometrium, but is studded with a variety of irregular crypts. The cervical epithelium produces glycoprotein-rich cervical mucus, the composition of which varies considerably throughout the menstrual cycle. The differing physical properties of cervical mucus make it easier or more difficult for spermatozoa to penetrate the cervix and find their way into the uterus.

Vagina

The vagina is a channel for sexual intercourse and serves as the birth canal. It is lined with a stratified squamous epithelium, but the epithelial cells contain deposits of glycogen, which vary in amount throughout the menstrual cycle. Glycogen breakdown products contribute to the acidity (pH 4.3) of the vaginal fluids. The low pH of the upper vagina serves a bacteriostatic function and prevents infectious agents from entering the upper genital tract through the cervix and ultimately spreading to the peritoneal cavity through the open ends of the uterine tubes.

Hormonal Control of the Female Reproductive Cycle

Reproduction in women is governed by a complex series of interactions between hormones and the tissues that they influence. The hierarchy of cyclic control begins with input to the hypothalamus of the brain (Fig. 1.15). The hypothalamus influences hormone production by the anterior lobe of the pituitary gland. The pituitary hormones are spread via the blood throughout the entire body and act on the ovaries, which are stimulated to produce their own sex steroid hormones. During pregnancy, the placenta exerts a powerful effect on the mother by producing several hormones. The final level of hormonal control of female reproduction is exerted by the ovarian or placental hormones on other reproductive target organs (e.g., uterus, uterine tubes, vagina, breasts).

Hormonal Interactions with Tissues during Female Reproductive Cycles

All tissues of the female reproductive tract are influenced by the reproductive hormones. In response to the hormonal environment of the body, these tissues undergo cyclic modifications that improve the chances for successful reproduction.

Knowledge of the changes the ovaries undergo is necessary to understand hormonal interactions and tissue responses during the female reproductive cycle. Responding to both FSH and LH secreted by the pituitary just before and during a menstrual period, a set of secondary ovarian follicles begins to mature and secrete 17β-estradiol. By ovulation, all of these follicles except one have undergone atresia, their main contribution having been to produce part of the supply of estrogens needed to prepare the body for ovulation and gamete transport.

During the preovulatory, or proliferative, phase (days 5 to 14) of the menstrual cycle, estrogens produced by the ovary act on the female reproductive tissues (see Fig. 1.15). The uterine lining becomes re-epithelialized from the just-completed menstrual period. Then, under the influence of estrogens, the endometrial stroma progressively thickens, the uterine glands elongate, and the spiral arteries begin to grow toward the surface of the endometrium. The mucous glands of the cervix secrete glycoprotein-rich but relatively watery mucus, which facilitates the passage of spermatozoa through the cervical canal. As the proliferative phase progresses, a higher percentage of the epithelial cells lining the uterine tubes becomes ciliated, and smooth muscle activity in the tubes increases. In the days preceding ovulation, the fimbriated ends of the uterine tubes move closer to the ovaries.

Toward the end of the proliferative period, a pronounced increase in the levels of estradiol secreted by the developing ovarian follicle acts on the hypothalamohypophyseal system, thus causing increased responsiveness of the anterior pituitary to GnRH and a surge in the hypothalamic secretion of GnRH. Approximately 24 hours after the level of 17β-estradiol reaches its peak in the blood, a preovulatory surge of LH and FSH is sent into the bloodstream by the pituitary gland (Fig. 1.16). The LH surge is not a steady increase in gonadotropin secretion; rather, it constitutes a series of sharp pulses of secretion that appear to be responding to a hypothalamic timing mechanism.

The LH surge leads to ovulation, and the graafian follicle becomes transformed into a corpus luteum (yellow body). The basal lamina surrounding the granulosa of the follicle breaks down and allows blood vessels to grow into the layer of granulosa cells. Through proliferation and hypertrophy, the granulosa cells undergo major structural and biochemical changes and now produce progesterone as their primary secretory product. Some estrogen is still secreted by the corpus luteum. After ovulation, the menstrual cycle, which is now dominated by the secretion of progesterone, is said to be in the secretory phase (days 14 to 28 of the menstrual cycle).

After the LH surge and with the increasing concentration of progesterone in the blood, the basal body temperature increases (see Fig. 1.16). Because of the link between an increase in basal body temperature and the time of ovulation, accurate temperature records are the basis of the rhythm method of birth control.

Around the time of ovulation, the combined presence of estrogen and progesterone in the blood causes the uterine tube to engage in a rhythmic series of muscular contractions designed to promote transport of the ovulated egg. Progesterone prompts epithelial cells of the uterine tube to secrete fluids that provide nutrition for the cleaving embryo. Later during the secretory phase, high levels of progesterone induce regression of some of the ciliated cells in the tubal epithelium.

In the uterus, progesterone prepares the estrogen-primed endometrium for implantation of the embryo. The endometrium, which has thickened under the influence of estrogen during the proliferative phase, undergoes further changes. The straight uterine glands begin to coil and accumulate glycogen and other secretory products in the epithelium. The spiral arteries grow farther toward the endometrial surface, but mitosis in the endometrial epithelial cells decreases. Through the action of progesterone, the cervical mucus becomes highly viscous and acts as a protective block, inhibiting the passage of materials into or out of the uterus. During the secretory period, the vaginal epithelium becomes thinner.

In the mammary glands, progesterone furthers the estrogen-primed development of the secretory components and causes water retention in the tissues. More extensive development of the lactational apparatus awaits its stimulation by placental hormones.

Midway through the secretory phase of the menstrual cycle, the epithelium of the uterine tubes has already undergone considerable regression from its midcycle peak, whereas the uterine endometrium is at full readiness to receive a cleaving embryo. If pregnancy does not occur, a series of hormonal interactions brings the menstrual cycle to a close. One of the early feedback mechanisms is the production of the protein inhibin by the granulosa cells. Inhibin is carried by the bloodstream to the anterior pituitary, where it directly inhibits the secretion of gonadotropins, especially FSH. Through mechanisms that are unclear, the secretion of LH is also reduced. This inhibition results in regression of the corpus luteum and marked reduction in the secretion of progesterone by the ovary.

Some of the main consequences of the regression of the corpus luteum are the infiltration of the endometrial stroma with leukocytes, the loss of interstitial fluid, and the spasmodic constriction and breakdown of the spiral arteries that cause local ischemia. The ischemia results in local hemorrhage and the loss of integrity of areas of the endometrium. These changes initiate menstruation (by convention, constituting days 1 to 5 of the menstrual cycle). Over the next few days, the entire functional layer of the endometrium is shed in small bits, along with the attendant loss of about 30 mL of blood. By the time the menstrual period is over, only a raw endometrial base interspersed with the basal epithelium of the uterine glands remains as the basis for the healing and reconstitution of the endometrium during the next proliferative period.