Hypothalamus

Hypothalamic neurons receive input to secrete GnRH from neurons in other parts of the brain such as the amygdala and both the visual and olfactory cortex. GnRH output is affected by three forms of rhythmicity: seasonal, circadian, and pulsatile. The seasonal rhythmicity is measured on a timescale of months and peaks in the spring. The circadian rhythmicity results in testosterone levels peaking in the early morning hours. The pulsatile rhythmicity peaks on average every 90–120 min. The seasonal and circadian rhythms are controlled by melatonin signaling. This signaling comes from, respectively, the pineal gland and neural connections arising from the suprachiasmatic nucleus. The suprachiasmatic nucleus functions in mammalian species as an internal 24-h clock. The neurons that compromise the GnRH pulse generator have not yet been identified. During embryonic development, the precursors of GnRH neurons migrate from the olfactory placode to their positions in the hypothalamus. In cases of congenital hypogonadotropic hypogonadism, the GnRH precursor neurons migrate abnormally to the hypothalamus, which results in the diminished ability of hypothalamic GnRH secretion. This condition is known as Kallman’s syndrome, when hypogonadotropic function exists together with midline defects such as olfactory deficiency (anosmia) cleft palate or other midline defects.

Pituitary

The pituitary is composed of the posterior and anterior lobes. The posterior lobe, or the neurohypophysis, arises during development as a ventral outpocket of the hypothalamus. Neural stimuli promote the secretion of oxytocin and vasopressin, two neurohypophysial hormones. The anterior pituitary, or adenohypophysis, is a glandular structure regulated instead by blood-borne factors. Gonadotropes within the anterior pituitary secrete LH and FSH. The anterior pituitary also contains specialized cells for the secretion of other glycoprotein hormones, four of which have detectable effects on some components of male reproductive function. These specialized cells and their respective secreted glycoprotein hormone are: corticotropes which secrete adrenocorticotrophic hormone (ACTH), lactotropes which secrete prolactin (PRL), sommatotropes which secrete growth hormone (GH), and thyrotropes which secrete thyroid stimulating hormone (TSH). A murine investigation of the effect of ACTH on fetal Leydig cell steroidogenesis indicated a common embryonic origin of adrenocortical and Leydig cells from mesenchymal stem cells in the mesonephros [5]. However, the natural function of ACTH in the adult human male reproductive system remains unclear. LH, FSH, PRL, and GH, are four glycoproteins with known significant effects on male reproductive function. For instance, in cases of chronic over secretion of prolactin due to pituitary adenomas, the suppression of spermatogenesis has been observed [6].

The normal secretion of LH occurs on average once every ninety minutes to two hours, and the amplitude of each pulse is six International Units per liter [7]. The tonic level of LH in the blood stream is 10 International Units per liter. Normal testosterone levels are thus maintained at approximately 5 ng/mL [7].

Steroid Feedback on the Hypothalamus Pituitary

GnRH levels are regulated by a negative feedback mechanism predominantly involving testosterone. Testosterone suppresses the release of GnRH through androgen receptors present in both hypothalamic neurons and the pituitary. Aromatase and 5α-reductase metabolize testosterone into estradiol and dihydrotestosterone, respectively. Steroid negative feedback likely results from testosterone and DHT binding to androgen receptors and estradiol binding to estrogen receptors. Testosterone provides feedback primarily at the hypothalamic level, whereas estrogen provides feedback predominantly at the pituitary to regulate gonadotropic secretion in response to GnRH surges [8]. These findings reflect the profound influence of selective estrogen receptor modulators (e.g., clomiphene or tamoxifen) as well as aromatase inhibitors on the pituitary. These medications decrease estrogen-associated feedback to the pituitary, resulting in increased LH and FSH secretion. In the male, effects of gonadotropin secretion varies depending on the steroid. For example, testosterone exerts negative feedback on LH secretion primarily in its androgenic form, whereas testosterone exerts negative feedback on FSH secretion primarily by its aromatized form, estradiol. In other words, FSH secretion is predominantly regulated in the male by estradiol. Steroid hormone receptors also exist in various isoforms. An investigation of the effects of the estrogen receptor ERβ on the negative feedback of estradiol found no correlation [9]. Moreover, the A and B forms of AR vary in their ligand binding and transcriptional activation properties, but it remains unknown whether these two forms are differentially expressed in the hypothalamus [10].

Development of the Male Reproductive Axis

The development of the male body plan is determined at fertilization when a Y chromosome from the father’s sperm combines with an X chromosome from the mother’s oocyte. At the start of embryogenesis, male and female embryos are morphologically indistinguishable. Gonadal differentiation will occur during embryogenesis in discrete steps and begins with the thickening (or placode) of the coelomic epithelium on the primitive kidney (mesonephros). The placode develops into a gonadal ridge, followed by the migration of primordial germ cells from the yolk sac (allantois) into the epithelium.

Primordial germ cells use pseudopodial motion to migrate to the genital ridge and locate their correct positions using chemotactic signals and tracks of extracellular matrix proteins. During the indifferent gonad stage, the gonadal ridge then develops medullary cords composed of epithelial tissue. Differentiation of the ovarian and testicular pathways begins with the movement of the primordial germ cells into the medullary cords, which occurs at about seven weeks post-conception. Sertoli cell precursors surround the primordial germ cells, which are henceforth referred to as seminiferous cords. This transition of seminiferous cords and intra-cord spaces establishes the two compartments within the testis: seminiferous and interstitial.

The SRY (Sex-determining Region on the Y chromosome) gene controls the morphogenetic events of early testis differentiation. The SRY gene encodes for a nuclear transcription factor that acts in unison with other transcription factors, including WT-1, SOX-9, and DAX-1 to promote male sexual differentiation [11, 12]. The discovery of the SRY gene product as the testis-determining factor resulted from studies of Y chromosome deletions involving the SRY gene. Males with such Y chromosome deletions were phenotypically female. Likewise, the translocation of the SRY gene into an X chromosome conferred a male phenotype in a genetic XX female. Additional studies of the involvement of the SRY gen in testicular development have found that approximately 10% of 46 XX males have no detectable SRY gene (SRY-). Evidence suggests that some men who are 4It 6,XX (SRY-) have endogenous activation of downstream targets of male gonadal development such as SOX9 that allow this developmental pathway to “bypass” SRY activation [13].

Cells from the mesonephros migrate into the testis and provide the source of mesenchymal stem cells that will eventually become Leydig cells. The regression of the anlagen of the female reproductive tract structures is mediated by hormone signaling [14]. The hormone, Mullerian inhibiting substance, or anti-Mullerian hormone, is secreted by Sertoli cell precursors. Fetal Leydig cells, meanwhile, secrete testosterone, which stimulates the differentiation of the Woflian duct system. Later on in embryological development, this system will develop into the epididymis, vas deferens, and sex accessory glands. SRY also mediates steroidogenic factor-1 (SF-1) production, a transcription factor that induces the expression of cytochrome P450 steroidogenic enzymes in Leydig cells, and is also involved in differentiation of Sertoli cells and pituitary gonadotropes [15].

Endocrinology of the Testis

The hormonal control of testis function was thought to be a simple, two-compartment model: FSH stimulates Sertoli cells to nurture germ cells as they undergo spermatogenesis, while LH stimulates Leydig cells to release testosterone. Though this understanding holds true with respect to Leydig cells, mutational studies of males with decreased FSH action suggest that FSH may not be essential for spermatogenesis. One study found that male mice remained fertile despite knockout mutations in the FSH and FSH receptor genes [16]. These findings are corroborated by the fact that some men with defective FSH receptors remain fertile, though it should be noted that their sperm output is quantitatively reduced [17]. Recent studies suggest a negative correlation between testes function and increased conversion of testosterone to estradiol (reflected as the T/E-2 ratio). Although some of this excess conversion may occur because of relative clumping of Leydig cells within dysfunctional testes with limited seminiferous tubular tissue. In addition, this condition can be treated by using an aromatase inhibitor to reduce the conversion of testosterone to estradiol [18]. Transgenic studies in the rodent model have also suggested the importance of stem cell factor in spermatogenesis [19]. Stem cell factor is a local, or paracrine, signaling molecule in the testis that is secreted by Sertoli cells. The factor binds to the cell surface receptors of spermatogonia, spermatocytes, and round spermatids. The cell-cell interactions that promote germ cell differentiation within the seminiferous tubules suggest the complexity of the endocrine control of spermatogenesis. In order to provide a framework for understanding endocrine and paracrine controls, the cellular organization of the testis will be reviewed.

The highly specialized spermatozoa are produced and transported by the testis, epididymis, and ductus deferens to the ejaculatory duct. A spermatozoa is ready for ejaculation and fertilization only after undergoing weeks of changes post the initial mitotic division. During this incredible transformation, certain highlights include (1) initial mitotic divisions that maintain a set of stem cells relatively resistant to external injury as well as a population of rapidly proliferating germ cells destined to become spermatozoa; (2) meiosis, that occurs inside tight junctions between Sertoli cells that produce a unique intratesticular environment shielding the forming haploid gamete from systemic influences; and (3) the dramatic differentiation of the prospective gamete into a specialized cell ideally suited for transit through the female reproductive tract and ultimately, fertilization.

The spermatozoon will obtain its overall shape and size in the testis, but it is further modified both structurally and functionally as it passes through the epididymis before acquiring the ability to naturally fertilize an oocyte and the capacity for substantial intrinsic motility. Of note, sperm retrieved directly from the testis may have twitching motility and capacity to fertilize an oocyte if injected intracytoplasmically into an oocyte.

By the seventh week of gestation, Leydig cells differentiate from mesenchymal precursor cells in the connective tissue stroma of the testis between the seminiferous tubules. This event is detected by the presence of androgens in circulation. Leydig cell steroidogenesis occurs in conjunction with androgen-dependent differentiation of the male reproductive system. A gonadotropin similar to LH, human chorionic gonadotropin (hCG) is secreted by the placenta and thought to stimulate the development of fetal Leydig cells based on observations that Leydig cells differentiate in abnormal, amencephalic fetuses in which there is no internal LH secretion.

Fetal Leydig cell differentiation regresses after birth due to a lack of continuous hCG stimulation. However, two to three months post-birth, a second wave of Leydig cell differentiation occurs, driven by gonadotropin production from the neonatal pituitary that briefly elevates male infant testosterone levels. This neonatal surge of LH and subsequent testosterone has been suggested from anecdotal observations to hormonally imprint the hypothalamus, liver, and prostate such that they respond appropriately to androgen stimulation later in life. It is also thought that androgen production during this time period in neonate development also hormonally imprints the phallus and scrotum. Therefore, the absence of an androgen surge in a male neonate may impair normal development [20]. After this second wave of Leydig cell differentiation in neonate development, the Leydig cells regress and the testis will remain dormant during childhood.

The hypothalamus develops the ability to emit pulses of GnRH at puberty , which is around twelve years of age. At this time, the pineal gland decreases its nocturnal melatonin production, providing partial explanation for why pubertal onset of GnRH pulses tends to initially occur at night. The pattern of GnRH pulses matures so that pulses remain more frequent at night than during the day. The “gonadostat” hypothesis for puberty proposes in addition to the suppression of melatonin inhibition, androgen negative feedback is also delayed by converting testosterone to androgens such as androstanediol, which have a weak affinity for ARs, via enzymes such as 5α-reductase and 3α-hydroxysteroid dehydrogenase. These reactions delay androgen negative feedback until sufficient development of steroidogenic capacity of the testes.

Other factors that influence puberty are the growth rate and nutritional status of the body. For instance, there are clear stimulatory effects of growth hormone and its paracrine mediator, insulin-like growth factor-I (IGF-1), on reproductive function [21]. It is now theorized that leptin, an adipocyte hormone, affects the timing of puberty [22]. This hormone regulates the size of fat stores in the boy.. Once the body has acquired sufficient nutritional resources, reproductive development may occur. Though it remains unclear the exact mechanism by which leptin exerts control over reproduction, leptin has been shown to exhibit stimulatory effects on gonadotropin secretion [23]. In addition, leptin receptors present in the testis appear to exert inhibitory effects [24].

Aging of the Hypothalamic and Pituitary Axis

Serum testosterone levels begin to decline in men beyond 50 years in age. This declined output may result from the declining health status in older men, but aging also exerts a specific effect on hormone levels. Though basal levels of LH in the blood increase as men age, LH pulsatility is dampened, indicating that aging has an effect on the GnRH pulse generator. Moreover, the steroidogenic capacity of Leydig cells also decreases with age. Men older than 40 years of age have a lower fecundity, measured as a 50% lower probability of achieving a pregnancy with their partners within one year, compared to men younger than 25, possibly related to the fact that high testicular concentrations of testosterone are essential for maintaining spermatogenesis [25]. Therefore, male fertility may be impacted by the age-related decline in Leydig cell steroidogenesis. Accumulated oxidative stress in aging men may affect both sperm production and sperm quality as well [26].

Gross Structures and Vascularization

The average ovoid testis in healthy, young men measures at 15–22 cm³ in volume and 4.5–5.1 cm in longitudinal length [27, 28]. Testicular parenchyma is enveloped in a capsule composed of three layers that encompass the tunica albuginea with vascular, and contractile capacity as well as notable sensory capacity for pressure. Due to the fact that the testicular artery traverses the capsule at an oblique angle, smooth muscle control of the capsule may impact blood flow into the testis in not only man but also several other species [29]. The removal of the testicular capsule in the rate rete testis did not inhibit seminiferous fluid flow; therefore, it is unclear the extent to which capsular smooth muscle contractions influences the flow of seminiferous tubule fluid out of the testis [30, 31]. The testicular arties penetrate the tunica albuginea just medial to the caput epididymis (allowing cooling of the epididymis in its lateral position) and the vessels then travel inferiorly along the posterior surface of the testicular parenchyma, which then branches passing anteriorly in a variable transverse fashion over the testicular parenchyma. The inferior pole of the testis therefore has major testicular artery branches that makes the lower pole a poor site to place orchiopexy sutures. The vessels then, passing anteriorly and branching out over the surface of the testis. There is clinical significance to knowing the location of these vessels, as they may be injured during orchiopexy or testis biopsy procedures [32, 33]. In comparison with the anterior and inferior sections of the testis, the medial and lateral midsection contain few vessels. Individual artieres to tubules travel parallel to and within the septae that contain each seminiferous tubule, making dissection between seminiferous tubules feasible with preservation of blood flow to the seminiferous tubules—a factor utilized during the sperm retrieval procedure of microdissection TESE (testicular sperm extraction).

The testis is organized into compartments within the capsule that are separated by the septae. Each septum separates seminiferous tubules. Within the septum, there is at least one centrifugal artery. This organization is clinically important, as it allows microdissection to occur of testicular tissue, preserving the integrity and blood supply of seminiferous tubules within these septa. This is the principal for microTESE, where nearly all testicular tissue can be examined under an operating microscope. The individual seminiferous tubules that contain the developing germ cells and interstitial tissue. Interstitial tissue contains Leydig cells, mast cells, and macrophages as well as nerves, blood and lymph vessels. In humans, 20–30% of the total testicular volume is comprised of interstitial tissue in normal men [34] whereas men with severely defective spermatogenesis have up to 60–70% of the testis composed of interstitial tissue [35].

The seminiferous tubules are extremely coiled and looped, and their ends typically meet at the rete testis. It has been estimated that the 600–1200 tubules in the human testis that, when combined, would reach a total length of about 250 m [36]. The 6–12 ductuli efferentes are tubules formed from the rete testis through which testicular fluid and spermatozoa travel to the caput epididymis [37]. The identification of the efferent ducts allows an ideal site for microsurgical epididymal aspiration for azoospermic obstructed patients, including those with congenital absence of the vas. In men with the disease of cystic fibrosis, often the only component of epididymis that is present for these men.

There is no somatic innervation in the testis, but the testis does receive autonomic innervation primarily from the intermesenteric nerves and renal plexus, that are associated with the testicular artery to the testis, providing abdominal sensation of testicular pressure or inflammation [38]. As androgens suppress immune system function, interleukins acting in the brain during an infection could stimulate an appropriate immune response through the inhibition of Leydig cells. Other investigations suggest the nervous system exerts control over the vascular tone in the testis [39]. In man, the functional significance of testicular innervation remains unclear.

Approximately 9 mL of blood per 100 g of tissue circulate per minute in the human testicular parenchyma [40]. An investigation of blood flow to the testes in man found that blood flow to the left testis varies from 1.6 to 12.4 mL/per 100 mg per minute. Interestingly, blood flow to the right testis varies from 3.2 to 38.5 mL/per 100 g per minute [41]. However, it remains unknown the clinical significance of variation if blood flow to the testes.

Three reviews are recommended as excellent, in-depth discussions of the vasculature of the mammalian testis [34, 42, 43]. The human testis and epididymis receives blood from three arterial sources: the internal spermatic testicular artery, the deferential vasal artery, and potentially from the external spermatic or cremasteric artery [44]. The spermatic artery enters the spermatic cord above the inguinal ring. The artery develops from the abdominal aorta just below the renal artery. It is closely associated with a network of veins that come together to form the pampiniform plexus. Within the pampiniform plexus, the artery and veins counterflow and are at time separated only by their vascular walls. This vascular arrangement permits the exchange of heat and small molecules, such as testosterone that diffuses passively from the vein (low concentration) to the artery (high concentration) [45, 46]. The vascular arrangement in the spermatic cord enables the counter-current exchange of heat, which lowers the temperature of blood circulating to the testis 2–4 °C in comparison to the rectal temperature of a normal individual [47]. As a result, the intratesticular temperatures are measured to be 3–4 °C lower than rectal temperatures in health individuals [48]. The loss of this temperature gap has been linked to testicular dysfunctions in humans such as idiopathic infertility as well as varicocele and cryptorchidism [49–51].

Though the internal spermatic artery branches in some males before entering the testis, the interconnections between the internal spermatic and deferential arteries facilitate the maintenance of testicular viability. In 56% of the cases, a single artery entered the testis; in 31% of cases, two branches entered the testis; and in 13% of cases, three or more branches entered the testis [52]. These findings offer practical insight into the number of testicular arteries present in the spermatic cord at the inguinal level. Jarow et al. compared the number of arteries found in intraoperative dissections versus cadaveric dissections and reported an average of 2 arteries and 2.4 arteries, respectively [53]. Another study investigating the number of internal spermatic arteries in intraoperative dissections found using ×10–15 magnification that in over 100 spermatic cords, a single internal spermatic artery was present in 50% of cases, two arteries were found in 30% of cases, and three arteries were found in 20% of cases [54]. The branching of the testicular artery typically occurs as it passes through the inguinal canal [55, 56]. The larger testicular artery broanches into a series of centrifugal arteries that run along the septa, penetrating the testicular parenchyma. The testicular artery derives its blood supply predominantly from internal spermatic artery with some support from the deferential arteries. There is limited evidence of any direct support of the external spermatic vessels to testicular blood supply. In certain cases, up to 90% of the testicular blood supply has been found to derive from the testicular artery. In such cases, a hindrance in testicular blood supply may result in testicular atrophy [57]. The intertubular capillaries are within the columns of interstitial tissue, whereas the rope ladder-like peritubular capillaries run near the seminiferous tubules.

Several mechanisms regulate testicular blood flow. Myogenic activity of the testicular capsular may play a role in autoregulation of arterial blood flow [58]. While the total testicular blood flow is thought to remain fairly constant, flow at the regional level of the testis varies substantially to fit varying local metabolic needs. At least in rodents, LH may affect testicular as well as cytokine release [59, 60]. In addition, the microvasculature of the testis appears to be capable of highly specialized functions [61].

Intratesticular veins are unusual in that they do not run alongside their corresponding arteries. The small veins within the parenchyma empty either to the veins under the capsule of the testis or to a group of veins near the mediastinum that run toward the region of the rete [34]. These veins together with the deferential veins form the pampiniform plexus. The testicular venous system was described by Ishigami et al. as stagnate due to the spermatic veins being thin-walled and poorly muscularized. Excluding the inflow points into the inferior vena cava or the renal vein, the venous system was also described as lacking effective valves [62], an observation that leads to clinical varicoceles due to reflux of blood within a substantial proportion of men, especially infertile men.

The human testis contains prominent lymphatic ducts [63, 64]. These ducts originate from lymph capillaries, which are within the intertubular space and do not penetrate the seminiferous tubules. The fact that the obstruction of the lymphatic ducts in the spermatic cord causes dilation of the interstitium but not the seminiferous tubules suggests the extracellular space of the interstitium, but not the seminiferous tubules, is drained via the lymphatics. The hindrance of lymphatic flow can also result in hydrocele formation, which is a recognized complication of inguinal or spermatic cord surgery, including non-microscopic varicocelectomy [65].

Extracellular fluid flows from the seminiferous tubules through the rete to form the rete testis fluid. This fluid is transported into the caput epididymis. Rete fluid was thought to have originated from both primary secretions with the seminiferous tubules and epithelial secretions [66–68]. However, Setchell proposed “the majority of the fluid leaving the rete, originates in the tubules” [34]. Though the origins of the rete testis fluid remain debated, the fluid is a dilute suspension that is isosmotic with plasma and contains spermatozoa. Estrogen seems to have a regulatory effect on the reabsorption in the rete testis and efferent ducts. A study of rodents found that the knocking out of estrogen receptors (ERKO) impaired intratubular fluid reabsorption [69]. In addition, the resulting build-up of fluid within the seminiferous tubules was observed to result in seminiferous tubular dysfunction.

Rete testis fluid contains a unique composition of ions, carbohydrates, amino acids, and proteins in comparison to those in blood plasma or lymphatics. Setchell and Waites remarked “differences in composition between the fluid inside the seminiferous tubules and excurrent ducts of the testis and blood plasma or testicular lymph make it clear that substances do not diffuse freely into and out of tubules.” These findings contributed to the conception of the blood-testis barrier, which has been observed not only in man [70] but also in numerous species. The blood-testis barrier will be reviewed in further detail later in this chapter.

Cytoarchitecture and Function of the Testis

The Sertoli Cell

Both the nanostructure and morphological features of the human Sertoli cell have been thoroughly reviewed [122–124]. The defining features of the Sertoli cell are the uniquely shaped nucleus, prominent nucleolus, low mitotic index, Sertoli-germ cell connections, and the tight junctional complexes between adjacent Sertoli cell membranes that define the predominant component of the blood-testis barrier. The Sertoli cell is located on the basement membrane of the seminiferous tubule and projects filamentous cytoplasmic ramifications toward the lumen. Germinal cells exist between these cytoplasmic extensions. The undifferentiated spermatogonia can be found near the basement membrane of the seminiferous tubule, while the more differentiated spermatocytes and spermatids are located towards the higher levels of the epithelium. The Sertoli cell therefore acts as a polarized epithelium with its base located in a plasma environment and its apex in an environment unique to the seminiferous tubule [109].

It is proposed that Sertoli cells assist germ cell development with the following features: (1) the creation and maintenance of the adluminal compartment of the seminiferous epithelium, which functions as a unique microenvironment (2) the existence of gap junctions between Sertoli and germ cells (3) the facilitation of germ cell migration in the seminiferous tubule. The microenvironment is actually a component of the previously mentioned “blood-testis barrier.” This barrier is in fact found at various levels within the testis. Gap junctions entail the cytoplasmic connection of Sertoli and germ cells, and the specific junctions between Sertoli and germ cells include an “opening” and “closing” mechanism that promotes communication amongst cells and migration of germ cells to the adluminal surface [125].

One of the molecules Sertoli cells secrete is androgen-binding protein (ABP) [126, 127]. This protein transports androgens intracellularly within the Sertoli cell. The protein may also store androgenic hormones for the seminiferous tubule and potentially the epididymis. ABP also serves as a useful marker for in vitro analysis of the hormonal regulation of Sertoli cell function. There has not yet been a correlation found between the production of ABP or other known Sertoli cell products that mark Sertoli cell function, and male infertility [128].

In addition to ABP, the Sertoli cell also secretes components of the extracellular matrix, including lamin, collagen type IV, and collagen type I. Other secretory products include proteins such as cerulopasmin, transferrin, glycoprotein 2, plasminogen activator, somatomedin-like substabces, T proteins, inhibin, H-Y antigen, clusterin , cyclic proteins, growth factors, and somatomedin (see [125] for an additional review of Sertoli cell function). Sertoli cells also produce steroids such as dihydrotestosterone, testosterone, androstenediols, 17β-estradiol, and other C-21 steroids [109, 129]. While the functions of each Sertoli cell secretory product have yet to be fully revealed, such information may clarify how Sertoli cells generate the microenvironment in which spermatogenesis occurs.

Both FSH and testosterone have been observed to contribute to Sertoli cell function regulation, including the production of ABP [109, 130–133]. Inhibin B, a secretory product of Sertoli cells, is responsible for the feedback inhibition of FSH secretion in the human male. Both the molecular forms and the clinical values of inhibin B have been reviewed in the beginning of this chapter. Other regulatory molecules that enable maximal ABP secretion in vitro include progesterone, hydrocortisone, insulin, EGF, transferrin, and vitamins A and E [129]. Lastly, as previously mentioned, Sertoli cell function has been shown in vitro to be stimulated by testicular peritubular cell products [134, 135]. However, the exact mechanisms by which these effector molecules regulate Sertoli cell function as well as their physiological roles remains unclear. The production of feedback molecules including Inhibin B, and subsequent FSH suppression, from Sertoli cells appear to reflect both the number of Sertoli cells and germ cell/Sertoli cell complexes. So, men with normal volume testes and maturation arrest or Sertoli cell-only may still have normal inhibin B and low FSH levels.

Sertoli Cell–Germ Associations

Investigations on laboratory animals have revealed cell communication within the testis to be a complex network. Interactions between Leydig cells and Sertoli cells, between Leydig cells and peritubular cells, between Sertoli cells and peritubular cells (see previous discussion on peritubular structures), and between Sertoli cells and germinal cells have all been identified in investigative studies. For a more in-depth review of these complex interactions and the paracrine factors that regulate them, see [142]. This discussion will focus exclusively on Sertoli cell-germ cell interactions.

Sertoli cell-germ cell specialized junctions were once believed not to exist; however, it is currently accepted that are multiple types of Sertoli cell-germ cell associations within the mammalian testes [143–148]. For a more extensive review of this topic, see [142, 149, 150].

Russell [150] noted “desmosome-like contacts function as attachment devices that maintain the integrity of the seminiferous epithelium and at the same time assure that germ cells are transported in an orderly fashion, toward the tubular lumen by virtue of configurational changes of the Sertoli cell.” He also added, “ectoplasmic specializations are complex surface specializations that appear to hold elongated spermatids within deep recesses of the Sertoli cell.” Moreover, he remarked that that “both the Sertoli cell and spermatid participate in the formation of tubulobulbar complexes that appear in the few days preceding sperm release.” Russell interpreted such observations to indicate that spermatids lose the majority of their cytoplasm by Sertoli cell phagocytosis of tubulobulbar complexes.

The associations between Sertoli cells and germ cells may aid in the migration of germ cells upward towards the lumen of the seminiferous tubule. In addition, the associations between the condensing spermatid and the apex of the Sertoli cell during spermiogenesis is associated with the loss of residual cytoplasm from developing spermatid. Overall, it is clear that the junctions between Sertoli-cells plays an important role in forming the blood-testis barrier.

The Germinal Epithelium

About 123 × 10⁶ spermatozoa are produced in the human male on a daily basis [151]. Sperm production, or spermatogenesis, entails the division of spermatogonia either to both maintain their number (stem cell renewal) as well as to make cells that will develop meiotically. Daughter cells will become spermatocytes and undergo reduction division, which results in the production of haploid spermatids. The spermiogenic phase includes the morphological changes associated with the size and shape of the spermatid as it develops into a mature spermatozoa. The following discussion of spermatogenesis will a general one and references will not necessary be made to original research due to space limitations, lack of information pertaining to human research, and the overall complexity of the topic. The discussion will rely on outstanding reviews [109, 110, 152, 153].

Histological evaluation of the human testis demonstrate substantive numbers of germ cells organized among Sertoli cells and developing as they migrate from the basement membrane to the lumen of the seminiferous tubule. Morphologic analysis indicated at least 13 types of identifiable germ cells present in the testis. These different types of germ cells were thought to depict the different stages of the developmental process. The germ cells were named in the order of least to the most differentiated. The order is dark Type A spermatogonia (Ad), pale Type A spermatogonia (Ap), Type B spermatogonia (B), preleptotene primary spermatocyte (R), leptotene primary spermatocytes (L), zygotene primary spermatocytes (z), pachytene primary spermatocytes (p), secondary spermatocytes (II), and Sa, Sb1, Sb2, Sc, Sd1, and Sd2 spermatids.

Hormonal Regulation of Spermatogenesis

Testosterone levels in man and other mammals increase around 100-fold in the testis in comparison to that measured in the peripheral circulation [164, 165]. Clinical and genetic data indicate that some 46,XX (SRY-) men have activation of downstream targets of STY including SOX9 that allow the normal gonadal developmental pathway to “bypass” SRY activation [13]. Though testosterone and other GnRH agonists have been explored as potential male contraceptives, limitations to these treatments include their failure to completely inhibit FSH secretion by the pituitary.

Hypophysectomy, or the surgical removal of the pituitary, in many species [166] including man [167–169] results in testicular atrophy. The fact that intratesticular injections of testosterone microsphere almost fully recovered spermatogenesis rates in rats previously treated with GnRH agonists further stresses the significant role that testosterone plays in supporting spermatogenesis. Testosterone regulates sperm production through its effects on the Sertoli cell [170]. Androgens not only initiate but also maintain human spermatogenesis. A case study of a 6-year-old boy with an androgen-secreting Leydig cell tumor affirmed testosterone’s impact on spermatogenesis [171]. Despite the lack of gonadotropin production, spermatogenesis was occurring in the testicular tissue exposed to the tumor. The contralateral testis was not exposed to the tumor, and no sperm production was observed in this area. Such findings suggest sperm production was initiated and maintained by the androgenic steroids secreted by the tumor.

For young men with congenital hypogonadotropic hypogonadism, early treatment with hCG may optimize testicular growth and subsequent spermatogenic potential, whereas treatment with exogenous testosterone and subsequent adult administration of hCG does not result in the same quantitative sperm.

For more in-depth reviews of pituitary gonadotropins and their effects on sperm production, see [109, 110, 166, 172]. The only known effect that luteinizing hormone has been observed to have on spermatogenesis is the stimulation of endogenous testosterone production.

In contrast, the effects of FSH on spermatogenesis have been more highly contested over the years. Cases of human males with severely defective FSH receptors have been reported to be fertile, though testicular volume, sperm concentration, and morphology were all significantly reduced [17]. In addition, cases of men with hypogonadism that do not produce FSH were also recorded to be fertile. Though the mechanism by which FSH exerts these effects remains unclear, it has been proposed that it aids in the initiation of spermatogenesis in pubertal males and also in the reinitation of spermatogenesis post germinal epithelium regression in animals who have undergone hypophysetomy [109, 110, 166]. Though spermatogenesis can begin without the presence of FSH in cases of human males with hypogonadotropic hypogonadism, FSH enhances quantitative and qualitative sperm production treatment. Spermatogenesis can occur without FSH, but it is the combination of FSH and testosterone that enables quantitatively normal sperm production.

Genetic Bases of Spermatogenesis

There are many ongoing investigations seeking to identify genes critical for spermatogenesis. The fact that about 5–10% of azospermic men have been found to also contain microdeletions on interval 6 of the Y chromosome led scientists to focus their attention on this area as the location of a critical factor (Azoospermic Factor) for spermatogenesis [173, 174]. A specific gene referred to as DAZ (deleted in azoospermia) was located in the AZFc region of the long arm of the Y chromosome [175]. Other regions of the Y chromosome where deletions are strongly associated with azospermia are AZFa and AZFb region deletions. Complete deletion of the entire AZFa region has been linked uniformly to Sertoli cell-only and azoospermia. DBY, a DEAD-box protein that functions as a regulator in transcription, is a gene in the AZFa region of the Y chromosome that is deleted in all cases of severe impairments of sperm production, suggesting its critical role in maintenance of spermatogenic cells [176]. Moreover, the AZFb region of the Y chromosome has been identified as crucial to the completion of spermatogenesis. No men with complete AZFb deletions have had full spermatozoal development usable for assisted reproduction [55, 56]. It is likely that other genetic defects will cause defects in sperm production, and studies, at least in limited populations, have demonstrated common defects [177].

Recent investigations have found compelling evidence to suggest that certain male gamete characteristics may have a significant effect on embryonic development. In addition, studies have identified important inter-species variation of embryonic growth. In the case of human embryonic development, mitotic activity of the embryo seems to organized normally by the paternally-inherited centrosome [178, 179]. Observations of human embryonic development support the notion that sperm not only provide genetic material but also assist in regulating mitotic activity via the male gamete centrosome. Cases in which embryos have not inherited the male gamete centrosomes have entailed chaotic mitotic activity and the lack of viable embryos [180]. As a result, further investigations are needed in order to identify the exact components of the male gamete that are vital for normal embryo development.