Abstract
The male reproductive system consists of organs that function to produce, transfer, and introduce mature sperm cells into the female reproductive tract, where fertilization can occur (Figure 3.1). The initial development of the male reproductive organs begins before birth when the reproductive tract differentiates into the male form. Several months before birth, the immature testes descend behind the parietal peritoneum into the scrotum, guided by the fibrous gubernaculum. The testes and other reproductive organs remain in an immature form. They remain incapable of providing reproductive function until puberty when levels of reproductive hormones stimulate the final stages of their development (Figure 3.2). Prepubertal boys have no spermatogenesis; however, spermatogonia preserve in their testicles. Sexual maturity and ability to reproduce are reached at puberty. A gradual decline in hormone production and testicular cell count during adulthood may decrease sexual desire and fertility.
The male reproductive system consists of organs that function to produce, transfer, and introduce mature sperm cells into the female reproductive tract, where fertilization can occur (Figure 3.1). The initial development of the male reproductive organs begins before birth when the reproductive tract differentiates into the male form. Several months before birth, the immature testes descend behind the parietal peritoneum into the scrotum, guided by the fibrous gubernaculum. The testes and other reproductive organs remain in an immature form. They remain incapable of providing reproductive function until puberty when levels of reproductive hormones stimulate the final stages of their development (Figure 3.2). Prepubertal boys have no spermatogenesis; however, spermatogonia preserve in their testicles. Sexual maturity and ability to reproduce are reached at puberty. A gradual decline in hormone production and testicular cell count during adulthood may decrease sexual desire and fertility.
Figure 3.1 Male reproductive system.
Figure 3.2 Male reproductive hormonal axis. FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone.
Testes
The testes measure about 4 × 2.5 cm, with a volume of ~25 ml and a weight of 30–45 g each. The left testis is generally located approximately 1 cm lower in the scrotal sac than the right testis. A dense, white, fibrous capsule called the tunica albuginea encases each testis and then enters the gland, sending out septa that radiate through its interior, dividing it into ~200–350 cone-shaped lobules (pyramids). Each lobule of the testis contains interstitial cells (Leydig cells, fibroblasts, and macrophages), lymphatic and blood vessels, and one to three tiny, coiled seminiferous tubules, which contain Sertoli cells and germ cells. The tubules from each lobule assemble to form a plexus called the rete testis. Efferent ductules then drain the rete testis and enter the genital duct, the head of the epididymis (Figure 3.3).
Figure 3.3 Testicular structure.
Leydig cells.
In 1850, histologist Franz von Leydig (1821–1908), a professor at the University of Bonn, was the first to describe Leydig cells, which he observed surrounding connective tissue septa. Only at 1935 testosterone was isolated from these cells.
The Leydig cell numbers decrease markedly from 3 to 6 months of age to the end of the first year of life, and from 6 years onwards the number of Leydig cells progressively increase. The Leydig cells are presented in clusters, contain an amount of smooth endoplasmic reticulum, and are responsible, under stimulation by luteinizing hormone (LH), for testosterone secretion. LH binds to a G protein-coupled receptor on the Leydig cells and induces increases in cAMP levels, which subsequently leads to activation of the side-chain cleavage of cholesterol, as well as other events which culminate in increased steroidogenesis and the production of testosterone and other androgens from cholesterol. The pathway begins with the conversion of cholesterol to pregnenolone, which takes place in the mitochondria. Pregnenolone is then converted to testosterone by microsomal enzymes, and the testosterone is released into the circulation (Figure 3.4) and regulates its own production by negative feedback on the hypothalamus and pituitary to modulate or suppress LH secretion.
Figure 3.4 Secretion pattern of testosterone.
In the plasma, 54% of the testosterone binds to sex hormone-binding globulin (SHBG, which is synthesized in the liver), 45% binds to human serum albumin (HSA), and only 1% circulates as free testosterone. Testosterone bioavailability is represented by the free androgen index. In many tissues, testosterone is reduced to two other potent androgens, dihydrotestosterone (DHT) and 5α-androstenediol.
Leydig cells also produce oxytocin [1] and small amounts of estrogens [2]. Follicle-stimulating hormone (FSH) and prolactin receptors have been identified on the Leydig cells. Prolactin has been shown to enhance LH-stimulated testosterone secretion, while FSH regulates LH receptors, rendering prolactin and FSH stimulators of the Leydig cell response to LH [3]. FSH induces rapid hypertrophy and hyperplasia of interstitial cells that transform into mature Leydig cells.
In both testes of a 20-year-old male the number of Leydig cells is up to 700 million and diminishes by one-half by the age of 60 [4], and consequently, plasma testosterone levels decline [5]. No division of Leydig cells has been observed in the testes of adult men.
Peripheral production of estrogen increases (adipose tissue is capable of aromatizing testosterone to estradiol) with male age, resulting in a decreased androgen/estrogen ratio and increased estrogenic (female) effect. This is believed to underlie the development of benign prostatic hypertrophy and gynecomastia in elderly men. Aromatase inhibitors, e.g., letrozole and anastrozole, can decrease estradiol levels. Tamoxifen, an estrogen receptor antagonist, can disrupt the interaction between estrogen and its receptor, thereby decreasing the estrogenic effect.
Sertoli cells.
A regular testicle contains 600–1200 seminiferous tubules with a total length of approximately 250 m. Seminiferous tubules are made up of layers of Sertoli cells, germ cells, and myoid cells. The adult seminiferous tubule contains an epithelium of five to eight layers of Sertoli cells. On their basal side, each Sertoli cell has tight junctions (TJs) and basal adherens junctions (AJs), with all Sertoli cells side by side, creating the blood–testis barrier (BTB) [6]. The TJs integrate membrane proteins, such as occludin, claudin, and junctional adhesion molecules, which are stimulated by testosterone. The AJs include complexes of membrane proteins, such as cadherin–catenin, nectin–afadin–ponsin, integrin–laminin–actinin. Loss of one of these junction proteins could lead to loss of cell–cell adhesion. Cadmium chloride and other smoking products have been known for decades to induce infertility in males [7], and this effect may be mediated by BTB damage in the testis. The BTB must open periodically to permit germ cell transport, to ensure the successful and continual production of sperm cells, and also plays a vital role in spermatogenesis [6, 8].
FSH binds to its receptor on the Sertoli cells of the seminiferous tubules, increases cAMP levels, protein synthesis, and androgen binding in the tubules. The Sertoli cells produce the androgen-binding protein (ABP), which displays a high affinity to testosterone. ABP is secreted into the lumen of the seminiferous tubule and then transported to the epididymis, where it is taken up, and 80% is degraded. The remaining 20% is released into the blood, likely from the base of Sertoli cells. Sertoli cells also produce inhibin (α- and β-subunits), which serves as a negative feedback in the pituitary to regulate FSH, but not LH. Increased inhibin B levels may be initiated by ductal obstruction or anomalies within the seminiferous tubules.
During aging, there is a reduction in Sertoli cell numbers (from 600 million/testis at 20–48 years to 300 million/testis at 50–85 years), which can affect the integrity of the BTB [9], reducing the sperm production rate, motility, and morphology [10]. Loss of BTB function during aging is apparently irreversible.
Spermatogenesis
Primordial germ cells give rise to germ type A cells. Type A stem cells form additional type A spermatogonia and differentiate into type B spermatogonia cells all through early puberty. Type B cells differentiate during late puberty and in the adult form primary spermatocytes, secondary spermatocytes, and spermatids. These events initially occur through mitosis, after which, chromosome numbers are halved through meiosis. The spermatogonium (diploid, 2 n) is a most immature male germinal cell, and is located at the basal membrane of seminiferous tubules and undergoes mitotic division (six cycles) before migration through the TJ. The number of spermatogonia in prepubertal testes is influenced by the rate of proliferation, apoptosis, and differentiation into more advanced germ cells. The number of spermatogonia per tubular cross-section tends to decrease from 2.5 to 1.2 (from 30 × 106/cm3 to 19 × 106/cm3, respectively) over the first 3 years of life, followed by a two-fold increase until it peaks at 2.6 at the age of 6–7 years, then plateaus (48 × 106/cm3) until the age of 11 years, after which it rises to values of 7 (100 × 106/cm3), marking the onset of puberty [11]. Proliferation and differentiation of spermatogonia are induced by elevated FSH and LH, as well as increased inhibin B and testosterone secretion. FSH fuels the proliferation of immature Sertoli cells, causing the dispersion of spermatogonia across the elongating seminiferous tubules. Rapid spermatogonia proliferation is associated with increased levels of gonadotropins [12], the last wave of Sertoli cell proliferation followed by maturation [13–14], and enhanced germ cell differentiation, resulting in complete spermatogenesis [12].
In the human testis, three types of spermatogonium are usually considered: dark type A spermatogonia (Ad), characterized by densely staining chromatin and which serve as reserve stem cells; these cells divide (mitotically) into daughter, active stem cells and pale type A spermatogonia (Ap), which are characterized by palely staining granular chromatin and which mature (mitotically divide) into type B or differentiating spermatogonia (Figure 3.5). The classification is based on the nuclear features, the presence of mitochondria aggregations, and the presence or absence of glycogen granules. The proliferation of Ap spermatogonia occurs constitutively, independent of gonadotropin stimulation, whereas the differentiation of these cells into B spermatogonia is gonadotropin dependent and driven by either LH or FSH [15].
Figure 3.5 Spermatogenesis. Ad, dark spermatogonia; Ap, pale spermatogonia; B, type B primary spermatocyte; R, resting primary spermatocyte; L, leptotene stage spermatocyte; Z, zygotene stage spermatocyte; P, pachytene stage spermatocyte; II, secondary spermatocyte; Sa–Sd, spermatids at various stages of differentiation.
Type B spermatogonia are the precursors (mitotically divide) of primary spermatocytes, which are recognized by their large nuclei (chromosome set: 44+XY), into which they transform before doubling internal DNA and before entering the first meiotic division (stages of preleptotene [R], leptotene [L], zygotene [Z], and pachytene [P]) to produce secondary spermatocytes. Primary spermatocytes differentiate from type B spermatogonia behind the BTB at the basal compartment of the seminiferous epithelium and must traverse the BTB at preleptotene and leptotene stages (transitional stage from primary to secondary spermatocytes) and move into the adluminal compartment for further development. The new TJs are formed under the migrating preleptotene and leptotene spermatocytes. Every 16 days, preleptotene and leptotene spermatocytes enter the TJ of the BTB and give 256 mature spermatozoa into the ejaculate. Secondary spermatocytes go directly through the second meiotic division to produce haploid spermatids. Since neither DNA reduplication nor recombination of the genetic material occurs, the second meiosis can occur quickly (lasting around 5 hours). From this stage, no further divisions take place, and each spermatid begins its transformation into a spermatozoon [16]. This spermatogenic cycle takes ~62–65 days (Figure 3.5).
Spermiogenesis.
Next, the spermatids undergo spermiogenesis, which involves the Sa (formation of acrosome), Sb1 and Sb2 (nuclear changes), Sc (development of flagellum), and Sd1 and Sd2 (reorganization of the cytoplasm and organelles) stages. Residual cytoplasm, including mitochondria, undergoes phagocytosis by Sertoli cells and spermatozoa are released from the seminiferous epithelium into the tubule lumen, in a process termed spermiation (Figure 3.6). A small residual cytoplasmic droplet may remain attached to the spermatozoa. As the cell undergoes further maturation during transit across the epididymis, this cytoplasmic droplet migrates along the tail and is finally lost.
Figure 3.6 Spermiogenesis (from spermatid to spermatozoa).
During the elongating spermatid stage of spermiogenesis, sperm chromatin undergoes a complex transition in which histones are extensively replaced first by transition proteins and then by protamines (a process called protamination). At the nuclear level, histones, which are associated with DNA and preserve the DNA chain in a coiled configuration, are replaced by protamines leading to a highly compact condensed chromatin structure (Figure 3.7). Protamines (protamine 1 [P1] and protamine 2 [P2]) are 27–65 amino acid-long proteins, rich in arginine and cysteine, amino acids that are highly positively charged. The replacement of most histones by P1 and P2 facilitates the high order of chromatin compaction and packaging, due to their stronger binding than histones to negatively charged DNA. This compaction is necessary for normal sperm function and may also be necessary for DNA silencing and imprinting changes within the sperm cell [17]. P1 and P2 are usually expressed in nearly equal quantities (1:1), but abnormal P1/P2 ratios are observed in some infertile men and are often associated with severe spermatogenesis defects, DNA fragmentation, lower fertilization rates, poor embryo quality, and reduced pregnancy rates [18]. Elevated P1/P2 ratios have been taken as evidence of nuclear immaturity. In contrast to most mammals, whose spermatozoa contain only one P1 type of protamine, humans contain a second type of protamine (P2), which is deficient in cysteine residues. Therefore, human sperm chromatin has a potentially less stable structure than that of species that contain P1 only [19]. Transition protein is important for DNA condensation. Approximately 20% of the DNA in spermatozoon remains decorated by histones. It remains unclear which genes are compacted by histones and which by protamines, and what the physiological significance of the different compaction may be. This histones/protamines rate cannot be altered by exogenous hormonal treatment. It has already been shown that some infertile patients have anomalies in protamine content [20–21].
Mature spermatozoa bear a centrosome, which contains a pair of centrioles. The oocyte has none. Sperm centrioles are absolutely essential for the formation of the centrosome, which will form a spindle, enabling the mitotic division of the zygote.
At the end of spermiation, the seminiferous tubule contracts by myoid cells, in a hormone-dependent manner. Tubular fluid builds up pressure, and spermatozoa are released from the tubule and pass to the epididymis.
Figure 3.7 Sperm cell DNA compaction process. Substitution of canonical histones (spermatogonia) to protamines (spermatids).
Epididymis
The epididymis is a 3–4 cm-long structure with a tubular length of 4–5 m, whose primary functions include post-testicular maturation (enabled by the unique microenvironment within the lumen of the duct that helps transform immotile, immature testicular spermatozoa into fully mature spermatozoa) and storage of fertile spermatozoa in a viable state within the cauda with their passage from the testis to the vas deferens. Testosterone and DHT are the major androgens controlling epididymal function. Other participant in this process include prolactin, which increases the number of LH receptors [22] and uptake of testosterone by the caput epididymis. The microenvironment and lower temperature of 30–32°C are thought to be major contributors to sperm cell survival.
The epididymis is divided into three functionally distinct regions: head, body, and tail or cauda. Different proteins exist in the functionally distinct regions of the epididymis, and during sperm cell maturation, membrane lipids undergo distinct physical and chemical alterations [23]. Two major functions of the epididymis have been described: adsorption and secretion. Much of the testicular fluid that transports sperm cells from the seminiferous tubules to the epididymis is resorbed in the caput, resulting in the concentration of the spermatozoa increasing by 10- to 100-fold. The epididymal plasma, in which the spermatozoa are suspended within the epididymis, is secreted by the epididymal epithelium. The epididymal fluid is rich in glycosyltransferases and glycosidases. Variations in the lipid composition of the plasma membrane of sperm cells during maturation are thought to underlie the sensitivity of ejaculated spermatozoa to cold shock, when compared with testicular sperm.
One of the most important changes in the spermatozoa during epididymal maturation is the tail axonemal complex “maturity” and the development of sperm cell motility. The motility of mature spermatozoa is dependent on the intracellular cAMP generated by adenylate cyclase and on subsequent successive phosphorylation of proteins, including protein kinase A, A-kinase anchor proteins, and many others [24].
As many as half of the spermatozoa released from the testis die within the epididymis and are routinely resorbed by the epididymal epithelium (principal cells) after spermiophagy by macrophages.
Sperm take roughly 2 weeks to get through the epididymis (from caput to cauda). Sperm cells are put in storage near the tail portion and in the vas deferens until they are ejaculated. After prolonged sexual inactivity, caudal spermatozoa first lose their fertilizing ability, followed by their motility and then their vitality. This process is one of the reasons for the maximal 3-day sexual abstinence limitation prior to the collection of ejaculates for semen analysis.
Inflammation of the epididymis, epididymitis, is often caused by infection or by sexually transmitted diseases such as chlamydia. Gonorrhea frequently destroys the distal section of the epididymis and spares the caput. The vas deferens may be affected (sclerosis), in addition to the epididymis. Patients with epididymal obstruction present with semen containing no sperm cells (azoospermia) or semen with low sperm cell counts (severe oligozoospermia), and elevated serum FSH levels.
Hormonal control.
The male reproductive system is under tight hormonal control. Negative feedback mechanisms operate between the hypothalamus, the anterior pituitary gland, and the hormone-producing cells of the testes, i.e., Leydig cells producing testosterone and Sertoli cells producing inhibin.
Inhibin secretion by the Sertoli cell is stimulated by FSH and inhibited by LH, the latter presumably acting via Leydig cell-derived testosterone. Inhibin acts in negative feedback control of FSH release at the pituitary level, whereas testosterone acts in a negative feedback loop inhibiting LH secretion at the hypothalamic and pituitary levels. Like gonadotropins, prolactin also plays an important role in male reproduction. Low prolactin levels (hypoprolactinemia) have been associated with reduced ejaculate (seminal vesicles) volume in infertile subjects [25], erectile dysfunction, and premature ejaculation. Men with abnormally elevated prolactin levels present with gynecomastia, diminished libido, and erectile dysfunction. Prolactin inhibits the production of gonadotropin-releasing hormone, LH, and FSH. Elevated titers of plasma prolactin were shown to induce spermatogonia apoptosis [26]. Blockade of the portal veins by adenoma may prevent the hypothalamic prolactin-inhibiting factor from reaching the hypophysis and induce elevation of plasma prolactin levels.
Vascular System
The testicular arteries have their origin from three different blood vessels: (1) the abdominal aorta, which elongates to form the testicular artery as the testes migrate, (2) the inferior epigastric artery, and (3) the internal iliac artery, which supplies blood to the vas deferens.
The veins leaving the testis divide near the dorsal pole to form a venous plexus called the pampiniform plexus. The venous blood then drains into the spermatic vein. The right spermatic vein collects blood from the right testis to the inferior vena cava, while the left spermatic vein drains blood from the left testis into the left renal vein. This anatomical difference is important for the detection of varicocele, with those on the left side easily detectable. The pressure in the right spermatic vein is around 10 mm Hg. The pressure in the inferior vena cava is on average 0 mm Hg. The patient cannot raise hydrostatic pressure in the inferior vena cava above the pressure in the right spermatic vein and right spermatic vein backflow cannot be evoked on the right side, so right varicocele cannot be detected by palpation; however, it can be clearly seen on venography.
Arterial blood is cooled from body temperature to scrotal temperature by the venous blood, which leaves the testis at a temperature similar to that prevailing under the scrotal skin (scrotal skin has a very thin epidermis and is well supplied by blood vessels resulting in scrotal blood cooling). In the healthy male, the temperature of the scrotal skin is symmetrically distributed and does not exceed 32.5°C. This temperature has importance in normal spermatogenesis. Impaired testicular thermoregulation is commonly implicated in abnormal spermatogenesis and impaired sperm function with outcomes ranging from subclinical infertility to sterility.
Structure of Spermatozoa
Spermatozoa in the seminal fluid were observed for the first time under the magnifying glass and drawn by Anton van Leeuwenhoek (1632–1723) in 1667. Overall, sperm cell length is ~50–60 µm, including the head, mid-piece, and elongated tail. The mature spermatozoon has an oval flat-shaped, 4.0–5.5 µm-long, and 2.5–3.5 µm-wide head, with a pale anterior part (acrosome, 40–70% of the head area) and darker posterior region. The head has a highly compact package of genetic chromatin material. The cylindrical midpiece is ~7–8 µm long and characterized by a helical arrangement of its mitochondria, which provide energy for sperm locomotion. Spermatozoa can be motile under anaerobic conditions without mitochondrial activity due to the utilization of fatty acids. The tail is ~10 times the length of the head (~45 µm), and is divided into a principal piece, about 40 µm long, and a short end piece (5–10 µm in length).
The central portion of the sectioned sperm tail is a cylinder composed of nine dense doublet microtubules (periaxoneme), arranged around two single microtubules in the center (axoneme). The 9 + 2 arrangement of microtubules is also associated with dynein, which is an ATPase motor protein that hydrolyzes ATP and then undergoes conformational changes, forcing the microtubules to move past each other. The dense fibers dictate the structure that limits the flexibility of the flagella and allows the progressive movement (ahead) of a spermatozoon.
Ejaculate
During sexual arousal before ejaculation, the Cowper’s glands (bulbourethral exocrine glands located posterior and lateral to the urethra at the base of the penis) secrete a clear mucoid alkaline pre-ejaculatory fluid that may appear at the tip of the penis [27]. This fluid aids in urethral lubrication and neutralizes the acidity of the urethra in preparation for the passage of sperm cells. During intercourse, a short burst of activity in sympathetic adrenergic nerves liberates norepinephrine that stimulates α1 adrenergic receptors on smooth muscle cells in the cauda epididymides and vasa deferentia. This results in the peristaltic transfer of the contents of the distal cauda to the prostatic part of the urethra. The same mechanism is responsible for the emission of prostatic fluid in which the emitted spermatozoa are suspended. It also causes the normally delayed emission of seminal vesicular fluid. The sympathetic autonomic nerves also stimulate contraction of the smooth muscle tissue of the bladder neck, hindering the reflux of urethral contents into the bladder. Dilation of the urethra evokes a reflex (ejaculation) caused by the somatic nervous system, leading to rhythmical contractions of the striated bulbo- and ischio-cavernous muscles. These contractions increase the pressure in the urethra, and with the bladder neck closed, the contents are ejected out of the penis. The expulsion is divided into several fractions: generally, ejaculate is composed of the first fraction of prostatic fluids (~25%), and a second fraction containing both vesicular fluids (~65%) (seminal vesicles and vasal ampullae) and fluids from bulbourethral glands (~10%). Others divided ejaculate into six fractions [28]. A typical ejaculate contains 300 × 106 spermatozoa in 3 ml (Figure 3.8). The daily production is estimated at 80–100 × 106 spermatozoa. The alkaline pH of semen protects spermatozoa from the acidic environment of the vagina.
