9.2.1 The male gamete: spermatogenesis, maturation, function

The testes fulfil two essential functions: the production and maturation of the male gametes and synthesis and the secretion of the sexual hormones. Unless otherwise specified, this chapter describes the situation in the human and provides the basis for understanding the endocrine and local regulation of testicular function. Data obtained in experimental animals are presented when the corresponding human mechanisms are not known or cannot be clarified for ethical reasons.

The term ‘spermatogenesis’ describes and includes all processes and events involved in the production of gametes, whereas ‘steroidogenesis’ refers to the enzymatic reactions leading to the production of male steroid hormones. Spermatogenesis and steroidogenesis take place in two morphologically and functionally distinct compartments (Figs. 9.2.1.1 and 9.2.1.2). Here ‘testicular compartment’ refers to the seminiferous tubules (tubuli seminiferi) and the interstitial compartment (interstitium) in between the tubules. Although anatomically divided, both compartments are closely interconnected in functional terms, and the integrity of both compartments is indispensable for normal production of sperm. Pubertal development and mature functions of the testis are controlled primarily by the brain, the hypothalamus, and the pituitary gland (endocrine regulation) and, at the secondary level, by local factors and mediators (paracrine and autocrine regulation) as described in Chapter 9.2.2. The key endocrine factors are lut-einizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone. Testicular spermatozoa are produced in the seminiferous tubules, which are long conduits that discharge into ducts located centrally in the human testis (rete testis).These ducts subsequently drain into the epididymis, a maturation and storage organ, through efferent ducts.

This compartment occupies 12–15% of the total testicular volume, 10–20% of which is occupied by Leydig cells. The number of Leydig cells is relatively low, but under the influence of luteinizing hormone this cell type produces two hormones, testosterone and insulin-like factor 3 (INSL3), which are important not only for spermatogenesis but also for body functions. Apart from Leydig cells, the interstitial compartments also contain connective tissue, cells of the immune system, fibroblasts, blood vessels, nerves, and lymph vessels.

Leydig cells were first described by Franz Leydig (1821–1908) in 1850. These cells produce and secrete the most important male sexual hormone, testosterone, under the influence of luteinizing hormone. From the developmental point of view, several successive types of Leydig cells can be distinguished: stem Leydig cells, progenitor Leydig cells, fetal Leydig cells, and adult Leydig cells (1). These developmental stages cannot be distinguished by their morphology alone. In a knockout mouse model, it was recently demonstrated that the protein COUP-TFII, a nuclear receptor, is a key regulator of differentiation in Leydig cells, although it is not essential in mature Leydig cells (2). Fetal Leydig cells become neonatal Leydig cells at birth and degenerate thereafter or regress into immature Leydig cells (3). Terminally differentiated Leydig cells are rich in smooth and rough endoplasmic reticulum, and in mitochondria with tubular cristae. These physiological characteristics are typical for steroid-producing cells and are very similar to those found in other steroidogenic cells, such as those in the adrenal gland and in the ovary. Other important cytoplasmic components are lipofuscin granules, the final products of endocytosis and lysosomal degradation, and lipid droplets, in which the preliminary stages of testosterone synthesis take place. Specific formations, called Reinke’s crystals, are often found in Leydig cells, and are probably formed by subunits of globular proteins with unknown function. Leydig cells can be seen adjacent to blood vessels (perivascular Leydig cells) and in close proximity to or within the tubular wall (peritubular Leydig cells). The functional significance of this topography is not yet understood. The proliferation rate of the Leydig cells in the adult testis is rather low and is influenced by luteinizing hormone. In addition to controlling testosterone production, luteinizing hormone also induces differentiation of Leydig cells. Lack of the hormone can lead to Leydig cell involution, evidenced by reduced cell size, accumulation of lipid droplets, and reduction of smooth endoplasmic reticulum abundance. Long-term deprivation of luteinizing hormone can lead to signs of Leydig cell dedifferentiation. In the prepubertal testis, FSH can stimulate Leydig cell testosterone production through an indirect effect mediated by the Sertoli cells (4).

As an immune privileged organ, the testis derives its immune competence from the interstitium which contains a variety of immunocompetent cells, e.g. leukocytes, macrophages, monocytes, dendritic cells, T and B lymphocytes, and mast cells (Fig. 9.2.1.2). For every 10–50 Leydig cells, one macrophage is to be found. Macrophages proliferate in the testis during postnatal life, probably under pituitary control, since human chorionic gonadotropin (hCG) is able to increase the mitotic index of testicular macrophages in rats. In the adult human testis, macrophages represent about 25% of all interstitial cells. Morphologically and biochemically, they are similar to macrophages resident in other tissues. In man and in seasonally reproducing animals, macrophages are found also within the seminiferous epithelium. The macrophages probably influence the function of the Leydig cells, in particular their proliferation, differentiation, and steroid production, through the secretion of cytokines. Macrophages secrete both stimulators and inhibitors of steroidogenesis. Proinflammatory cytokines, reactive oxygen species, nitric oxide, and prostaglandins can inhibit Leydig cell function (5). There is also evidence for the involvement of neurotransmitters and related signalling factors in the regulation of Leydig cell functions, including their proliferation, differentiation, and steroid production(6). Testicular macrophages have a reduced capacity to excrete some cytokines such as interleukin-1β (IL-1β) and tumour necrosis factor α (TNFα) compared to macrophages from other tissues (7). Furthermore, when lipopolysaccharides (LPS), resembling the surface of bacteria, were given to immature and mature mice, enhanced levels of the testicular cytokine IL-6 and constitutive elevation of the production of other anti-inflammatory mediators were observed (8, 9). Interestingly, the expression of proinflammatory cytokines such as IL-1β and TNFα by testicular macrophages demonstrates the testicular capability of an inflammatory response. To date, two macrophage types have been distinguished in the adult testis that differ in the expression of markers such as ED1 and ED2, and of inflammatory mediators. In the rat, ED2⁺ expressing macrophages do not participate in promoting inflammatory processes, but they may take part in maintaining the immune privilege as an immunoregulatory team player. However, ED1⁺/ED2⁻ macrophages are involved in testicular inflammatory responses. During acute and chronic inflammation the influx of ED1+ monocytes changes the equilibrium of the macrophage population. The number of mononuclear cells increases in cases of testicular disease.

The contribution of other immune cells such as mast cells to the immune system has often been underestimated, but the complexity of these cells and their involvement in the innate and adaptive immune system was recently shown (10, 11). Mast cells also release factors that act as mediators and as such are capable of influencing disease induction and progression. As a functional example, mast cells in the brain can change vascular permeability through factor release, thereby opening the blood–brain barrier and allowing the entry of activated T lymphocytes and inflammatory cell traffic. Their main secreted product, a serine protease tryptase, is a mitogen for fibroblasts and thus enhances the synthesis of collagen, resulting in fibrosis, thickening, and hyalinization of tubular walls.

T lymphocytes also migrate through tissues as a part of the normal process of immune surveillance. In the testis, the activation of the immune system is thought to be inhibited by locally produced factors. For example, Leydig cells are able to adhere to lymphocytes and suppress their proliferation. An important role in the immune control of the testis is played by the endothelium. Testicular endothelial cells are less permeable to dyes than other organs and the uptake of many substances from the circulation is cell mediated. Maturation of testicular microvessels occurs at puberty and is hormone dependent. Remarkably, endothelial cells express the hCG and luteinizing hormone receptor, and hCG and luteinizing hormone influence vascular permeability in the rat (12). Most probably, the immune privilege of the testis is brought about by the interstitial cells rather than the so called blood–testis barrier, which is discussed in the Sertoli cell section.

Spermatogenesis takes place in the tubular compartment. This compartment represents 60–80% of the total testicular volume. It contains the germ cells and two different types of somatic cells: the peritubular cells and the Sertoli cells. The testis is divided by septae of connective tissue into 250–300 lobules, each one containing between one and three seminiferous tubules. Overall, each human testicle contains about 600 seminiferous tubules. The length of individual seminiferous tubules is 30–80 cm. Considering an average number of about 600 seminiferous tubules per testis and an average length of the tubuli seminiferi of about 60 cm each, the total length of the tubuli seminiferi is about 360 m per testis, that is, 720 m of seminiferous epithelium per man. Both tubular length and tubular diameter determine testicular size.

The human seminiferous tubular wall (lamina propria) is a highly complex structure composed of several layers. The germinal epithelium rests upon a basal lamina (basement membrane), followed by a layer of all collagen fibres, up to six layers of the so-called peritubular cells (myofibroblasts), each of them separated by extracellular collagen fibres. At the outermost periphery, fibroblasts can be present. Myofibroblasts are poorly differentiated myocytes with the capacity for spontaneous contraction. These cells express factors typical for contractile cells such as α-smooth muscle actin, panactin, desmin, smooth muscle myosin, and gelsolin; however, they also express factors characteristic of connective tissue cells, such as vimentin, collage, laminin, fibronectin, fibroblast protein, and adhesion molecules (13, 14). Testicular myofibroblasts are stratified around the tubule and form up to six concentric layers (Fig. 9.2.1.2). The human testicle differs from the organization of other mammals, whose seminiferous tubules are surrounded only by two to four layers of myofibroblasts. The contractile capacity of peritubular cells to transport sperm towards the exit of the seminiferous tubules is influenced by various factors. Several regulators of cell contractions are reported, such as oxytocin, oxytocin-like substances, prostaglandins, androgenic steroids, endothelins, endothelin converting enzymes, and endothelin receptors. Peritubular contractility is mediated by endothelin and this effect is modulated by the relaxant peptide, adrenomedullin, produced by Sertoli cells (15). Mice with selective peritubular cell androgen receptor deficiency revealed defects in genes related to contractility, e.g. endothelin-1 and endothelin receptors A and B, adrenomedullin receptor, and vasopressin receptor 1a (16). Whether the peritubular cells, besides their contractile properties, also possess other functions in the testis is not yet clear, but tubules challenged by inflammation-driven diseases show irreversible thickening of the lamina propria (17).

Androgens play an important role in the physiological differentiation of myofibroblasts in primate testes, because they induce the production of actins, and thereby the contractility of the tubular cells, during testicular development (18). Tubular walls with thickening of the layer of collagen fibres, and condensation of the extracellular material present between the peritubular cells, are denoted as fibrotic or less severely hyalinized. Thickening of the tubular wall reduces the exchange of metabolic substances, thereby disturbing the function of the germinal epithelium or even leading to its destruction. Hypogonadotrophic hypogonadism, experimentally provoked by withdrawal of endocrine hormones in nonhuman primates, causes testicular involution that is associated with extreme reversible thickening of the tubular wall. The decrease of testicular volume involves the folding of the wall along the length of the tubuli seminiferi, thereby causing an enlargement of the tubular diameter. This becomes particularly evident when fluid is injected into regressed seminiferous tubules: tubular diameter increases and tubular wall thickness decreases (19). Additionally, an interaction between testicular mast cells and peritubular cells leading to fibrotic changes of the seminiferous tubular wall has been suggested (20). Peritubular and interstitial fibrosis was shown to correlate progressively with spermatogenic damage in testes from vasectomized men (20).

Sertoli cells are somatic cells located within the germinal epithelium and are named after Enrico Sertoli (1842–1910), who first described these cells in 1865 and, due to their prominent cytoplasmic projections and ramifications, called them cellulae ramificate. These cells rest on the basal membrane, and extend through the lumen of the tubules’ seminiferous epithelium (Fig. 9.2.1.3). In a broader sense, they are the supporting structures of the entire height of the germinal epithelium. All morphological and physiological differentiation and maturation events of the germinal cell, up to the mature sperm, take place here. Special ectoplasmic structures sustain the alignment and orientation of the developing sperm cells during differentiation. Approximately 35–40% of the volume of the germinal epithelium is represented by Sertoli cells. The intact testis with complete spermatogenesis contains approximately 25 × 10⁶ Sertoli cells per gram testis (20).

The Sertoli cells of most species, including humans, synthesize and secrete a large variety of factors, including cytokines, growth factors, opioids, proteases, steroids, prostaglandins, and regulators and modulators of the cell cycle and cell survival. Sertoli cells provide a three-dimensional framework along which germ cells develop, mature and are gradually transported towards the tubular lumen. Given this scenario, it is assumed that the Sertoli cells guide the germ cells along their long and complex development from a stem spermatogonium into elongated spermatids. Sertoli cells and germ cells are in fact intimately associated functionally and morphologically, and Sertoli cells possess specialized processes for interaction with germ cells. Sertoli cell cytoplasm contains endoplasmic reticulum both of the smooth (steroid synthesis) and rough type (protein synthesis), a prominent Golgi apparatus (elaboration and transport of secretory products), and lysosomal granules (phagocytosis), as well as microtubuli and intermediate filaments (for adaptation of the cell shape during the different phases of germ cell maturation). Sertoli cells, but not germ cells, contain receptors for androgen and for FSH. The trophic effects of these hormones on spermatogenesis are therefore mediated via Sertoli cells, supporting the idea of a governing role for this cell type in the spermatogenic process. However, the elimination of specific germ cell types by administration of specific testicular toxins provoked stage-dependent changes in the secretion of inhibin from Sertoli cells. More recent data support the contention that germ cells control Sertoli cell functions. For example, the time pattern of germ cell transitions and development during the spermatogenic cycle seems to be autonomous, as suggested from heterologous germ cell transplantation studies (21). One spermatogenic cycle lasts about 8 days in mice and 12—13 days in rats. Notably, the cycle duration of rat germ cells transplanted into mouse testis remained 12–13 days. Moreover, male germ cell differentiation seems not to be limited to the strict structure provided by the Sertoli cells, as germ cell differentiation has been achieved in in vitro experiments without any close contact between the somatic and germinal cell types (22). On the basis of these observations, it becomes likely that it is actually the germ cells that govern the spermatogenic process, whereas the function of the Sertoli cell is to nurse germ cells in response to their metabolic needs. This could also explain why both FSH and luteinizing hormone or testosterone alone can stimulate sperm production (qualitatively normal spermatogenesis) and why the combination of both yields fully normal sperm production (quantitatively normal spermatogenesis). In the first instance, the Sertoli cell ‘stores’ are just sufficiently filled to enable the production of some sperm; in the second instance the ‘stores’ are completely filled and full numbers of sperm can be produced.

Another important function of Sertoli cells is that their number is responsible for final testicular volume and the amount of sperm production in the adult. Stereological investigations suggest that the number of Sertoli cells in boys increases until the fifteenth year of life. In prepubertal macaque monkeys, Sertoli cells exhibit little mitotic activity; however, their proliferative activity can be clearly stimulated experimentally with trophic factors such as androgens and FSH (23). In adulthood, these cells are mitotically inactive and their number does not increase any further. Each individual Sertoli cell is in morphological and functional contact with a defined number of sperm. The number of sperm per Sertoli cell depends on the species. In men we observe about 10 germ cells or 1.5 spermatozoa per each Sertoli cell (24). In comparison, every macaque monkey Sertoli cell is associated with 22 germ cells and 2.7 sperm (25, 26). This suggests that within a certain species a higher number of Sertoli cells results in greater production of sperm and testis size, assuming that all the Sertoli cells are functioning normally. In contrast, testicular cell numbers were very similar across several primate species, as determined by flow cytometry, suggesting that testis size is the main determinant of total germ cell output (27).

Sertoli cell proliferation is markedly activated in the immature testis when exposed to gonadotropin activity (23, 28). Both Sertoli cell number and expression of markers of cell division are stimulated by these hormones. The expression of Sertoli cell markers such as transferrin, androgen-binding protein and junction proteins such as N-cadherin, connexin-43, gelsolin, laminin-γ3, occludin, testin, nectin, zyxin, and vinculin is androgen dependent (16). It appears that several of these components are involved in establishing the blood–testis barrier, and also in the release of sperm and subsequent remodelling of the Sertoli cell to germ cell junctions (29). The division of Sertoli cells ends when the first germ cells undergo meiotic division. By this point, Sertoli cells have built tight junctions between each other, forming the so-called blood–testis barrier. Lack of connexin-43, a predominant gap junction protein, prevents Sertoli cell maturation and is associated with continued division of Sertoli cells and spermatogenic arrest beyond spermatogonial development (30, 31). Experimentally induced prolongation of the division phase of Sertoli cells, produced for example by thyroid hormone deprivation, results in an increase of testicular weight and sperm production of more than 50% in the rat model. Patients with Laron’s syndrome have a disturbed thyroid function and insulin-like growth factor 1 (IGF1) deficiency, and often have larger than normal testicles (32).

Through the production and secretion of tubular fluid, Sertoli cells create and maintain the lumen of the tubules. More than 90% of Sertoli cell fluid is secreted into the tubular lumen. The structural elements of the blood–testis barrier prevent its reabsorption. This results in a certain pressure that maintains the patency of the lumen. Sperm are transported in the tubular fluid; unlike blood this contains a high concentration of potassium ions and low concentration of sodium ions. Other constituents are bicarbonate, magnesium and chloride ions, inositol, glucose, carnitine, glycero-phosphorylcholine, amino acids, and several proteins. The germ cells are thus contained in a fluid of unique composition. Sertoli cells are capable of phagocytosis and can degrade abnormal germ cells and cellular remnants shed from the elongating and condensing germ cells.

The basolateral aspect of neighbouring Sertoli cells comprises specialized membranes that form a band, sealing the cells from each other and obliterating the intracellular space (using occluding tight junctions). Closure of the blood–testis barrier coincides with the beginning of the first meiosis in the germinal cells (preleptotene, zygotene) and with the arrest of proliferation of Sertoli cells, and has been demonstrated in the species investigated to date. The blood–testis barrier divides the seminiferous epithelium into two regions which are anatomically and functionally completely different from each other. Early germ cells are located in the basal region, while later stages of maturing germ cells are found in the adluminal region. During development germ cells are displaced from the basal to the adluminal compartment. This is accomplished by a synchronized dissolution and reassembly of the tight junctions above and below the migrating germ cells. Two important functions are covered by the blood–testis barrier: the physical isolation of haploid, and thereby antigenic, germ cells to prevent recognition by the immune system (prevention of autoimmune orchitis) and the preparation of a particular environment for the meiotic process and sperm development. The constitution of the blood–testis barrier and its selectivity in excluding certain molecules means that the cells localized in the adluminal compartment have no direct access to metabolites deriving from the periphery or from the interstitium. Therefore, these cells are completely dependent on Sertoli cell nourishment.

Spermatogenesis starts with the division of stem cells and ends with the formation of mature sperm (Figs. 9.2.1.3 and 9.2.1.4). The entire process can be divided into four phases: (1) sustainment of committed cell types (stem cell maintenance), mitotic proliferation, and differentiation of spermatogonia; (2) the meiotic division of tetraploid germ cells (spermatocytes) yielding haploid spermatids; (3) the transformation of haploid germ cells (spermatids) into testicular sperm (spermiogenesis); and (4) the release of sperm from the germinal epithelium into the tubular lumen (spermiation).

Spermatogonia lie in the basal part of the seminiferous epithelium and are classified as type A and type B spermatogonia. Spermatogonia divide mitotically and are ontogenetically derived from gonocytes. Two major subtypes of A spermatogonia can be distinguished from a cytological and a physiological point of view: the Ad (dark) spermatogonia and the Ap (pale) spermatogonia. The Ad spermatogonia do not show proliferating activity under normal circumstances (36) and are considered to represent testicular stem cells (37). These germ cells, however, become mitotically active when the overall spermatogonial population is drastically reduced, for example after radiation exposure (38). In contrast, the Ap spermatogonia also divide and renew themselves but can additionally differentiate into two B spermatogonia. Detailed studies in nonhuman primates led to a revised model for spermatogonial expansion in men (36): only Ap spermatogonia divide and give rise to Ap spermatogonia (to replenish this cell pool) as well as to B-type spermatogonia for further development (Fig. 9.2.1.5). The human testis contains a single generation of B-type spermatogonia. Germ cells then develop into preleptotene spermatocytes before the beginning of meiotic division. Progeny cells remain in close contact with each other through intercellular bridges. This ‘clonal’ mode of germ cell development—also confirmed for primates (39)—is possibly the basis of, and probably the prerequisite for, the coordinated maturation of gametes in the seminiferous epithelium.

After a phase of DNA synthesis resulting in duplication of their DNA content, the spermatocytes undergo the different phases of meiotic division, giving rise to haploid germ cells. The meiotic process, divided into two prophases, metaphase, anaphase and telophase, is a critical event of gametogenesis during which recombination of genetic material, reduction of chromosome number, and development of spermatids are accomplished. The first prophase, the actual recombination phase, is subdivided into the leptotene, zygotene, pachytene, diplotene, and diakinesis stages that follow each other sequentially. In the earliest meiotic cells (preleptotene spermatocytes), intensive DNA synthesis takes place. Pairing and unpairing in the first meiotic prophase involves substantial structural modifications of the chromosomes. These modifications commence with chromosome synapsis and the beginning of the development of the synaptonemal complex during the zygotene stage. Completion of synapsis and crossing-over occur during the pachytene stage. Dissolution of the synaptonemal complex and separation of the chromosomes, except in those regions where chiasmata are present, take place during the diplotene stage. RNA synthesis is pronounced in the diplotene stage.

Secondary spermatocytes derive from the first meiotic division. These germ cells contain a double haploid chromosomal complement, that is, a diploid amount of DNA but a haploid chromosomal number, because the sister chromatids are regarded as a single chromosome (22 duplicated autosomal chromosomes and either a duplicated Y or X chromosome). During the second meiotic division, secondary spermatocytes split into haploid spermatids. No DNA synthesis takes place at this time. The daughter cells remain interconnected through intercellular bridges. The prophase of the first meiosis lasts 1–3 weeks, whereas the other phases of the first meiosis and the entire second meiosis are concluded within 1–2 days. Hence, meiosis is controlled in a cell-autonomous manner.

Spermatids derive from the second meiotic division, and start as round cells remaining mitotically inactive indefinitely. These cells undergo a remarkable and complicated transformation before the final production of differentiated elongated spermatids (testicular sperm). These processes include the condensation and structural shaping of the cell nucleus, the development of the acrosome, the formation of a flagellum and the expulsion of a large part of cytoplasm. The overall process is called spermiogenesis and, from a qualitative point of view, is identical in all species. During this process, the nuclear chromatin is successively rearranged and histones are replaced by transition proteins followed by protamines. The process of spermiogenesis is divided into four phases: Golgi, cap, acrosome, and maturation phase.

During the Golgi phase, acrosomal bubbles and the craniocaudal symmetry is established. In the cap phase, the spermatids become elongated and the acrosome develops, covering the cranial half to two-thirds of the spermatid. In the acrosomal phase, the cell nucleus becomes further condensed and elongation of the cell continues. During the fertilization process, enzymes are released by the acrosome, allowing the sperm to penetrate the egg. During nuclear condensation the majority of histones are lost and gene transcription stops. Nuclear chromatin is now extremely compact, implying that the proteins necessary for spermiogenesis have to be transcribed before this point of time and justifying the observation of RNA species with a very long half-life. Histones, transition proteins, and protamines are important factors involved in the condensation of nuclear chromatin. The mRNA translational control mechanisms are currently being unravelled, and RNA-binding proteins seem to play an important role. The flagellum is now mature. The principal event during the maturation phase of the spermatids is the extrusion of the rest of the cytoplasm as a so-called residual body. Residual bodies are phagocytosed by Sertoli cells and have a regulatory role.

Elongated spermatids and their residual bodies influence the secretory function of Sertoli cells (production of tubular fluid, inhibin, androgen-binding protein and IL-l and -6). When the residual bodies are degraded, a new spermatogenic cycle begins. The release of sperm in the tubular lumen is named spermiation. This process can be particularly affected by hormonal modifications, temperature, and toxins. The reasons for this sensitivity are, however, not known. In the gonadotropin-deficient testis, some elongated spermatids are not released but are phagocytosed by Sertoli cells. These sperm are transported to the basal part of the Sertoli cell and are degraded. Round and elongated spermatids already contain all the information necessary for fertilization; since the advent of intracytoplasmic injection of testicular sperm into oocytes it has been possible to induce pregnancies successfully. Haploid germ cells express a number of gene products specific to these cells (40) and altered expression of these genes or only a mismatch of the epigenetic methylation is associated with disturbances of spermatogenesis and fertility.

Spermatozoa are the final product of a complex differentiation series of precursor germ cells, and represent structurally unique cells. The human spermatozoon is approximately 60 µm in length, with the tail measuring about 55 µm. The head contains the genetic information and is enveloped by the acrosome. During the fertilization process, some enzymes are released by the acrosome, allowing the sperm to penetrate the egg. Mitochondria are present in the midpiece and provide the energy for sperm motility. The sperm tail is mainly composed of dense fibres and the axonemal complex (9 + 2 microtubule doublet arrangement) and provides the structural and functional basis for motility. The main function of the complex structures of the finally differentiated male germ cell is to provide an appropriate vehicle for the DNA to reach the ovulated oocyte in the oviduct. Further functions, not to be disregarded, include the male centromere, needed for the first division of the fertilized egg, and possibly the spatial information for the body axes.

The complex process of division and differentiation of germ cells follows a precise pattern. All germ cells pass through several stages characterized by particular cellular associations. Recognizing that the acrosome development is stage-dependent was crucial for the understanding of germ cell maturation. The number of stages of spermatogenesis differs according to the species. In man, spermatogenesis covers six stages (I–VI; Fig. 9.2.1.5) and the succession of these stages over time is called the spermatogenic cycle. The duration of the spermatogenic cycle is also species-specific and lasts between 8 and 17 days, with the human spermatogenic cycle requiring 16 days for completion. For the development and differentiation of an A spermatogonium into a mature sperm, at least four spermatogenic cycles are necessary, resulting in the overall duration of human spermatogenesis of around 64 days. The duration of the spermatogenic process is not influenced by reproductive hormones (41), but can be modified as a function of age or following exposure to heat or to toxins. (42) However, a recent review suggests a cycle of 74 days by including time for spermatogonial renewal (33). Investigations carried out in the 1960s led to the conclusion that the duration of spermatogenesis is genetically determined, does not vary throughout life and cannot be influenced experimentally. However, many indirect experimental findings oppose this hypothesis. For example, the first spermatogenic cycle during puberty proceeds faster than during adult age. It has also been demonstrated in the rat that the duration of germ cell maturation can actually be manipulated by exogenous factors.

The spermatogenetic stages follow a precise order, not only in time but also in space. In the rat, serial transverse sections through the seminiferous tubules show that stage I is always followed by stage II, stage III always by stage IV and so on. This is described as the spermatogenic wave. In the entire human testis and in parts or whole testis of various other nonhuman primate species, each tubular cross-section simultaneously shows different stages. Quantitative analysis of the germ cell population has suggested that the distribution of spermatogenic stages in these species does not follow an irregular pattern but a helical topography of spermatogenic stages (43). Other investigations of human spermatogenesis confirmed the principle of helical patterns, but not the presence of a complete spermatogenic wave, i.e. the complete succession of all stages (44). Germ cell transplantation into testicular tubules revealed that one spermatogenic stage represents a single clone of germ cells (21). Therefore, variation in clonal size could lead to the appearance of several stages per cross-section (see Wistuba et al. (45) for further discussion), and species differences with regard to the number of spermatogenic stages are related, at least in part, to clonal size. A comparative and quantitative analysis of the incidence of tubules with one or more spermatogenic stages in 17 primate species yielded that in men, great apes, and New World monkeys, multi-stage tubules are more common, whereas in prosimians and Old World monkeys single-stage tubules predominate (27, Fig. 9.2.1.6).

Earlier investigations considered human germ cell production as being rather inefficient. Human germ cell production results in comparatively low sperm numbers per Sertoli cell. When expressed in millions of sperm per gram of testis over 24 hours, the rat has values of 10–24, nonhuman primates values of 4–5, and men values of 3–7 million. Stereological germ cell counts failed to detect meiotic germ cell losses in primates including men (24–26). More recent work using flow cytometric quantization including meiotic cells and spermatids showed that human germ cell yields are comparable to other primates (27, Fig. 9.2.1.7). The observed germ cell yields for the transitions from spermatogonia into spermatocytes and from spermatocytes into spermatids (meiosis) matched those expected from theoretical computations. Every day, approximately 400 × l0⁶ sperm are produced by men with intact spermatogenesis. The turning point of primate spermatogenesis and the outcome of germ cell production is determined by spermatogonia and their entry into meiosis. Human spermatogenesis is more efficient than assumed earlier. Differences in germ cell number per cell or tissue unit are rather related to the number of spermatogonial divisions (39), with men considered to have only a single generation of B-type spermatogonia (Fig. 9.2.1.5).However, some nonhuman primates can have four such generations since their testicular organization more closely resembles that of other mammals.

All phases of germ cell proliferation and development are prone to disturbances. Germ cell production and development are entirely dependent on the hormones luteinizing hormone, testosterone, and FSH which stimulate spermatogenesis indirectly by acting on somatic testicular cells. The stimulatory effects of these hormones result in increased proliferation of spermatogonial cells, followed by production of meiotic and haploid germ cells resulting from the increased availability of precursor cells. A classification system based upon the histoarchitecture of the epithelium and the relative number of germ cells is described in Chapter 9.3.6, and the clinical consequences of spermatogenic defects are reviewed in Chapter 9.4.2. Pathological conditions and criteria are: the absence of any cellular elements within the seminiferous tubules (tubular atrophy), lack of germ cells including spermatogonia (Sertoli-cell-only syndrome), spermatogenic arrest at the level of primary spermatocytes, and round or elongating and elongated spermatids. Currently, very little is known concerning the factor(s) that are responsible for these specific spermatogenic disturbances except for cases with reproductive hormone deficiency or chromosomal imbalance, e.g. Klinefelter’s syndrome patients, in whom meiosis is disturbed, leading to germ cell loss. Lack of appropriate hormonal support provokes progressive involution of the seminiferous epithelium. In the final state, the seminiferous tubules are populated by Sertoli cells and spermatogonia or a few spermatocytes are the only germ cells remaining. Leydig cell atrophy and dedifferentiation and/or involution of the seminiferous epithelium, germ cell degeneration, dedifferentiated Sertoli cells, and pronounced thickening of the tubular wall are characteristic morphological and cytological features of disturbed spermatogenesis (46). Sperma-togenic involution is fully reversible, as seen from histological analysis of recovering testes following experimentally induced hypogonadotrophic hypogonadism in nonhuman primate models, and from the restoration of sperm production and fertility by gonadotropin treatment patients with secondary hypogonadism (Chapter 9.5.1).

During spermatogenesis, a series of mitotic and meiotic cell divisions leads to the production of haploid germ cells, which undergo dramatic morphological changes to give rise to spermatozoa. To date, several infertility-related genes have been investigated in an attempt to identify reliable molecular markers that can predict the presence of haploid cells in the testes of infertile men. The highly complex process of spermatogenesis requires the expression and precise coordination of a number of genes. Dysfunction of such genetic factors is associated with disturbed spermatogenesis, and is suspected to be a frequent cause of male infertility (47–50). Among these candidate genes is the DAZ (Deleted in AZoospermia) gene family consisting of two autosomal genes, BOULE and DAZL (DAZ-like), and the Y-chromosomal DAZ gene cluster. All DAZ members are RNA-binding proteins specifically expressed in the germline, and are essential for germ cell development (reviewed by Reynolds and Cooke (50)). In flies, such as Drosophila, male boule mutants are sterile and their germ cells are arrested at the spermatocyte stage, demonstrating the requirement of boule for meiosis (51). Abnormal function of such genes is frequently associated with male infertility. Testicular biopsy samples stained for BOULE with complete meiotic arrest seem to be independent of the etiology of the spermatogenic damage, which identifies this factor as a possible fundamental mediator of meiotic transition also in humans. The lack of BOULE expression and, possibly, of other important regulators of meiosis, might represent a common key molecular mechanism involved in meiotic arrest.

Deletions of the AZF (azoospermia factor) subregions on the Y chromosome are also accompanied by a diverse spectrum of spermatogenic disturbances ranging from hypospermatogenesis to total depletion of germ cells, causing infertility. The AZF region encodes gene products that are candidates for controlling spermatogenesis genetically. Although it is known which genes are affected, a general principle of cause and effect cannot yet be deciphered, and the deletion type has nonuniform histological phenotypes. Future studies should focus on understanding the biological function of AZF genes, which is an essential step for the development of more appropriate and knowledge-based therapies.

The development of germ cells relies on the appropriate balance between germ cell proliferation, differentiation, survival and controlled cell death. Programmed cell death (apoptosis) comprises a coordinated sequence of signalling cascades leading to cell suicide. Unlike necrosis, this form of cell death occurs under physiological conditions (spontaneous apoptosis) but can also be induced by exposure to toxins, disturbances of the endocrine environment, and so on. In the human testis spermatogonia, spermatocytes, and spermatids undergoing apoptosis have been detected, and ethnic differences in the incidence of testicular apoptosis have been suggested. Apoptotic germ cells are present in the intact human testis and in the testes of ageing men (52), and apoptotic cell numbers are elevated in men with spermatogenic disorders (53). Endocrine imbalance or heat treatment induces testicular apoptosis via intrinsic and extrinsic pathways in nonhuman primates (54).

Immotile sperm leave the seminiferous tubules passively in fluid that enters the rete testis and is drawn into the efferent ducts, by ciliary activity and absorption of the fluid. Sperm are moved through the epididymis, in part by hydrostatic pressure originating from fluids secreted in the seminiferous tubules. They also migrate through the epididymis by peristaltic motion of the duct. The epididymis fulfils several functions: sustenance of sperm and their protection from cells of the immune system, fluid reabsorption, and protein secretions that modify the luminal fluid and mediate sperm maturation and storage. Under normal circumstances, the human epididymis has a daily transport capacity of around 150 × 10⁶ sperm and a storage capacity of about 600 × 10⁶ sperm. Although sperm can pass through the human cauda within a couple of days, fertile sperm can be stored for several weeks in men. How long effective storage may be in the human is uncertain, but sperm motility in the ejaculate of young men can be preserved for up to 78 weeks after the last ejaculation (55). Many epididymal functions are dependent on an adequate supply of androgenic hormones.

The epididymis comprises a highly convoluted duct of approximately 5 m in length and is separated into three major segments: caput, corpus and cauda. The epididymal tubule is surrounded by peritubular myoid cells. A variety of different epithelia line the epididymal duct in men: for example, seven different epithelial types were described for the human caput epididymis. The main epididymal tubule in the distal caput and corpus has a columnar epithelium with microvilli. These microvilli provide a huge increase in luminal membrane surface area that may be important in providing area for cell surface receptors, transport channels, and even membrane for endocytic events. At the level of the corpus epididymis, the lumen contains readily evident concentrations of spermatozoa. The main cell-types are so-called principal cells. The apical borders of epididymal epithelial cells exhibit cell to cell tight junctions (56) composed of a number of cell adhesion molecules (57, 58), which impose a blood-epididymal barrier similar in effect to the blood–testis barrier; that is, the blood-epididymal barrier provides a specialized, immune-privileged microenvironment in which sperm remain isolated from other body compartments (59).

Gene expressions and protein secretions vary in distinct patterns along the human epididymal duct (60). Complex associations of proteins in membranous vesicles are also secreted in some species, and those vesicles have been shown to transfer specific proteins directly to luminal spermatozoa. Similar vesicles have also been reported in the human epididymis (61), but their role remains unresolved.

Among the epididymal cells, the principal cells are most abundant. Other types present are basal cells, narrow cells, clear cells, apical cells, and halo cells, which are scattered along the duct in lesser numbers, all of which differ in relative abundance depending on the epididymal region. Principal cells are involved in many secretory and absorptive functions. The proluminal transport system is responsible for fluid secretion, possibly to reduce fluid viscosity and to support sperm transport, and the secretion of low molecular weight compounds such as l-carnitine, glycerophosphocholine and myo-inositol, which form part of macromolecules such as glycoproteins and growth factors. Electrolytes and small organic molecules also change in characteristic patterns along the epididymis, and it is the exposure of sperm to this ever-changing microenvironment that is necessary for their full maturation (62). For instance, α-glucosidase is localized to the brush-border of the microvilli of principal cells. Cell products and enzymes are presumed to interact with spermatozoa during epididymal maturation and storage. Both l-carnitine and α-glucosidase are present in the ejaculate and serve as marker substances for the detection of excurrent duct obstructions.

Basal cells adhering to the basement membrane form a network beneath the principal cells. This cell type is usually not present at birth. High levels of glutathione S-transferase and superoxide dismutase are found, suggesting a role in detoxification. Basal cells may regulate electrolyte and water transport by the principal cells, involving two proteins which are exclusively expressed by the basal cells: transient receptor potential proteins, which serve as transmembrane pathways for Ca²⁺influx, and cyclo-oxygenase 1 (COX1), a key enzyme in the formation of prostaglandins (63). Basal cells are also engaged in immune reactions, as they express macrophage antigens. A similar function is assumed for the intraepithelial lymphocytes (also denoted the ‘halo’ cells as mentioned). Dependent on the species, apical cells and clear cells—where the nomenclature is based upon nuclear localization within the epithelium and on cytological features—are also present to various extents. These cells lack true apical cilia but can engulf particulate matter and remove contents of the cytoplasmic droplets following its dissolution. Macrophages and mast cells can be present but are rare under normal conditions.

The bilateral absence or the complete obstruction of the epididymal ducts are associated with azoospermia and male infertility. A cause of epididymal dysgenesis is mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene that occurs in cystic fibrosis, the most common inherited disease. Over 500 different mutations of CFTR have been identified, explaining the wide spectrum of the cystic fibrosis phenotype (64). One constant in the disease is that approximately 95% of men with clinical cystic fibrosis have congenital absence of the vas deferens (64), which is commonly accompanied by absence of the cauda and corpus epididymis as well. Testicular sperm are barely motile and are incapable of reaching the egg following insemination. Thus, epididymal maturation is not necessarily required for assisted fertility, although it facilitates the development of sperm-egg interactions. A major function of the epididymis is to endow the sperm with the capacity for appropriate mobility in order to enable them to reach the ovulated oocyte in the female reproductive tract. Hence, epididymal dysfunction can be associated with male infertility even when testicular function is normal (65). Transgenic mice bearing a targeted inactivation of c-ros tyrosine kinase receptor fail to develop the initial epididymal segment and are infertile despite testicular germ cell production, and sperm transit through the epididymis appears unaffected (66). Mice lacking a functionally active retinoic acid receptor alpha are devoid of the distal epididymal epithelial cells and are infertile. Much remains unknown about the epididymis generally. The epididymis is important for the development of a fertile ejaculate and a functioning organ depends on both endocrine and lumicrine secretions from the testis.

The epididymis is a target organ for androgenic steroid hormones, but does not contain receptors for luteinizing hormone or FSH. A new study shows that although FSH receptors have not been demonstrated in monkeys, deprivation of FSH leads to decrease in epididymal weight (67). 5α-dihydrotestoterone is required for the ontogenetic development of epididymal structures. During adulthood, androgens control the synthesis, secretion and transepithelial transport of glycerophosphocholine, l-carnitine and myoinositol. Androgens also regulate the size of the epididymis. The development of sperm motility and velocity patterns is reduced under conditions of androgen deficiency (68), as is the time of passage of sperm through epididymal transit. Orchidectomy-induced loss of epididymal functions can be restored only partially by androgen supplementation, indicating the involvement of other additional luminal testicular factors influencing the epididymis. Other hormones such as aldosterone, progesterone, prolactin, oestrogens, endothelin1, oxytocin, melatonin, and vasopressin have been implicated in the regulation of the epididymis. Vasopressin enhances myoid cell contractions and aldosterone increases water resorption. Sperm emission and the ejection phases are regulated by an integrated and time-coordinated activity of the parasympathetic and sympathetic systems, which ultimately lead to sperm propulsion from the urethra. Endothelin1and oxytocin, with their receptors, act in an oestrogen-dependent autocrine and paracrine loop to regulate epididymal contractile activity at least partially (69). Oestrogen receptor α deficient mice show impaired fluid resorption in the efferent ducts.