The Testis

The testis lies within the scrotum and is covered on all surfaces, except its posterior border, by a serous membrane called the tunica vaginalis. This structure forms a closed cavity representing the remnants of the processus vaginalis into which the testis descends during fetal development (Figure 1). Along its posterior border, the testis is loosely linked to the epididymis which at its lower pole gives rise to the vas deferens.

Figure 1.

The relationships of the tunica vaginalis to the testis and epididymis is illustrated from the lateral view and two cross sections at the level of the head and mid-body of the epididymis. The large arrows indicate the sinus of the epididymis posteriorly. Reproduced with permission from de Kretser et.al.1982 in ‘Disturbances in Male Fertility’ Eds K Bandhauer and J Frick, Springer - Verlag Berlin.

The testis is covered by a thick fibrous connective tissue capsule called the tunica albuginea. From this structure, thin imperfect septa run in a posterior direction to join a fibrous thickening of the posterior part of the tunica albuginea called the mediastinum of the testis. The testis is thus incompletely divided into a series of lobules.

Within these lobules, the seminiferous tubules form loops, the terminal ends of which extend as straight tubular extensions, called tubuli recti, which pass into the mediastinum of the testis and join an anastomosing network of tubules called the rete testis. The seminiferous tubules are very long and coiled, with the average human testis containing approximately 500 tubules, each ranging from 30-70cm long (1). The adult mouse testis contains an average of 12 tubules, each with an average length of 134 ± 79mm, as measured by 3D image reconstruction techniques (2). Each tubule is connected to the rete testis at either end, however the tubules often contain branch points that arise prior to birth (2), likely during morphogenesis of the embryonic spermatogenic cords.

From the rete testis, in the human, a series of six to twelve fine efferent ducts join to form the duct of the epididymis. This duct, approximately 5-6m long in the human, is extensively coiled and forms the structure of the epididymis that can be divided into the head, body and tail of the epididymis (3). At its distal pole, the tail of the epididymis gives rise to the vas deferens (Figure 2).

Figure 2.

The arrangement of the efferent ducts and the subdivisions of the epididymis and vas are shown. Reproduced with permission from de Kretser et.al.1982 in ‘Disturbances in Male Fertility’ Eds K Bandhauer and J Frick, Springer - Verlag Berlin.

The arterial supply to the testis arises at the level of the second lumbar vertebra from the aorta on the right and the renal artery on the left and these vessels descend retroperitoneally to descend through the inguinal canal forming part of the spermatic cord. The testicular artery enters the testis on its posterior surface, sending a network of branches that run deep to the tunica albuginea before entering the substance of the testis (4). The venous drainage passes posteriorly and emerges at the upper pole of the testis as a plexus of veins termed the pampiniform plexus (Figure 3). As these veins ascend they surround the testicular artery, forming the basis of a countercurrent heat exchange system which assists in the maintenance of a temperature differential between the scrotally placed testis and the intra-abdominal temperature (5).

Figure 3.

The arrangement of the vasculature of the testis in the region of the distal spermatic cord and testis is shown. Reproduced with permission from de Kretser et.al.1982 in ‘Disturbances in Male Fertility’ Eds K Bandhauer and J Frick, Springer - Verlag Berlin.

The Distal Reproductive Tract

The vas deferens ascends from the testis on its posterior surface as a component of the spermatic cord passing through the inguinal canal. It descends on the posterolateral wall of the pelvis to reach the posterior aspect of the bladder, where its distal end is dilated forming the ampulla of the vas (Figure 4). At this site it is joined by the duct of the seminal vesicle, on each side, to form an ejaculatory duct that passes through the substance of the prostate to enter the prostatic urethra. The seminal vesicles and the prostate, the latter of which opens by a series of small ducts into the prostatic urethra, contribute approximately 90-95% of the volume of the ejaculate. During the process of ejaculation, these contents, together with sperm transported through the vas, are discharged through the prostatic and penile urethra. Retrograde ejaculation is prevented by contraction of the internal sphincter of the bladder during ejaculation. Failure of this sphincter to contract results in retrograde ejaculation and a low semen volume.

Figure 4.

The diagram depicts the relationship between the vas deferens, the seminal vesicles, the posterior aspect of the bladder and the prostate gland. The cytological features of the epithelium of the seminal vesicles is shown: this tissue is androgen dependent. Reproduced with permission from de Kretser et.al.1982 in ‘Disturbances in Male Fertility’ Eds K Bandhauer and J Frick, Springer - Verlag Berlin.

Spermatogonial Renewal and Differentiation

Spermatogonia are precursor male germ cells that reside on the basement membrane of the seminiferous epithelium. Spermatogonial stem cells (SSCs) divide to renew the stem cell population and to provide spermatogonia that are committed to the spermatogenic differentiation pathway. SSC are pluripotent and have the ability to differentiate into derivatives of all three germ layers (13,14). SSCs and spermatogonia exist within a niche that is responsive to cues from surrounding somatic cells and germ cells (15-17).

In general, two main types of spermatogonia, known as Type A and B, can be identified in mammalian testes on the basis of nuclear morphology (7). Type A spermatogonia exhibit fine pale-staining nuclear chromatin and are considered to include the SSC pool, the undifferentiated spermatogonia (A_undiff) pool, and spermatogonia that are committed to differentiation (A_diff). The A_undiff pool is comprised of the SSC, single A spermatogonia (A_s), and interconnected cysts of either 2 (known as A paired, or A_pr) or more (aligned or A_al) undifferentiated spermatogonia that remain connected by intercellular bridges. Once per cycle (see section Spermatogenesis: cycles and waves), the A_undiff cells transform into A_diff cells, which are then designated A₁, A₂, etc. A_diff spermatogonia ultimately divide to produce type B spermatogonia. Type B spermatogonia show coarse chromatin collections close to the nuclear membrane (18) and represent the more differentiated spermatogonia that are committed to entry into meiosis (19).

Research has focused on dissecting the molecular properties of the various type A spermatogonial subtypes to identify the SSC of the testis and to investigate their clonal behavior as they divide and differentiate. The pioneering technique of spermatogonial transplantation (20-23) has been key to understanding the stem cell potential of different spermatogonial subtypes. A_s can divide to renew their population or to produce A_pr cells, which represents an initial step towards differentiation. The A_pr cells divide to produce A_al cells which then divide, but do not undergo cytokinesis, to produce chains (or cysts) of spermatogonia, termed A_al4-16 (16). As the A spermatogonia subtypes progress through these steps and the chain length increases, there are changes in their molecular signatures, the expression of cell surface markers and other characteristics (24,25). Importantly, their propensity to become committed to differentiation also increases, with data in mice suggesting a linear relationship between chain length and ability to commit to differentiation (26) and that the microenvironment within the SSC niche is key to determining cell fate decisions in spermatogonia (15). A_undiff cells can reversibly transition between self-renewing and differentiation-primed states in response to cues from the niche (16).

There are likely different functional stem cell subtypes within the A_s population, such as active and reserve stem cells (16). There are several models of spermatogonial stem cell division and differentiation that have been gleaned from studies in rodents (16). In the more simplistic model, a subset of A_s cells are proposed to divide completely to produce single SSC with regenerative and self-renewal properties. However other models propose that syncytia of A_al spermatogonia can fragment into single cells which can then exhibit SSC behavior (27), suggesting that most or all A_undiff spermatogonia may be able to retain stem cell potential, particularly under conditions where regeneration of the spermatogonial population is required (16,27), such as during radiation or chemotherapy.

In humans and other primates, type A spermatogonia are classified into two subtypes, A dark (A_d) and A pale (A_p), based on morphology (19) but can be classified into 4 different states based on transcriptional differences (28). Some investigators have proposed that the A_d spermatogonia are similar to A_s in the rodent and thus represent the SSC or reserve spermatogonial population (29-31). Others have suggested that the A_p spermatogonia are the true stem cell of the testis (32), and type A_p spermatogonia show characteristics of A_s spermatogonia in rodents (33). Transcriptomics (e.g. (34-36)) and protein expression (e.g. (37)) of the spermatogonial subpopulations in humans may provide more information on the kinetics of SSC renewal and differentiation in men. For example, recent studies suggest that, in shorter lived species such as rodents, spermatogonia differentiate into SSC at birth, whereas a subpopulation of fetal-like SSC is maintained in humans, acting as a reserve population that are less susceptible to mutagenesis during the longer lifespan (38).

Meiosis

Meiosis is the process by which gametes undergo reductive division to produce a haploid spermatid, and in which genetic diversity of the gamete is assured via the exchange of genetic material (39). During meiosis I, DNA synthesis is initiated, resulting in a tetraploid gamete. The exchange of genetic information is achieved during meiotic recombination, which involves the induction of DNA double-strand breaks (DSBs) during pairing of homologous chromosomes and the subsequent repair of DSBs using homologous chromosomes as templates. Once the exchange of genetic material is complete, the cells proceed through two successive reductive divisions to yield haploid spermatids. This process is governed by genetically programmed checkpoint systems.

Meiosis commences when Type B spermatogonia lose their contact with the basement membrane and form preleptotene primary spermatocytes. Preleptotene primary spermatocytes commence DNA synthesis and the condensation of individual chromosomes begins, resulting in the appearance of thin filaments in the nucleus which identify the leptotene stage (40). At this stage, each chromosome consists of a pair of chromatids (Figure 6). As the cells move into the zygotene stage, there is further thickening of these chromatids and the pairing of homologous chromosomes. The further enlargement of the nucleus and condensation of the pairs of homologous chromosomes, termed bivalents, provides the nuclear characteristics of the pachytene stage primary spermatocyte. During this stage, there is an exchange of genetic material between homologous chromosomes derived from maternal and paternal sources, thus ensuring genetic diversity of the gametes. The sites of exchange of genetic material are marked by the appearance of chiasmata and these become visible when the homologous chromosomes separate slightly during diplotene. The exchange of genetic material involves DNA strand breakage and subsequent repair (41).

Figure 6.

The diagrammatic representation of the events occurring between homologous chromosomes during the prophase of the first meiotic division shows the period of DNA synthesis, the formation of the synaptonemal complex and the processes involved in recombination. Reproduced with permission from de Kretser and Kerr (1994) in “The Physiology of Reproduction” Ed E Knobil & J D Neill, Lippincott, Williams & Wilkins (7).

The diplotene stage is recognized by partial separation of the homologous pairs of chromosomes that remain joined at their chiasmata and each is still composed of a pair of chromatids. With dissolution of the nuclear membrane, the chromosomes align on a spindle and each member of the homologous pair moves to opposite poles of the spindle during anaphase. The resultant daughter cells are called secondary spermatocytes and contain the haploid number of chromosomes but, since each chromosome is composed of a pair of chromatids, the DNA content is still diploid. After a short interphase, which in the human represents approximately six hours, the secondary spermatocytes commence a second meiotic division during which the chromatids of each chromosome move to opposite poles of the spindle forming daughter cells that are known as round spermatids (19,42). Meiotic maturation in the human takes about 24 days to proceed from the preleptotene stage to the formation of round spermatids.

It is well known that advancing maternal age is associated with increased meiotic errors leading to reduced gamete quality. Studies suggest that paternal age is associated with increased defects in chromosome pairing, however no increase in aneuploidy is observed at metaphase II, suggesting that such errors are corrected during metaphase checkpoints in males (43). Since female gametes are arrested in meiosis I for many years, but paternal gametes only take days to complete meiosis, it is likely that age has more of an impact on aneuploidy arising during meiosis in females compared to males (39).

Spermiogenesis and Spermiation

The transformation of a round spermatid into a spermatozoon is a complex sequence of events accomplished by the process of spermiogenesis. No cell division occurs, but a conventional round cell is converted into a spermatozoon with the capacity for motility. The basic steps in this process (Figure 7) are consistent between all species and consist of (a) the formation of the acrosome (b) nuclear changes (c) the development of the flagellum or sperm tail (d) the reorganization of the cytoplasm and cell organelles and (e) the process of release from the Sertoli cell termed spermiation (7,44-48).

Figure 7.

The changes during spermiogenesis involving the transformation of a round spermatid to a mature spermatozoon are shown. Redrawn with permission from de Kretser and Kerr (1994) in “The Physiology of Reproduction” Ed E Knobil & J D Neill, Lippincott, Williams & Wilkins (7).

Acrosome formation begins when a series of granules from the Golgi complex coalesce. These granules migrate to the nuclear membrane where they form a cap-like structure which spreads over approximately 30-50% of the nuclear surface (44). Acrosome biogenesis begins with the formation of the acrosomal vesicle early in round spermatid development and progressively extends as a “cap” over the nucleus as round spermatids differentiate further (49) (Figure 7). The acrosome attaches to the nuclear membrane via the actin-based acroplaxome, and its biogenesis involves Golgi-derived proteins and endocytic vesicles being transporting along microtubule tracks and delivered to the developing acrosome as it spreads across the nuclear surface(50).

Once the acrosome is fully extended, round spermatids begin the elongation phase of spermiogenesis. At the beginning of this phase, the nucleus polarizes to one side of the cell (Figure 7) and comes into close apposition with the cell membrane in a region where it is covered by the acrosomal cap. Soon after, the spermatid’s chromatin starts to visibly condense, forming progressively larger and more electron dense granules together with a change in the shape of the condensed nucleus. This change in nuclear shape varies significantly between species. The condensation of chromatin is achieved by the replacement of lysine-rich histones with transitional proteins which in turn are replaced by arginine-rich protamines (51,52). The spermatid chromatin becomes highly stabilized and resistant to digestion by the enzyme DNAse. Associated with these changes is a marked decrease in nuclear volume and, importantly, the cessation of gene transcription (53). Therefore, the subsequent spermatid elongation phase proceeds in the absence of active gene transcription (47).

At the commencement of spermatid elongation, a microtubule-based structure known as the manchette is formed (50). This is a microtubule network that emanates from a perinuclear ring at the base of the acrosome and extends outwards into the cytoplasm. The manchette is closely opposed to the nuclear membrane, and facilitates nuclear head shaping, likely by exerting a force on the nucleus as it progressively moves distally towards the posterior portion of the nucleus (49,54-56).

The formation of the sperm flagella commences early in spermiogenesis (Figure 7) in the round spermatid phase, when a filamentous structure emerges from one of the pair of centrioles which lie close to the Golgi complex. The developing flagellum and the pair of centrioles become lodged in a fossa in the nucleus at the opposite pole to the acrosome. The central core of the flagella’s axial filament, called the axoneme, consists of nine doublet microtubules surrounding two single central microtubules, which represents a common pattern found in cilia (Figure 8). These doublet microtubules are comprised of various tubulin heterodimers and are associated with axonemal dyneins that hydrolyze ATP to generate axoneme movement, as well as other regulatory complexes that cooperate to regulate motility (57). The central axoneme extends throughout the flagellum (Figure 7).

The sperm flagellum is a fascinatingly complex structure (50,58-60). The connecting piece, also known as the head-tail coupling apparatus, is a small yet complex structure that connects the spermatid nucleus to the flagellum. In the middle piece, the axoneme is surrounded by outer dense fibers and the mitochondrial sheath (Figure 7). The outer dense fibers are important for axoneme function, including maintaining tensile strength and elasticity, and the helically arranged mitochondria provide ATP to facilitate motility and other metabolic support necessary for sperm function. The septin-based annulus forms during early spermiogenesis (Figure 7) and traverses the axoneme as it guides flagella development, until it ultimately resides between the middle piece and principal piece and functions as a membrane diffusion barrier (61). The majority of the flagellum is comprised of the principal piece (Figure 7) and is surrounded by the fibrous sheath that has multiple roles in sperm motility and function (62).The development of the flagellum during spermiogenesis is a complex, multiple-step process that involves a mechanism known as Intra-Manchette Transport (IMT), similar to Intra-Flagellar Transport (IFT) used in other ciliated cells. IMT involves proteins being “shuttled” from the spermatid nucleus down to the developing flagellum via molecular motors travelling along “tracks” of microtubules and filamentous actin and is essential for normal spermatid development (56,63).

The mature elongated spermatids undergo a further complex remodeling during spermiation, the process by which the mature spermatids are remodeled and then released from the supporting Sertoli cells (see section SERTOLI CELLS ) prior to their passage to the epididymis (46). This remodeling includes the removal of specialized adhesion junctions that have ensured tight adhesion of the spermatid to the Sertoli cell during its elongation process, further remodeling of the spermatid head and acrosome and removal of the extensive cytoplasm to produce the streamlined spermatozoon. The spermatid nucleus is “pushed” towards the lumen by the Sertoli cell extending its apical processes. Simultaneously, the spermatid cytoplasm remains stationary, so that the cytoplasm appears to flow down around the spermatid nucleus. Some observations suggest that prolongations of Sertoli cell cytoplasm send finger-like projections which invaginate the cell membrane of the spermatid cytoplasm and function to ‘pull’ the residual cytoplasm off the spermatid (44). This final removal of the spermatid cytoplasm during spermiation is important for determining the normal morphology of the released spermatids.

The final release of elongated spermatids at the end of spermiation is an instantaneous event and likely involves phosphorylation-dependent signaling cascades within the Sertoli cell resulting in changes in the adhesive nature of cell adhesion molecules (46), culminating in the Sertoli cell “letting go” of the mature spermatid (64). The morphological features of spermiation are relatively conserved between species, particularly among mammals (65). Spermiation is highly susceptible to perturbation by pharmacological modulators and by agents that suppress gonadotropins, reviewed in (46), and failure of spermiation can be recognized by the presence of mature elongated spermatid nuclei being phagocytosed by the Sertoli cells (66). Mutations in some genes expressed in spermatids can impair the ability of the Sertoli cell to remove the spermatid’s cytoplasm, leading to an inability of Sertoli cells to release the spermatid (46).

After spermatid disengagement, the remnants of the spermatid cytoplasm form the residual body. The residual bodies contain spermatid proteins and subcellular particles such as mitochondria, lipid and ribosomal particles. Residual bodies are phagocytosed and transported to the base of the Sertoli cell where they are broken down by lysosomal mechanisms. Some components of residual bodies are transported out of the Sertoli cell and across the basement membrane of the seminiferous tubule (67) into the interstitial fluid. This deposition of residual body components into the interstitial fluid likely facilitates the delivery of many spermatid-derived proteins into the interstitium, and, ultimately, into the circulation (68). An important function of these sperm proteins may be to interact with interstitial immune cells and promote peripheral tolerance against sperm-specific proteins (69). Because sperm appears in the body during puberty, long after establishment of immune tolerance, this promotion of peripheral tolerance to a variety of sperm proteins may provide a level of protection against an immune response in the case of damage or injury to the testis (68-70) (see section TESTICULAR IMMUNE CELLS).

Figure 8.

A cross-section through the developing mid-piece of the sperm tail shows the aggregation of mitochondria (arrows) surrounding the outer dense fibers (labelled 1-9) which in turn surround the axoneme composed of 9 doublet microtubules surrounding two central microtubules. Reproduced with permission from “Visual atlas of human sperm structure and function for assisted reproductive technology” Ed A.H. Sathanathan 1996.

Spermatogenesis: Cycles and Waves

Spermatogenesis occurs in a precisely ordered manner within the seminiferous tubules. A cross section of one seminiferous tubule reveals that a particular spermatogonial cell type is always associated with cells in a particular phase of meiosis and of spermatid development. These ordered cellular associations are referred to as stages and are denoted by Roman numerals (see Figure 9) (71). The stages are distinguished from one another based on recognizable differences in cell morphology. In mice there are 12 distinguishable stages, whereas in rats there are 14 (Figure 9). Human spermatogenesis was previously classified as having 6 stages (42,72), but a more recent study was able to discern 12 stages (73).

The completion of all recognizable stages is termed the spermatogenic cycle; e.g. the time taken for a single point in the seminiferous epithelium to progress through all stages. Another important consideration is that the stages are organized along the length of an entire seminiferous tubule in a largely consecutive manner; that is, one stage will usually be followed by the next, with the stages generally following a descending order distal from the rete testis (74). The space along the tubule occupied by a series of all stages, following one another sequentially, is called the spermatogenic wave (74) (Figure 9). Multiple waves can be visualized along one entire seminiferous tubule (2), with each wave releasing mature elongated spermatids during spermiation. The purpose of the wave is to generate continual pulses of sperm release to ensure the testes are constantly producing sperm. These pulses of sperm release along the length of the seminiferous tubules allow the testes to continually produce sperm, with the average normo-spermic man able to produce approximately 1000 sperm per heartbeat.

Interestingly though, the spermatogenic wave along the tubule is not perfectly arranged. In most seminiferous tubules, there are multiple sites at which the stages abruptly reverse, and continue in the opposite order, or even begin at a completely different stage (2,74,75). The direction of flow of released spermatids also shows abrupt changes in direction along the tubule, likely in part due to contractions of the surrounding peritubular myoid cells (76). Nevertheless, the constant waves of spermatid release and flow of fluid through the tubules towards the rete testis ensures the constant delivery of sperm to the epididymis.

Another important consideration is that the wave along the seminiferous tubules in marmoset and human testes is arranged in a more complex manner than lower order species. In lower order species, such as mice and rats, one wave is seen along the tubule, and one stage is observed at a cross-section in that tubule (Figure 9). In marmoset and human testes, however, several stages can be observed in one tubule cross section, because multiple waves at different stages intertwine along the length of the tubules, arranged in an irregular (77) or helical pattern (78,79).

Over the last decade, much has been revealed about the initiation and maintenance of spermatogenic cycle in mice, see (80) for a recent review. The spermatogenic cycle is initiated during the early postnatal period by the commitment of immature spermatogonia to differentiation, specifically the differentiation of A_undiff to A_diff spermatogonia. This key step is governed by a local pulse of retinoic acid (RA) within the seminiferous epithelium (80,81). During the initiation of spermatogenesis at puberty, the commitment of spermatogonia to meiosis is regulated by RA derived from the Sertoli cells (80). Spermatogonia differentiate and become committed to meiosis at various points along the tubule, determined by the local production of RA by Sertoli cells at that site (81,82). Therefore, the spermatogenic cycle is initiated by the timed, local production of RA by Sertoli cells. However, during subsequent cycles, once meiotic and post-meiotic germ cells appear in the epithelium, germ cells contribute to local RA synthesis within the tubule and thus can contribute to regulating the timing of entry of spermatogonia into the cycle (80,81). The transplantation of rat germ cells into mouse testes drives the spermatogenic cycle to proceed according to the kinetics of the rat (83), highlighting that germ cells contribute to the timing of the spermatogenic “clock”. Therefore, RA generated by Sertoli cells “sets the clock” during the first wave, however differentiating germ cells “fine tune” the timing of the clock during adult spermatogenesis (80,81,84,85).

It is thus evident that a cycle of spermatogenesis within a particular area, or segment, of a seminiferous tubule is driven by the RA-mediated differentiation of spermatogonia. Yet how the different segments along the length of a tubule co-operate to drive the orderly, sequential wave of spermatogenesis along the length of the tubule remains somewhat mysterious. In order to visualize how spermatogenesis becomes established, Ventela and colleagues transplanted green fluorescent protein (GFP)-expressing mouse spermatogonia into infertile mouse testes and examined the generation of GFP-labelled colonies for several months (86). They showed that spermatogonia could colonize a particular area of tubule and establish the spermatogenic cycle within that region. Along the length of the tubule, different colonies were established, with each colony cycling through the entire spermatogenic process. As time progressed, the colonies grew along the length of the tubules, each, continuing through their own cycle. Eventually, adjacent colonies fused and then appeared to begin to co-operate with each other, until the tubules began to exhibit the synchronous wave (86). These observations suggest that adjacent, independently generated colonies may communicate with each other to establish the spermatogenic wave, with RA being a key factor driving the cycle within each colony (80,81,85,87). However, given the ordering of stages along the tubule is often disrupted (2,74,75), the co-operation between adjacent colonies is likely imperfect.

A fascinating study suggested that Sertoli cells exhibit cyclic function along the length of the tubules in the embryonic testis, prior to the initiation of spermatogenesis, and even in the absence of germ cells (88). This study suggested that Sertoli cells along the tubule show innate variation in the expression of various proteins, including a retinol binding protein (88). Thus, it is possible that there could be a “pre-figured cycle” inside the Sertoli cells, generated prior to birth during the development of the fetal testis cords, and that this prefigured variation may drive local conditions that determine where spermatogonia become committed to meiosis at various sites along the tubule after birth. When and how this Sertoli cell clock is pre-figured in the embryonic testis is unclear (88) and its importance in contributing to the establishment of the spermatogenic wave in adulthood is yet to be established.

In summary, the spermatogenic cycle is initiated by a pulse of RA from Sertoli cells at particular sites along the tubules, and this RA stimulates undifferentiated spermatogonia to become committed to differentiation (80). Once meiotic and post-meiotic germ cells appear in the epithelium, they also regulate the timing of the spermatogenic cycle, likely by contributing to the regulation of the RA pulse (80,85), and perhaps via the production of other factors. The establishment of the spermatogenic wave along the length of the tubule involves the synchronization of cycles in adjacent segments via unknown mechanisms. It is possible that Sertoli cells exhibit subtle functional differences along the length of the tubules, determined prior to birth (88), that could contribute to the establishment of the spermatogenic wave.

Figure 9.

The top panel shows a diagrammatic representation of the stages of the seminiferous cycle in the rat and shows the types of germ cell associations which form the stages. The stage is denoted by roman numerals. These stages follow one another along the length of the seminiferous tubule, as illustrated in the diagram in the middle panel, however most seminiferous tubules exhibit areas where the stages do not precisely follow one another according to this cycle. Examples of the histology of the seminiferous epithelium at two different stages are given in the bottom panel.

Sertoli Cell Differentiation and Proliferation Determines Adult Spermatogenic Output

The testis originates from the genital ridge, a thickening of the ventral coelomic epithelium, during early embryonic development, and the indeterminate gonads become specified to differentiation into either the female or male pathway. The gonads become committed to testis differentiation via the expression of the Y chromosome-linked gene Sry (92). The SRY target gene, Sox9, then becomes expressed in pre-Sertoli cells and, along with other mechanisms, directs transcriptional cascades that promote Sertoli cell differentiation and further specification of the gonad to the male fate (92). Fetal Sertoli cells aggregate around the primordial germ cells and begin to form the seminiferous cords that become the seminiferous tubules after birth (93) and the proliferation of fetal Sertoli cells directs seminiferous cord development and length (94). Various studies have demonstrated that fetal Sertoli cell survival, proliferation and function is an important determinant of adult testis size and spermatogenic potential, reviewed in (70).

In rodents, Sertoli cells proliferate in both fetal and early postnatal life prior to puberty to establish the adult population (95). In humans there are two waves of proliferation; during the fetal and early neonatal period when the population increases 5 fold, and again prior to puberty when the population increases more than two fold (96), reviewed in (95,97). Postnatal Sertoli cell proliferation is regulated by numerous endocrine and paracrine factors, including follicle stimulating hormone (FSH), androgens, thyroid hormone, activin and growth factors (98-100) (also see section Endocrine Regulation of Sertoli cells) and is thus vulnerable to disturbance. Alterations to postnatal Sertoli cell proliferation or the cessation of proliferation at puberty alters adult testis size, (70,100). If Sertoli cell numbers are reduced during fetal or postnatal development, they do not appear to regenerate or increase their proliferation rate to compensate (101,102). Mouse models of Sertoli cell ablation demonstrate that the size of the Sertoli cell population prior to puberty is a strict determinant of adult testis size and sperm output (101,103).

Once Sertoli cells cease pre-pubertal proliferation, they begin to differentiate to attain the so-called “terminally differentiated” phenotype that will support germ cell development. The transition from proliferation to differentiation is thus a key switch in Sertoli cell development and can determine the timing of the onset of spermatogenesis as well as the number of Sertoli cells and germ cells (104,105). However, Sertoli cells can de-differentiate in certain conditions of impaired spermatogenesis, reviewed in (95). For example, a loss of claudin 11 (a protein involved in Sertoli cell tight junctions) causes Sertoli cells to remain proliferative during development and to lose their epithelial phenotype (106). De-differentiated Sertoli cells in cell cycle are not observed in normospermic men but are present in men after 12 weeks of gonadotropin suppression (107). Intriguingly, adult Sertoli cells can even trans-differentiate into granulosa cells in the absence of the Sertoli cell transcription factor Dmrt1; this activates Foxl2-mediated female somatic cell programming (108). Therefore, the maintenance of adult Sertoli cell differentiation is essential for normal spermatogenesis.

Sertoli Cell Structure and Function

Sertoli cells are highly specialized epithelial cells (Figure 10) that are indispensable for germ cell development, providing physical, metabolic and nutritional support at precisely timed intervals as dictated by the spermatogenic process, see (70,89-91,109) for reviews. Sertoli cells reside on the basement membrane of the seminiferous epithelium and are characterized by a basally located nucleus that shows deep indentations and a tripartite nucleolus (109). The Sertoli cell cytoplasm is large and complex, rich in microtubules and actin and intermediate filaments, with numerous cytoplasmic processes/projections that contact the four to five different types of germ cells (109).

Figure 10.

The general architecture of the Sertoli cell is shown. Note the thin cytoplasmic processes that extend between the germ cells. The Sertoli cell is in contact with a variety of germ cells and adjacent Sertoli cells when three dimensional perspectives are considered.

Sertoli cells interact with one another via a specialized junctional complex that forms between adjacent Sertoli cell plasma membranes towards the base of the cells (110,111). This complex contains different types of adhesion junctions, including gap, tight/occluding and adhesion junctions, along with a specialized actin-based structure termed the basal ectoplasmic specialization. The tight/occluding junctions prevent the diffusion of substances from the interstitium into the inner part of the seminiferous tubule (Figure 11). Because of the location of the junctions, spermatogonia have free access to substances from the interstitium (including the vasculature), however the germ cells “above” this junction, including meiotic and post-meiotic germ cells, have restricted access to factors from the interstitium. These junctions effectively divide the seminiferous epithelium into a basal compartment containing spermatogonia, which has free access to factors from the interstitium and an adluminal compartment containing meiotic and post-meiotic germ cells, the environment of which is strictly determined by the Sertoli cell. As preleptotene spermatocytes migrate from the basement membrane of the tubule into the adluminal compartment, the tight junctions open to allow them to migrate towards the lumen. At the same time, tight junctions reform beneath the preleptotene spermatocytes which have now left the basement membrane and are termed leptotene spermatocytes (Figure 11). The remodeling of these junctions to facilitate spermatocyte translocation coincides with the release of spermatids by the Sertoli cell during spermiation (112).

These inter-Sertoli cell junctions contribute to the so-called “blood-testis barrier” which creates a specialized environment within the seminiferous tubules. The Sertoli cell junctions and the blood-testis barrier are required for fertility (113). These junctions allow the environment of meiotic and post-meiotic germ cells to be precisely controlled by the Sertoli cell, enabling the timed delivery of factors uniquely required for germ cell development. The inter-Sertoli cell junctions exhibit differential permeability and can exclude different sized molecules depending on their stage and functional status (114), contributing to the stage-specific regulation of germ cell development.

Meiotic and post-meiotic germ cells develop at puberty, long after the establishment of immune tolerance after birth, and thus these germ cells could be recognized as “foreign” by the immune system. Germ cells in the adluminal compartment of the seminiferous tubules are protected from immune system recognition by the creation of an immune-privileged environment. This environment is in part mediated by specialized anti-inflammatory immune cells within the interstitium (115) (also see section Immune Cell Regulation of Immune Privilege and Tolerance) and the production of immunomodulatory factors, such as anti-inflammatory cytokines, by Sertoli cells (110). Junctions between Sertoli cells likely play some part in immune-privilege because a loss of androgen receptor in Sertoli cells results in increased permeability in the inter-Sertoli cell junctions and the immune-privileged status of the seminiferous tubules is compromised (116), However the seminiferous tubules continue to exclude immune cells when Sertoli cell junctions are absent (113) or even when Sertoli cells are ablated (103), suggesting that these junctions are not entirely responsible for immune privilege and that peritubular myoid cells may also play a role (70). Sertoli cells also release many germ cell-specific proteins into the interstitial fluid, likely via multiple mechanisms (68,117). While some germ cell-specific proteins remain sequestered within the seminiferous epithelium, others are released and can promote Treg-mediated immune tolerance (69,118). Therefore, Sertoli cells release germ cell-specific proteins to tolerize the immune system and to provide another layer of immune protection to the developing germ cells, reviewed in (117).

Figure 11.

Overview of the contribution of inter-Sertoli cell junctions to the blood-testis barrier. The diagram indicates the seminiferous epithelium with Sertoli cells (blue) and developing germ cells (orange). The position of the inter-Sertoli cell junctions is indicated (purple). Outside the seminiferous tubule is the interstitum (green shaded area), which contains interstitial cells and blood and lymphatic vessels. The inter-Sertoli cell junctions create a molecular weight barrier, preventing passive movement of proteins across them. These junctions effectively divide the testis into two compartments. The basal compartment has free access to substances from the interstitium and vasculature, whereas the adluminal compartment is strictly controlled by the Sertoli cell. The completion of meiosis and spermiogenesis occurs within the adluminal compartment. The inter-Sertoli cell junctions transiently remodel (junction remodeling) to allow leptotene spermatocytes to translocate from the basal to the adluminal compartments, whilst protecting the functionality of the barrier. Abbreviations: Sg = spermatogonia, Sc = spermatocytes, St = spermatids, eSt = elongated spermatids.

Sertoli cells continually change shape and structural features in response to germ cell maturation and the stage of the spermatogenic cycle. They respond to the needs of the developing germ cells by constantly changing the expression of many mRNA transcripts according to the stage of germ cell development (119). Adhesion junctions between germ cells and Sertoli cells change as germ cells mature (120) and the Sertoli cell cytoskeleton remodels as the stages progress (121). A specialized Sertoli cell adhesion junction termed the apical ectoplasmic specialization facilitates tight adhesion to the newly elongating spermatids, facilitating their translocation down towards the base of the epithelium as they elongate, and then transports them back up to the luminal edge as they complete spermiogenesis (121,122). At the end of spermatogenesis, Sertoli cells effectively push the mature elongated spermatid towards the luminal edge, stripping off the spermatid’s excess cytoplasm during the process of spermiation (see section Spermiogenesis and Spermiation), ultimately releasing the spermatid, into the lumen via changes in a specialized adhesion junction known as the disengagement complex (46).

Regulation of Germ Cells

Sertoli cells support germ cell survival, development and function during spermatogenesis (70). They provide structural and nutritional support, for example by providing lactate which is the preferred substrate for germ cell glycolysis (123). Sertoli cells produce many RNAs and proteins that direct the development of germ cells from their early specification in the fetal testis, through to the production of elongated spermatids in adulthood. The endocrine regulation of germ cell development is covered in other sections (Endocrine Regulation of Spermatogonia; Endocrine Regulation of Meiosis; Endocrine Regulation of Spermiogenesis and Spermiation) but some examples of the specific roles of Sertoli cells in regulating spermatogenesis are given below.

A key cell-fate decision in the early embryonic testis is the entry of germ cells into either meiosis and the female pathway of development, or the suppression of meiosis and the entry into mitotic quiescence and the male pathway of development. Sertoli cells direct the specification of germ cells into the male pathway by the expression of various factors that suppress the entry of germ cells into female meiosis (124,125). The Sertoli cells then produce factors that promote the entry of the male-specified germ cells (gonocytes) into mitotic arrest and maintain gonocyte mitotic quiescence throughout fetal life (97,126).

After birth, the gonocytes transition into proliferating spermatogonia that will ultimately provide the germ cell “reservoir” for adult spermatogenesis. Germ cell mitosis is re-activated and the gonocytes migrate to the periphery of the cords and acquire a spermatogonia-like transcriptional signature. This process is regulated by a fine balance of Sertoli cell-derived factors including stem cell factor (SCF) and platelet-derived growth factors (PDGF) (126). A subset of spermatogonia is then triggered to enter spermatogenesis by Sertoli cell-derived factors including RA and estradiol (81,126). Throughout spermatogenesis, the maintenance of the SSC niche is essential for continued sperm production (127). Sertoli cells are major contributors to this niche because increasing the number of Sertoli cells increases the number of SSC niches (128). Sertoli cells maintain the SSC niche by producing different factors, notably glial cell-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) (89,127).

Sertoli cells support the entry of spermatogonia into meiosis by producing RA in the first wave of spermatogenesis, and by contributing to a pulse of RA throughout adulthood (81,85,87). Sertoli cells also support the survival of spermatocytes as they proceed through the long meiotic prophase (129,130) and the final meiotic division into round spermatids (130). During spermiogenesis, Sertoli cells support the survival of round spermatids, particularly in the mid-spermatogenic stages (129,130). As spermatids undergo complex morphogenesis, they are supported by the formation of specialized adhesion junctions with the Sertoli cells (109). Interestingly, Sertoli cells project small cytoplasmic processes, known as penetrating processes, directly into the spermatid cytoplasm (131). The function of these processes is not known but they could presumably be involved in supporting the later phases of spermiogenesis when the spermatids cease transcription. The final phase of spermatogenesis, spermiation, is mediated entirely by the Sertoli cell (46). The Sertoli cell apical cytoplasm pushes the spermatid towards the lumen, removes the excess spermatid cytoplasm and ultimately releases the elongated spermatid (46).

Regulation of Other Testicular Cells

While it has long been known that a healthy Sertoli cell is required for germ cell development, it is now clear that Sertoli cells support the development and function of other testicular cells. Mouse models of acute and specific ablation of Sertoli cells have revealed the role of Sertoli cells in supporting the function of other testicular somatic cells (101-103).

Sertoli cells are essential for the fate and function of the peritubular myoid cells (PMC) (see section PERITUBULAR MYOID CELLS). During fetal and early postnatal life, Sertoli cells maintain the differentiation of PMC which in turn is necessary for the formation of seminiferous tubules (102). Once PMC have formed before puberty, they remain in the testis even after Sertoli cells have been ablated for up to a year, however Sertoli cells are required to maintain gene expression and function in adult PMC (103). Sertoli cells also produce endothelin in a stage-specific manner, which regulates contractility of the PMC at specific stages along the tubule (132). In turn, PMC supports various Sertoli cell functions (133).

Sertoli cells likely regulate the function of cells in the testicular vasculature, because the specific ablation of Sertoli cells reduces the total vascular volume of the adult testis as well as the number of vascular branches and microvessels (134). These defects result in a reduced fluid exchange between the testicular vasculature and the interstitium and reduced gonadotropin-stimulated circulating testosterone. These studies suggest that Sertoli cells maintain testicular vasculature functions important for the ability of Leydig cells to receive gonadotropin stimuli and/or the release of testicular testosterone into the circulation (134).

During fetal testis development in mice, Sertoli cells are required for the recruitment of monocytes and their differentiation into testicular macrophages (135). Sertoli cells are well known to produce immunomodulatory factors that contribute to testicular immune privilege (110,136,137). The ability of Sertoli cells to create an immunosuppressive environment is well established and underlies their potential therapeutic use in cell and tissue transplantation technologies, where they may be able to suppress host rejection (138). As well as depositing germ cell-derived antigens into the interstitium (see section Sertoli cell Structure and Function), Sertoli cells produce a vast array of cytokines and factors to regulate the phenotypes of testicular macrophages, dendritic cells and T cells to promote an immunosuppressive environment within the testis (138) and promote tolerance and innate immunity (137,139) (also see section TESTICULAR IMMUNE CELLS ).

Finally, it is now well understood that Sertoli cells have a major impact on the development and function of the testosterone-producing Leydig cells (70,140,141). In the fetal testis, the newly specified Sertoli cells produce factors required for the commitment of interstitial progenitors to the steroidogenic cell fate (142,143). After birth, adult Leydig cells (ALCs) differentiate under the influence of Wt1 expression in Sertoli cells (143,144). During the neonatal and pre-pubertal period, Sertoli cells direct the number of ALCs in the testis (101,102). In adulthood, Sertoli cells are important for ALC survival and the pulsatile release of testosterone (103,140).

Fetal Leydig Cell Development

Fetal Leydig cells (FLC) first appear after testis determination and, soon after, pre-Sertoli cells organize into the seminiferous cords, at approximately E12.5dpc in mice and week 8 of gestation in humans (126,148-150). FLC originate from the coelomic epithelium and mesonephros and are specified as NR5A1/SF1-expressing steroidogenic cells, that are prompted to differentiate by various signaling events, including factors produced by fetal Sertoli cells and endothelial cells (149-151). FLCs populate the interstitium where they reside in clusters, surrounded by basement membrane, in the interstitial space between the seminiferous cords. FLCs rarely proliferate, thus they populate the testis by differentiation from progenitors (147,150). Many factors regulate fetal Leydig cell specification, development and function (149-151). Fetal Leydig cells are transcriptionally and functionally distinct from adult Leydig cells (147,152).

In both mice and humans, FLC differentiation is regulated by Desert hedgehog (Dhh) and Platelet-derived growth factor A (Pdgfa). These factors are Sertoli cell-derived and act in a paracrine fashion via their respective receptors, Patched1 (Ptch1) and platelet-derived growth factor receptor A (Pdgfra), on fetal Leydig cells to stimulate differentiation and steroidogenesis (147,153-155). Fetal Leydig cells produce steroids necessary for androgen-dependent masculinization (see section Leydig Cell Steroidogenesis) as well insulin-like protein 3 (INSL3) that regulates testis descent (147,156).

Postnatal and Adult Leydig Cell Development and Regulation

After birth, the interstitium is repopulated postnatally by cells that become the adult Leydig cells (ALC) (147,151). Some FLC persist in the adult testis (149,151,157). These FLC constitute a minor percentage of Leydig cells and do not express the gene responsible for testosterone biosynthesis, HSD17B3, (158). Some of the FLC may contribute to vascular pericytes and seminiferous tubule-associated cells that have the potential to differentiate into ALC (149,151,157) (also see below).

ALC are generated from stem cells during puberty (149,151,157). These stem Leydig cells (SLC) are multipotent, capable of giving rise to other cell lineages (157). Distinct cell types can be discerned during postnatal development, including the SLC, progenitor Leydig cells, immature Leydig cells that do not make testosterone, and ALC with the capacity to synthesize androgens (157,159-161). An additional generation of Leydig cells appears during puberty in primates; these may contribute to a brief neonatal rise in androgens that could be involved in imprinting androgen-dependent target organs (157,160,162,163). The Leydig cells that appear during the early neonatal period are functionally immature until puberty, when they acquire an ability to produce testosterone and become ALC (164,165). The postnatal SLC arise from different fetal cell lineages, including FLC that de-differentiate prior to birth and persist in the postnatal testis (149,157,166). Under normal conditions, ALC are very long lived and do not appear to divide, but the population can fully regenerate when it is ablated (165). After ablation in adulthood, ALC regenerate from SLC, including those that are located around the outside of the seminiferous tubules and blood vessels (157). The SLC associated with the seminiferous tubules exhibit differences depending on the stage of the spermatogenic cycle, suggesting that their function can be influenced by factors from the seminiferous tubules (157).

In the adult testis, ALC exists within an interstitial cell niche, composed of ALC and abundant immune cells, particularly interstitial macrophages (see section Testicular Macrophages, Leydig Cells and Steroidogenesis). The function of ALC is highly dependent on the immune cells in this niche (see section Testicular Macrophages, Leydig Cells and Steroidogenesis). The niche is surrounded by telocytes in multiple species including humans (167). Telocytes are of mesenchymal origin and possess a small cell body and multiple long projections, and they are speculated to play multiple roles in maintaining functions of ALC and the interstitial cell niche (167).

As ALC are long lived, highly metabolic cells necessary for androgen production, studies have investigated the consequences and mechanisms of Leydig cell aging in various species. In the aged testis, ALC show morphological and functional changes, including elevated ROS, reduced mitochondrial function and reduced steroidogenic enzyme expression and output (168). Studies in mice suggest that it is not Leydig cell aging per se causing these changes, but rather age-related changes in the surrounding microenvironment (169), involving reduced LH signaling from the pituitary (168), altered functions of telocytes (170) and a subset of aged macrophages with a pro-inflammatory phenotype that could impact on the function of ALC in the interstitial cell niche (171). Testicular macrophages exhibit functional differences in aging men (172), suggesting they could contribute to ALC dysfunction during human aging.

Leydig Cell Steroidogenesis

The primary function of ALC is steroidogenesis to produce androgens. Leydig cells have the capacity to synthesize cholesterol, the substrate for steroidogenesis, from acetate or to take it up from lipoproteins (145,173). ALC contain abundant smooth endoplasmic reticulum and mitochondria which have tubular cristae that are unique to steroidogenic cells. The enzymes required for steroidogenesis are located in the mitochondria and in endoplasmic reticulum, thus intracellular transport of substrates between these organelles is necessary for androgen production.

Adult men typically produce around 7 mg testosterone daily but also produce lesser amounts of weaker androgens such as androstenedione and dihydroepiandrosterone. In addition to testosterone, through the actions of steroid 5α-reductase enzymes, the more potent androgen dihydrotestosterone (DHT) is produced by the testis in smaller amounts. Fascinatingly, a study using spatial mass spectrometry to localize androgens in the mouse testis revealed that testosterone was primarily localized within the seminiferous tubules but DHT was predominantly localized in the interstitium (174), however the mechanism responsible for this is unclear. The testis also contributes approximately 25% of the total daily production of 17β-estradiol through the local action of the enzyme aromatase which converts androgenic substrates to this estrogen (175), also see elsewhere in Endotext (176)). The remainder of the circulating estradiol is produced by the adrenal and peripheral tissues through the actions of aromatase.

The biosynthesis and regulation of testosterone production is covered elsewhere in Endotext (177). Briefly, LH produced by the pituitary (see section The Hypothalamic-Pituitary-Testis Axis) activates the G protein-coupled LH receptor (LHCGR) on Leydig cells to activate adenylate cyclase, leading to increased cAMP production and the subsequent activation of kinases (178). LHCGR activation regulates many signaling and transcriptional events in the ALC, including the expression of many transcriptional activators and repressors, to ultimately stimulate steroidogenesis (178). Upon LHCGR activation, cholesterol is mobilized into the mitochondria where the cytochrome P450 CYP11A1 enzyme produces pregnenolone in a key rate-limiting step. Testosterone is ultimately synthesized in a series of enzymatic steps via the so-called canonical, or classic, pathway of androgen biosynthesis (159). The final conversion of the androgen precursor, androstenedione, to the androgen testosterone is performed in the endoplasmic reticulum and is achieved by the rate-limiting reaction catalyzed by 17β-hydroxysteroid dehydrogenase enzyme (HSD17B) activity. Testosterone is converted to DHT by two steroid 5α-reductase enzymes, SRD5A1 and 2. Both enzymes contribute to the pre-pubertal production of DHT in the testis when testosterone levels are low, and then 5α-reductase activity declines as testosterone is produced at very high levels in the adult testis (179). Testosterone release is periodic, with substantial diurnal variation seen in adult men (180).

In ALC, HSD17B3 is the primary enzyme converting androstenedione to testosterone, and humans deficient in HSD17B3 exhibit phenotypes of disordered sexual development (181). However, mice lacking HSD17B3 exhibit sexual development, are fertile and continue to produce testosterone due to the activity of HSD17B1 in Sertoli cells during fetal testis development (182). In the adult mouse testis, HSD17B1 is no longer present, and HSD17B3 is the major enzyme present in ALC (159). However, in mice congenitally deficient in HSD17B3, the adult testis likely produces testosterone via the action of other hydroxysteroid dehydrogenases such as HSD17B7 and HSD17B12 (183).

Other pathways of androgen biosynthesis also exist in the testis. The so-called alternate pathway was first discovered in the tammar wallaby (184) and has since been reported in multiple species including mice and humans (185-187). This pathway utilizes various enzymes, including SRD5A1 and 2, to produce DHT independently of testosterone (188). Alternate pathway precursors are detected in the mouse testis and circulation and are up-regulated in the circulation in the absence of HSD17B3, indicating that the canonical and alternate pathways co-operate in adult male mice to maintain androgen bioactivity (188). The 11-keto androgens 11-keto testosterone and 11-keto DHT also exhibit androgen bioactivity (189). These may contribute to androgen action in the neonatal mouse testis (190) and 11-keto DHT is detected in the adult mouse testis (188). Interestingly 11-keto DHT is upregulated in the circulation of mice lacking HSD17B3 (188). Thus, the canonical, alternate and 11-keto androgen pathways of androgen biosynthesis co-operate, at least in mice, to maintain androgen bioactivity necessary for male fertility and sexual function (188).

It is also relevant to mention here that there are differences between FLC and ALC steroidogenesis. Fetal Leydig cell steroidogenesis is essential for androgen-dependent masculinization of the developing fetus. Fetal Leydig cells become steroidogenic around E13 in mice and 7-8 weeks of gestation in the human (150,191). In mice and humans, FLC produce the androgen precursor androstenedione, but the conversion to testosterone is accomplished by hydroxysteroid dehydrogenase activity in the Sertoli cells (147,150,161,192-194). Human fetal Leydig cells favor the Δ5 pathway of steroidogenesis, whereas rodent Leydig cells favor the Δ4 pathway (150). Like in ALC, human FLC steroidogenesis requires LHCGR stimulation, however in mice, fetal steroidogenesis is initiated prior to LHCGR expression (150) and testicular androgens can be produced in the absence of gonadotropins (195). Thus, fetal mouse steroidogenesis is less reliant on LHCGR-stimulation, however it could be stimulated by other G protein-coupled receptors as well as paracrine factors (150,152). Finally, it should be noted that, in mice, both HSD17B1 and 3 contribute to testosterone synthesis in the fetal testis (182), however the phenotype of disordered sexual development in human XY males with mutated HSD17B3 suggests that HSD17B3 is primarily responsible for testosterone production in humans (159).

Regulation of Leydig Cell Function by Other Testis Cells

Leydig cell development and function is critically dependent on other testicular cells. Early studies in rodents suggested that the seminiferous tubules influence Leydig cell number, maturation and testosterone production (196-198). As described earlier (see section Regulation of Germ Cells), Sertoli cells are an important regulator of Leydig cells, influencing the commitment of interstitial progenitors to the steroidogenic cell fate (142,143), the postnatal development and recruitment of ALC (101-103,143,144), ALC survival and the pulsatile release of testosterone in the adult testis (103,140).

In contrast, the ablation of germ cells from the testis has little effect on ALC steroidogenesis (141). However, when ALC are ablated from the adult testis, Leydig stem cells colonize the testis differently in the presence or absence of germ cells (199). Also, germ cell transplantation into mice lacking germ cells since birth stimulates ALC steroidogenesis (200). Thus, it is possible that germ cell-dependent mechanisms could support some aspects of ALC development and function (117)

Peritubular myoid cells (PMC) also appear to influence Leydig cell development and function. The deletion of AR from ~40% of PMC resulted in the adult testis containing two ALC populations that arose during postnatal development and could be differentiated from one another structurally and functionally (201). One population showed a marked reduction in the expression of various genes and other functional changes and appeared to undergo initial differentiation to immature Leydig cells, but did not complete their development into fully functional ALC. In contrast, the other population expressed steroidogenic enzymes and were functioning in a compensated state to maintain testosterone production. Thus, this study suggests that androgen-dependent signals in PMC influence postnatal ALC development and function (201).

Finally, the development and function of Leydig cells is highly dependent on the types of immune cells present in the interstitium, and particularly on interactions with the specialized interstitial macrophages (see section Testicular Macrophages, Leydig Cells and Steroidogenesis).

Types of Testicular Immune Cells

Resident immune cells in the testis are exclusively found within the interstitium and are prevented from entering the seminiferous epithelium under normal conditions. The testis contains both monocytes and lymphocytes. In mice, immune cells (CD45+ cells) comprise ~8% of the total testis cell population (223). Of these, approximately 80% are macrophages (monocytes) in mice and rats (136,224) and macrophages are the most abundant immune cell type in the human testis (136,225).

In mice, macrophages appear very early in testis development (226) and in the adult, the majority reside in the interstitium, associated with Leydig cells and the vasculature (termed interstitial macrophages) and the remainder associate with the outside of the seminiferous tubules (termed peritubular macrophages) (224,227). The interstitial and peritubular macrophages are morphologically, functionally and transcriptionally distinct and can be distinguished by protein markers, (115,136,219,228,229), for example, CSF1R is high in interstitial macrophages whereas MHCII is high in peritubular macrophages (227). It is now understood that multiple transcriptionally, developmentally and functionally distinct subpopulations of testicular macrophages exist (222,228-230). The majority of macrophages in the testis are of an anti-inflammatory phenotype, secreting high levels of anti-inflammatory cytokines and low levels of pro-inflammatory cytokines, whereas a smaller subset are pro-inflammatory macrophages that secrete IFNγ and TNFα cytokines and can induce an inflammatory response (137,219). Testicular macrophages are also highly phagocytic (231). Transcriptome analyses show that macrophages undergo functional changes and features of senescence in the ageing human testis (172) and studies in mice suggest that a novel macrophage subset is increased during aging and may contribute to a hostile proinflammatory microenvironment in the aging testis (171).

After macrophages, the next most common immune cell types in the mouse testis are lymphocytes, predominantly T cells and Natural Killer (NK) cells, however B cells are not present under normal conditions (136,224). T cells are also present in the human testis (136,225). In the mouse, CD3+ T cells are present in the testis at birth, in both interstitial regions and adjacent to the seminiferous cords (232). In adult mice, there are various subsets of T cells including cytotoxic T cells, T helper and regulatory T (Treg) cells (136,137). Mast cells are also observed in the testis of some species (136), and are particularly prevalent in humans (217). Dendritic cells are also found in smaller numbers throughout the interstitium (136). Finally, telocytes are extremely large cells with long cellular projections found in many tissues and have been described in the testicular interstitium of many species, including mice and humans (167) but their functional role is unclear.

Immune Cell Regulation of Immune Privilege and Tolerance

The testis is a distinct immune-privileged site where certain antigens are tolerated without inducing a detrimental immune response (139). The maintenance of testicular immune privilege is essential for testis homeostasis and the prevention of autoimmune responses, and testicular immune cells have an important role to play (115,137). Disruption to immune privilege leads to autoimmune diseases, orchitis (infection of the testis) and impacts on male fertility. Testicular immune privilege is a complex, active process that involves multiple levels of regulation (137) and both central and peripheral immune tolerance contribute to testicular immune privilege (136). See (136,137,139,220,222,228) for comprehensive reviews on this topic.

Briefly, the immune privileged environment within the testis is maintained by cooperation between various testicular cells, including the Sertoli cells (see section SERTOLI CELLS), germ cells, Leydig cells and immune cells (137). The testicular interstitium contains many immunosuppressive factors, including high levels of corticosterone, that may contribute to immune privilege (231). Approximately 80% of testicular macrophages secrete immunosuppressive factors and exhibit an immunosuppressive phenotype that contributes to testicular immune privilege (136,137). The interstitial macrophages secrete immunosuppressive factors such as IL10, whereas the peritubular-associated macrophages function to present Sertoli cell-derived sperm antigens to promote tolerance (228).

Dendritic cells, specialized antigen-presenting cells, function to minimize responses to autoantigens while maximizing responses against foreign pathogens, depending on their maturational status (137,233). In the testis under normal physiological conditions, dendritic cells are tolerogenic and contribute to the maintenance of immune privilege (136,137,233). Tregs are the main lymphocytes contributing to immune privilege in the testis that function to inhibit antigen-specific immune responses (137,139). Testicular macrophages may induce Treg expansion (231).

In summary, multiple cell types, including different immune cells, contribute to the maintenance of tolerance and immune privilege in the testis.

Testicular Immune Cell Response to Infection and Injury

As well as maintaining immune privilege, testicular immune cells must also be ready to respond to pathogens. To elicit a local response to pathogens, the testis has to overcome immune privilege and adopt effective innate immune responses. The testes have limited exposure to the outside environment, however they are a reservoir for microbes and viruses, particularly because they are predominantly comprised of seminiferous tubules that do not contain immune cells (234). Thus, the testes must find a balance between maintaining self-tolerance and being susceptible to infection.

Various insults to the male reproductive tract can compromise the immunological balance within the testis (136). Physical damage and injury can compromise immune privilege and lead to sperm antibody formation, as can surgical interventions such as vasectomy and testis biopsy. Reproductive tract infections involving the testis (orchitis) and/or epididymis (epididymitis) are also associated with sperm antibody formation. The formation of autoantibodies to sperm is the most common immunological dysfunction of the male reproductive tract and can impede fertility (136,235). In humans, isolated orchitis is usually associated with viral infections, such as mumps, whereas bacterial orchitis is usually caused by urethral pathogens and is almost always associated with inflammation of the epididymis (136). During orchitis, the immune response to infection causes a range of damage to the testis, such as fibrosis, ischemia and obstruction, reduced androgen production and increased risk of further testicular autoimmune reactions (136).

Although the testis is an immune-privileged site, it is remarkable in its ability to respond to infection or injury, and pathogens that invade from the blood or via the ascending male genitourinary tract are usually eliminated (136,139). The testis possesses highly effective local innate immunity systems that are a first line response to eliminate pathogens. These systems are complex and involve a multitude of factors, including defensins and pattern recognition receptors (136,139) as well as co-operation between testicular somatic cells, particularly Sertoli cells and Leydig cells, immune cells and germ cells (139). While innate immunity plays a fundamental role in the response of the male reproductive tract to infections, it also plays a wider role in male reproduction and various regulatory mechanisms are shared by the innate immune and the reproductive systems (136).

If innate immunity systems fail, the adaptive immune response pathway is activated (136). Broadly, these responses involve the appearance of antibodies and immune complexes in the testis, which is then followed by the infiltration and activation of macrophages and dendritic cells, and increases in various T cells (T helper, Treg and cytotoxic T cells) are also observed (115,136,219). Activation of the innate and adaptive immune systems can have ongoing detrimental effects on the male reproductive tract and can lead to tissue damage, autoimmunity and sub- or infertility (136).

Testicular Macrophages and the Regulation of Seminiferous Tubule Development and Function

Macrophages are found in all organs and perform both immune- and non-immune-related functions (222,236). As described above, macrophages comprise the majority of immune cell types in the testis and contribute to testicular homeostasis by their involvement in immune privilege and response to inflammation and infection. However, tissue-resident macrophages can also play important local roles in tissue development and function and thus it is pertinent to consider other roles for testicular macrophages.

Macrophages are first observed in the testis at embryonic day 11.5 in the mouse, at a time when all macrophages originate from the early yok sac (226). Fetal liver-derived monocytes appear later during embryonic development in the mouse testis (237). Some studies suggest that macrophages arising during the fetal period seed the interstitial macrophage population, and bone marrow-derived macrophages seed the peritubular macrophages during postnatal development (228,230) and others suggest fetal liver-derived macrophages predominantly seed both interstitial and peritubular cells (229). A more recent study suggests that fetal hematopoietic stem cell-derived monocytes colonize the murine testis later in fetal development and differentiate into adult testicular macrophages, and the peritubular macrophages differentiate later than interstitial macrophages (135). Fetal Sertoli cells, but not Leydig or germ cells, regulate testicular macrophage recruitment and differentiation during embryonic development (135). Various hypotheses exist in terms of the mechanisms governing the origin and steady state maintenance of testicular macrophages in the adult testis (222). In the adult testis, macrophages are extremely long lived with a slow turnover rate and low rates of proliferation (228,230).

Testicular macrophages appear to play important roles during embryonic testis development. In the fetal mouse testis, macrophages are of an M2, anti-inflammatory type, and are actively proliferating (226). These macrophages associate with the developing vasculature, with large numbers accumulating around areas of vascularization, and are present both inside and outside of the developing seminiferous cords (226,232). Their localization suggests a role in the clearance of germ cells as they migrate from the mesonephros, of Sertoli cells that fail to be incorporated into the developing cords and of endothelial cells during vascularization (226). Consistent with these roles, macrophage depletion during early embryonic testis development in mice causes defects in testicular vascularization and abnormal seminiferous cord development (135,226).

The roles of macrophages in regulating seminiferous tubule function in the postnatal and adult testis is less well understood (228). Peritubular macrophages preferentially associate with areas of seminiferous tubules containing differentiating spermatogonia and the transient depletion of most testicular macrophages impairs the proliferation and development of differentiating spermatogonia (227). Other studies suggest that the postnatal depletion of macrophages, via strategies that deplete all macrophages in mice, does not have a major effect on adult testis function under homeostatic conditions (229,238) though the global depletion of macrophage production from early in fetal life causes marked defects to spermatogenesis, with very few sperm produced in adult mice (229,239). Further studies using methods that exclusively deplete only macrophages in the testis, as well as specific subtypes of testicular macrophages (such as interstitial vs peritubular), are needed to better define the roles of these cells in normal seminiferous tubule homeostasis (222,228).

Testicular Macrophages, Leydig Cells, and Steroidogenesis

It has been known for many years that testicular interstitial macrophages and Leydig cells reside together within niches in the interstitial space (136,221,228,240-242). Estimates suggest one macrophage to approximately four Leydig cells, though this varies with species (136). Recent studies suggest the interstitial Leydig cell-macrophage niche is bordered by telocytes; these are extremely long cells that form a net-like structure enclosing the interstitial cell niche (243), however their functional relevance in the interstitium is not yet understood.

The Leydig cells and macrophages make close physical contact in the interstitial cell niches (136,221,228,240-242) and form gap junctions with one another (244). Strikingly, long tubular cytoplasmic processes of Leydig cells penetrate deeply into coated membrane invaginations within adjacent macrophages (242,245,246) and these are first observed between the cells at puberty (245) when Leydig cell steroidogenesis is initiated. These Leydig cell penetrating processes are not observed in ageing testes, however close contact remains between the Leydig cells and macrophages (247). Fascinatingly, a recent study showed that stem Leydig cells form long nanotubes with adjacent testicular macrophages and, under stimulation from ROS released from activated macrophages during inflammation, the stem Leydig cells transfer mitochondria along the nanotubes to the macrophages to support their function (248). Whether adult Leydig cells form such nanotubes and use them to communicate with macrophages is not known.

It is well known that Leydig cells and macrophages are functionally coupled in both the normal and inflamed testis. Many studies show that macrophages can influence Leydig cells, and vice versa, in the normal testis, during cell regeneration and in models of inflammation and injury, reviewed in (136,221,228,240-242).

Macrophages are important for Leydig cell proliferation and differentiation (136,141,221,228,240-242,249). The acute depletion of macrophages in prepubertal, but not post-pubertal, rats leads to reduced Leydig cell numbers, suggesting that testicular macrophages are important for prepubertal Leydig cell development (250). When adult Leydig cells are destroyed and then allowed to repopulate the testis, the depletion of testicular macrophages markedly inhibits Leydig cell repopulation from stem cells (251). Testicular macrophages produce factors that stimulate the proliferation and differentiation of Leydig cells, including 25-hydroxycholesterol, interleukins and transforming growth factor alpha (TGFα) (221,241).

In the adult testis, the transient depletion of testicular macrophages in vivo does not have an overt effect on Leydig cells numbers, but causes reduced testicular testosterone production (227), suggesting a role for macrophages in maintaining steroidogenesis in the normal testis. Testicular macrophages produce cytokines that can enhance Leydig cell steroidogenesis under certain conditions (141,221). Recent studies suggest that interstitial, but not peritubular, macrophages from the adult testis stimulate Leydig cell steroidogenesis (135). Testicular macrophages also produce 25-hydoxycholesterol, which can support Leydig cell steroidogenesis and is hypothesized to be used as a substrate for testosterone production (249). More recent studies have shown that testicular macrophages produce progesterone that is a precursor steroid in androgen biosynthesis (244). Thus, there is evidence to suggest that interstitial macrophages may support, and perhaps even contribute to, adult Leydig cell steroidogenesis under homeostatic conditions, however further research is needed (141).

In situations where there is infection or inflammation in the testis, macrophages become activated and can have suppressive effects on Leydig cells and steroidogenesis. Activated macrophages produce pro-inflammatory cytokines and reactive oxygen species (ROS) that can inhibit Leydig cell steroidogenesis (136,219,241). Acute and chronic inflammation in the testis results in excessive activation of macrophages and reduced Leydig cell testosterone production (136). In rodents, inflammation causes testicular macrophages to produce increased levels of pro-inflammatory molecules that inhibit steroidogenesis, including IL1, TNFα, nitric oxide, ROS and PGE2 (136). It is also important to note that inflammation can negatively affect steroidogenesis via the elevation of pro-inflammatory factors in the circulation and alterations to the hypothalamic-pituitary-testis axis (136). Thus, infection and inflammation can reduce Leydig cell steroidogenesis, and this suppression is in part regulated by the testicular macrophages.

Leydig cells also regulate testicular macrophages, consistent with the hypothesis that these cells are functionally coupled within the interstitial cell niche (136,249). Evidence in rodent models suggests that Leydig cells produce factors responsible for recruiting and maintaining testicular macrophages, and that the mechanisms responsible are dependent on stimulation of the LH receptor (LHCGR) on Leydig cells (136,249). For example, the suppression of LH in rodents, which reduces LHCGR activation in Leydig cells and suppresses steroidogenesis, leads to a marked reduction in testicular macrophages (136,252-254). The mechanisms by which Leydig cells influence macrophages are not known but are likely to be LHCGR-dependent, non-androgenic products and via direct cell-cell contacts (136,249,254).

Leydig cells also play a role in maintaining immune privilege and promoting innate immunity, either by producing immunomodulatory factors themselves, or by influencing the function of macrophages and other immune cells. Leydig cells secrete multiple immunosuppressive factors and produce androgens that regulate immune privilege and suppress immune responses to auto-antigens (139). They also exhibit innate antiviral abilities in response to viral infection, and express Toll-like receptors that trigger innate immune responses (139,255). Recent studies suggest focal localization of AR on the membrane of macrophages within the interstitial cell niche, suggesting they may be able to respond directly to Leydig cell-derived androgens (135). Androgens may regulate the activation of testicular lymphocytes via effects on testicular macrophages (137,256) and can directly regulate Treg cell expansion (257). Under inflammatory conditions, Leydig cell-derived androgens play a protective role by suppressing the production of pro-inflammatory cytokines by testicular immune cells and restricting macrophage and T cells migration to the testis (137).

In summary, testicular macrophages and Leydig cells are functionally coupled within the interstitium in the normal and inflamed testis. They communicate via direct interactions, including via unique structures that Leydig cells project into the macrophages, and via the production of different immunomodulatory factors. They co-operate to support their differentiation and survival, and, in the adult testis, they exhibit co-operativity in terms of Leydig cell steroidogenesis and LH-responsiveness, the maintenance of an immunosuppressive and immune-privileged environment, and in promoting innate immunity and the response to infection and inflammation.

The Hypothalamic-Pituitary-Testis Axis

Successful male fertility is dependent on the function of the hypothalamic-pituitary-testis axis (Figure 12). This axis is initiated with the pulsatile release of gonadotropin-releasing hormone (GnRH) from neurons in the hypothalamus and is mediated by kisspeptin/neurokinin B/dynorphin A (KNDy) neurons (259,260). The decapeptide GnRH is released into the hypophyseal portal system and is transported to the anterior pituitary. GnRH binds to its receptor (GNRHR) on gonadotrope cells in the pituitary to stimulate production of the gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH) (259,260). The pattern of GnRH-dependent secretion of FSH and LH are not identical (259). These gonadotropins stimulate testicular production of hormones that in turn regulate gonadotropin production via a negative feedback loop. The following section briefly explains the regulation of the hypothalamic-pituitary-testis axis in adult males and the reader is referred to more comprehensive reviews that also discuss the pre- and postnatal development and function of this axis (261-264).

Figure 12.

The endocrinology of the hypothalamic-pituitary-testis axis. GnRH is released from the hypothalamus and stimulates the production of the gonadotropins LH and FSH by the anterior pituitary. LH and FSH enter the testis via the circulatory system. LH targets receptors on Leydig cells to stimulate steroidogenesis and the production of testosterone (T). Testosterone acts on receptors within the seminiferous epithelium (including receptors in PMCs and Sertoli cells) to promote spermatogenesis. Testosterone is also released into the bloodstream where it feeds back on the hypothalamus and pituitary to negatively regulate gonadotropin production; this feedback is in part mediated by the conversion of testosterone to estradiol. FSH targets receptors within Sertoli cells to support multiple functions that are important for quantitatively normal sperm production. Abbreviations: PMC = peritubular myoid cells, Sg = spermatogonia, SC = spermatocytes, St = spermatids, eSt = elongated spermatids, Mɸ = interstitial macrophages. Image of brain courtesy of brgfx on Freepik.

LH acts on the LH receptor (LHCGR) on Leydig cells to stimulate the production of the androgen testosterone. Testosterone can be converted to estradiol via the activity of the aromatase enzyme. Testosterone can inhibit LH secretion directly without metabolism to estradiol or dihydrotestosterone (265,266) and it can act at the hypothalamus to decrease GnRH pulse frequency without a change in pulse amplitude (267). The non-aromatizable androgen DHT can partially suppress circulating LH and FSH in men (268) highlighting androgen-dependent negative feedback effects on both LH and FSH. Approximately a quarter to half of circulating estradiol is produced by the testis and the rest is produced via local aromatization of testosterone in various peripheral tissues (258,269). Estradiol has a negative feedback effect on pituitary gonadotropin production by regulating GnRH pulse frequency in the hypothalamus and LH pulse amplitude in the pituitary, the latter by reducing the sensitivity of gonadotropes to GnRH (269). A wide range of evidence suggests that both testosterone and estrogen mediate negative feedback effects of gonadotropin production, but that estrogen is the primary mediator, with testosterone having a minor but determinant role (258). Studies in mice suggest that androgen feedback on the pituitary influences prolactin but not gonadotropin production (270).

FSH acts on FSH receptors (FSHR) that are specifically expressed in Sertoli cells to regulate spermatogenesis (271). Testosterone and estradiol can negatively regulate FSH via effects on the hypothalamus (see above) however there are considerable mechanistic differences in the negative feedback regulation of FSH and LH. In response to FSH, the testis produces the peptide hormone inhibin B which enters the circulation and negatively regulates FSH, but not LH, production by the gonadotropes (272). FSH stimulation in the pituitary has been thought to predominantly involve the peptide hormone activin, produced within the pituitary and binding to activin family receptors in the gonadotropes, stimulating FSHβ subunit gene transcription (272). In turn, inhibin B from the testis is a negative regulator of FSH production via acting as an antagonist of activin receptors, thus preventing activin’s stimulatory effect FSHβ subunit gene transcription in gonadotropes (272).

However, a very recent study suggests that activin may not be the primary driver of FSH synthesis, at least in rodents (273). In mice, muscle-derived myostatin binds to a complex of TGFβ and activin receptors and stimulates FSH production in gonadotropes. Data from transgenic mouse models suggests that myostatin is a primer driver of FSH regulation in both males and females, with activin having a more minor role (273). Male mice lacking muscle-derived myostatin exhibited significantly reduced levels of FSH, but not LH, and reductions in testis weight and serum testosterone. This fascinating study thus suggests the existence of an endocrine feedback loop involving muscle effects on the pituitary to control FSH and provides a mechanism by which muscle signals to the pituitary to indicate reproductive fitness (273). Other endocrine systems impacting on the hypothalamic-pituitary-testis axis include adipose tissue-derived leptin impacting on FSH production (274) and the existence of a pancreas-bone-testis axis in mice (275).

Endocrine Regulation of Sertoli Cells

Sertoli cells are the orchestrators of spermatogenesis and many testis functions, playing key roles in establishing and maintaining the spermatogenic cycle and wave (see section Spermatogenesis: Cycles and Waves) and regulating the development and function of germ cells and other testicular cells (see section SERTOLI CELLS). The proliferation of Sertoli cells during pre- and postnatal development is a key determinant of testis size and ultimate spermatogenic output (see section Sertoli Cell Differentiation and Proliferation Determines Adult Spermatogenic Output). Sertoli cells respond to, and regulate, many endocrine stimuli arising from outside the testis. The following section briefly covers the main endocrine factors regulating Sertoli cells. The reader is referred to (258) for the roles of estrogen.

As described in section Spermatogenesis: Cycles and Waves, retinoic acid (RA) is key regulator of the spermatogenic cell cycle acting via Sertoli cells, reviewed in (80,85,276). Retinol stored in the liver is transported in the circulation via binding proteins. The actions of RA at a given site are regulated by a delicate balance between RA synthesis and degradation enzymes, RA binding proteins and receptors (80,85,276). Retinoic acid signaling is mediated through nuclear RA receptors (RARs) that bind to DNA and either activate or suppress target genes.

Within Sertoli cells, retinol is converted to the active metabolite RA via a series of enzymes. The first role of retinoic acid in Sertoli cells is during embryonic development where the expression of the retinoic acid degrading enzyme Cyp26b1 and other factors by early Sertoli cells (E12.5 in the mouse) controls the specification of primordial germ cells to commit to the male pathway of gene expression and meiosis (277). In the postnatal testis, RA is synthesized within the Sertoli cells, with a “pulse” of RA generated in stages VII-IX of the spermatogenic cycle (80,276) Mice lacking RARα expression in Sertoli cells exhibit a disrupted spermatogenic cell cycle, whereas the administration of exogenous RA to testes without advanced germ cells causes all Sertoli cells to “reset” to stage VII of the spermatogenic cycle (84). RA exerts its effects primarily via RAR alpha in Sertoli cells (278,279). This pulse of RA in the mid-spermatogenic stages is necessary for the entry of spermatogonia into meiosis (see below) but also regulates other important Sertoli cell functions occurring in these stages, notably sperm release (see (280) and references therein) and the formation and maintenance of the blood-testis-barrier (e.g. (281-283), reviewed in (85)). Thus, RA generation and action in Sertoli cells directs many Sertoli cell functions which are essential for spermatogenesis.

Thyroid hormone has an important role in the regulation of Sertoli cell proliferation which is a key determinant of testis size and sperm output. Thyroxine released by the thyroid gland is converted locally in the testis to the active hormone 3, 5, 3′-tri-iodothyronine T3 that activates its alpha1 isoform receptor (THRA) in Sertoli cells to control their proliferation during puberty (284). Specifically, T3 is a key signal to Sertoli cells to cease proliferation and commence terminal differentiation. This role of T3 is illustrated by the fact that hypothyroidism during puberty prolongs Sertoli cell proliferation, leading to increased Sertoli cell numbers, testis size and sperm output, whereas pubertal hyperthyroidism causes a premature cessation of proliferation, leading to reduced Sertoli cell numbers and sperm output in adulthood, reviewed in (284,285).

Sertoli cells, unlike germ cells, express receptors for androgens and FSH, and thus “transduce” the effects of these hormones to the developing germ cells. Spermatogenesis does not proceed in the absence of androgens, whereas spermatogenesis can proceed but is quantitatively reduced in the absence of FSH (reviewed in (198,285-287)). It is well established that both of these hormones are needed for quantitatively normal spermatogenesis. Both androgens and FSH have independent effects on Sertoli cells but also act co-operatively and synergistically to initiate and maintain normal spermatogenesis (see section Quantitatively Normal Spermatogenesis Requires Both Independent and Synergistic Actions of Androgens and FSH ) and, by inference, optimal Sertoli cell functions.

FSH acts as a mitogen for pubertal Sertoli cell proliferation and in the absence of FSH or its receptor, testes are smaller and Sertoli cell populations are reduced, as is sperm output (286,288,289). Thus, FSH supports postnatal Sertoli cell proliferation to establish a quantitatively normal population and, since Sertoli cell number determines sperm output (see section Sertoli Cell Differentiation and Proliferation Determines Adult Spermatogenic Output), is necessary for the production of normal numbers of sperm. During the establishment of spermatogenesis, a wave of germ cell apoptosis is important for establishing future spermatogenesis, perhaps by achieving a balance in the Sertoli cell:germ cell ratio (290). Since reductions in FSH at this time cause even greater apoptosis, it is possible that FSH acts on Sertoli cells to limit this apoptotic wave and establish normal spermatogenesis, reviewed in (286). FSH supports various Sertoli cell functions and their ability to support normal numbers of germ cells, as evidenced by reduced Sertoli cell-germ cell ratios in mice lacking FSH production or action (289,291,292). FSH can maintain germ cell development in gonadotropin-deficient men for 6 weeks (293) and has permissive effects on spermatogenesis in non-human primates and men, see (294,295). Therefore, FSH action on Sertoli cells is not essential for spermatogenesis but is required for normal Sertoli cell number and function.

Androgens, including testosterone and DHT, act on androgen receptors (AR) in the testis to support normal spermatogenic function. Androgens can act on the AR and produce the so-called classical signaling pathway, whereby ligand-bound AR translocates to the nucleus, binds to Androgen Response Elements (AREs) in the promoter region of androgen-dependent genes, and modulates transcription. This pathway produces a response hours after androgen stimulation. However, androgens can have much more rapid effects via non-classical pathways, involving AR-mediated intracellular calcium influx or activation of SRC and the ERK phosphorylation pathway, reviewed in (296,297). Both classical and non-classical pathways are active in Sertoli cells (296) and both are necessary for spermatogenesis (297,298).

In the absence of AR in Sertoli cells, no sperm are produced and spermatogenesis is arrested at the end of meiosis (130,299), highlighting that androgen action on Sertoli cells is needed for the completion of meiosis and spermiogenesis. Androgens regulate Sertoli cell number during pubertal development, reviewed in (286), and are a driver of Sertoli cell maturation; this latter requirement was demonstrated in transgenic mice with premature activation of AR expression in postnatal Sertoli cells, causing Sertoli cells to prematurely exit the proliferative phase and enter the maturation phase, leading to a reduction in Sertoli cell numbers (300). Thus, the precise timing of AR expression in Sertoli cells is important for normal testis development and optimal sperm output. Androgens are necessary for the formation of tight junctions between Sertoli cells which contribute to the blood-testis-barrier, reviewed in (286), and they drive the expression and translation of many genes expressed in the Sertoli cells themselves, and indirectly modulate the expression of genes in germ cells (e.g. (301)). Importantly, Sertoli cell morphology, function and androgen-dependent gene expression is impaired when AR is ablated from PMC (133), indicating that androgen action via PMC cells also supports Sertoli cell function and spermatogenesis.

Androgens and FSH have co-operative and synergistic effects on spermatogenesis (198,285,286,288,289) (see section Quantitatively Normal Spermatogenesis Requires Both Independent and Synergistic Actions of Androgens and FSH) and, since Sertoli cells are the only testicular cells to express both FSH and androgen receptors, some synergistic actions likely occur within the Sertoli cells themselves. Their ability to support germ cells is impaired when Sertoli cells lack expression of either FSH receptors or AR, however the effect is exacerbated when both receptors are depleted (302). Similar synergistic actions of FSH and androgen are apparent in terms of the ability of Sertoli cells to release mature sperm at spermiation (303). FSH and androgen signaling pathways can converge in Sertoli cells, for example in activating the MAP kinase pathway and elevating intracellular Ca²⁺ levels, reviewed in (304) and both hormones co-operate to modulate the Sertoli cell expression of particular miRNAs (305).

Endocrine Regulation of Leydig Cells

The primary endocrine regulator of adult Leydig cells (ALC) is LH, released by the pituitary, acting on LHCGR receptors ((178) and see section Leydig Cell Steroidogenesis). LH stimulation activates cAMP signaling and regulates intracellular calcium levels and other intracellular signaling cascades (178). Ultimately, LHCGR activation increases transcription of key proteins and enzymes that enable the conversion of cholesterol into testosterone via a series of enzymatic steps in the mitochondria and, subsequently, in the endoplasmic reticulum.

Fetal Leydig cells (FLC) produce the precursor androgen androstenedione which is converted to testosterone in the fetal Sertoli cells (147,150,161,192-194). Fetal androgen production is initiated prior to the activation of the hypothalamic-pituitary-testis axis in both rodents and humans, however this axis plays a more important role in steroidogenesis later in gestation (150). Various observations suggest species-dependent differences in terms of the signals that initiate FLC steroidogenesis, with human steroidogenesis more reliant on activation of LHCGR than mouse steroidogenesis, reviewed in (150). Interestingly, studies in mice suggest that the stimulation of steroidogenesis during the fetal period could also involve Corticotropin-releasing hormone (CRH) and Adrenocorticotropic Hormone (ACTH) action on their respective receptors that are expressed in FLC but not ALC (152).

Like Sertoli cells, Leydig cell proliferation and differentiation can also respond to alterations in thyroid hormone levels during development, as demonstrated in animal models of neonatal hypo- and hyperthyroidism (306,307). Broadly, transient neonatal hypothyroidism arrests postnatal Leydig cell proliferation but promotes proliferation of precursor cells, whereas hyperthyroidism stimulates the differentiation of Leydig cell precursors, thus leading to reduced or increased Leydig cell numbers and steroidogenic output, respectively (306,307). The timing of thyroid hormone manipulation during the early postnatal period is an important variable (308). Manipulation of thyroid hormone action on Leydig cells in vitro can also modulate steroidogenesis (306,307). Whether the effects of thyroid hormone on Leydig cells are direct or are mediated by Sertoli cells is not entirely clear (306). Thyroid hormone directly regulates neonatal Sertoli cell proliferation and differentiation (see section Sertoli Cell Differentiation and Proliferation Determines Adult Spermatogenic Output) and the number of Sertoli cells directs Leydig cells numbers and development (101-103). Thus, it seems likely that at least part of the impacts of altered thyroid function on Leydig cells during development are via Sertoli cells (306). Finally, it is worth noting that most hypothyroid men with concomitant hypogonadism have normal LH and FSH levels, suggesting their hypogonadism is not due to a primary defect in Leydig cells (309).

Finally, it is worthwhile considering the effects of glucocorticoids on Leydig cells. Elevated circulating levels of glucocorticoids, as a result of illness or pharmacological administration, are well known to be associated with reduced testosterone production. Stress-induced glucocorticoids can impact the hypothalamic-pituitary-testis axis, and the ability of excessive glucocorticoid activity to suppress Leydig cell function, steroidogenic gene expression and testosterone production is well established (310). Conversely, Leydig cell steroidogenesis requires endogenous glucocorticoid action, because a reduction in glucocorticoid receptors (GR) specifically in Leydig cells leads to reduced expression of the LH receptor (LHCGR) and steroidogenic enzymes (311). Interstitial fluid of adult mice contains high levels of corticosterone, which is also produced by testicular macrophages (231), and thus local testicular production of glucocorticoids could modulate Leydig cell function. Taken together, the evidence suggests that Leydig cell steroidogenesis is responsive to glucocorticoids but requires a delicate balance of GR activation, and that excessive GR activation leads to reduced Leydig cell function and steroidogenesis.

Endocrine Regulation of Spermatogonia

Vitamin A from the circulation is metabolized to RA within the seminiferous tubules to produce a pulse of RA that acts on receptors within target cells. As discussed earlier (see section Spermatogenesis: Cycles and Waves), this pulse of RA stimulates spermatogonial differentiation. RA from Sertoli cells acts on receptors within spermatogonia at stages VII-VIII of the mouse spermatogenic cycle, directly stimulating undifferentiated spermatogonia to become committed to differentiation (80,81,312). As A_s spermatogonia divide to produce chains of spermatogonia, joined by intercellular bridges, they show an increasing ability to respond to RA and become committed to differentiation, whereas SSCs are protected from the RA pulse by cells within the SSC niche (15,25,26). The pulse of RA is generated within the Sertoli cells (313) and activates receptors in spermatogonia to co-ordinate a complex gene expression program (276). This includes the induction of STRA8 that directly controls the transcription of many genes and is required for spermatogonial differentiation (276).

Evidence suggests that GDNF promotes SSC self-renewal while RA promotes spermatogonial differentiation (127,314,315). Because RA directly suppresses Sertoli cell GDNF expression (316), there is likely co-operativity between these two factors during the spermatogenic cycle to maintain the balance between spermatogonial renewal and differentiation that is essential for continued spermatogenesis.

Spermatogonia lack receptors for both FSH and androgen and therefore actions of these hormones must be indirect, via Sertoli cells and/or other testicular somatic cells. In rodents, spermatogonial development is not particularly susceptible to a loss of androgens and spermatogonia can enter meiosis in the absence of androgen action on Sertoli cells (e.g. (130,317)). Ablation of AR within PMC results in reduced numbers of spermatogonia (133). This reduction could be due to a loss of androgen-dependent PMC cell-mediated effects, perhaps PMC-derived GDNF (318) and/or a consequence of the high testicular testosterone concentrations in this model having inhibitory effects on spermatogonia, as has been noted in other studies (319). Interestingly, SSC renewal is enhanced when LH and testicular androgens are suppressed, an effect mediated by Sertoli cells (320); perhaps this mechanism could preserve the SSC pool in situations where endocrine factors are temporarily compromised.

In contrast to androgens, spermatogonia are more sensitive to FSH in rodents and primates (289,317,321,322). FSH can prevent spermatogonial apoptosis (323), however observations in mice lacking FSH production or action suggest that FSH is not essential for spermatogonial differentiation (291). FSH can also stimulate GDNF, which regulates SSC renewal (127), however FSH is not likely to be the major driver of Sertoli cell-derived GDNF (126,320).

When both LH and FSH are suppressed via inhibition of GnRH production/action, there are relatively small decreases in spermatogonial populations in rodents but a major block in spermatogonial development in primates and humans, reviewed in (271). Thus, primate spermatogonia are more sensitive to gonadotropin deprivation than rodent. Androgens and FSH appear to have supportive effects on spermatogonia via their actions on Sertoli cells, but there is species-specific variability in the sensitivity of these cells to each of these hormones, reviewed in (271,288,324).

Endocrine Regulation of Meiosis

Meiosis technically begins with the differentiation of type B spermatogonia into preleptotene spermatocytes which begin DNA synthesis. However, spermatogonia become committed to further differentiation and entry into meiosis during the A to A1 transition; this commitment to meiosis is an irreversible step leading to the production of preleptotene spermatocytes that undergo meiosis (81,276). The induction of the RA-inducible gene Stra8 in spermatogonia stimulates their differentiation, with STRA8 inducing a complex transcriptional program in differentiating spermatogonia (276). Recent studies have shown that, in contrast to female germ cell development, the initiation and completion of meiosis in males does not require RA (325). This study demonstrated that RA is required for a meiosis transcriptional program that is induced in differentiating spermatogonia; once this program has been initiated by RA, male germ cells can enter and complete meiosis in the absence of RA (325).

It is well known that the completion of meiosis requires androgen. Meiosis arrests at the pachytene/diplotene stage in mice lacking AR in Sertoli cells, and no haploid spermatids are produced (130,299). However, spermatocyte numbers are even further reduced when AR is ablated from PMC (133), suggesting that androgenic support of meiosis is mediated via both Sertoli cells and peritubular myoid cells. Meiosis was disrupted in pubertal rats when the non-classical AR pathway was blocked, suggesting that meiosis requires rapid actions of androgen on testicular somatic cells (298). Interestingly, while the completion of meiosis is absolutely dependent on androgen, it requires comparatively lower levels of androgen than the later process of spermiogenesis (285,326,327).

Mice lacking FSH show a modest but significant reduction in the progression of meiosis (291), perhaps via effects on spermatocyte survival. Both FSH and androgens support meiotic cell survival, particularly in the hormone-sensitive mid-spermatogenic stages (VII and VIII in rodents). Preleptotene and pachytene spermatocytes in stages VII and VIII are particularly vulnerable to FSH and/or androgen suppression, and apoptosis of these cells is a feature of gonadotropin suppression, reviewed in (285). The replacement of either FSH or androgen prevents spermatocyte loss/apoptosis in rodents (129) and humans (293), highlighting the fact that both of these hormones can support spermatocyte survival.

Endocrine Regulation of Spermiogenesis and Spermiation

RA regulates both spermiogenesis and spermiation. The pulse of RA generated within the adult seminiferous epithelium (see section Spermatogenesis: Cycles and Waves) initiates a transcriptional program in spermatocytes that is ultimately required for the completion of spermiogenesis (325). Sertoli cell-derived RA acting on RARα/RXRβ heterodimers in Sertoli cells is essential for spermiation (46,85). Deletion of the gene encoding the RA receptor RARα from Sertoli cells causes abnormalities in both spermiogenesis and spermiation (85). Progression through spermiogenesis and spermiation also requires the expression of the RA receptor RARα within the spermatids (328).

Both spermiogenesis and spermiation are well known targets of androgen action in the testis. While the complete ablation of androgen action in Sertoli cells causes an arrest at the end of meiosis (130,299), androgen insufficiency causes a failure of round spermatids to attach to Sertoli cells and enter the elongation phase of spermiogenesis, and the failure of mature spermatids to be released at the end of spermiation (285,287,303,329,330). Spermiation failure is an early feature of androgen suppression during adult spermatogenesis, however continued suppression eventually causes the death and/or detachment of round spermatids from Sertoli cells so that they are unable to elongate into mature spermatids (303).

Spermiation failure is observed when gonadotropins are suppressed in rodents, monkeys and men (271). It is induced rapidly after gonadotropin suppression and is the first morphological disturbance to spermatogenesis (46). In men undergoing gonadotropin suppression for the purpose of male contraception, spermiation failure can occur early in some men, leading to a rapid decline in sperm counts (331). Whether or not spermiation failure is achieved could determine whether male hormonal contraceptive-mediated gonadotropin suppression induces azoospermia (zero sperm in the ejaculate) or oligospermia (low but detectable levels of sperm in the ejaculate) (46).

It seems likely that androgens and FSH co-operate to regulate spermiation. Acute suppression of FSH alone causes spermiation failure in rats (303), whereas the administration of FSH to men undergoing gonadotropin suppression can support spermiation (293). Suppression of either FSH or testosterone alone causes significant spermiation failure in rats, but the suppression of both has a synergistc effect, indicating that both hormones co-operate to promote spermiation (303). Thus, the action of both testosterone and FSH on Sertoli cells supports the normal release of sperm at the end of spermatogenesis.

Androgens and Sperm Production

The primary stimulus for the initiation of spermatogenesis is the LH-induced rise in testosterone at puberty as androgens are required for the initiation of spermatogenesis (332). Once spermatogenesis has been initiated during puberty, androgens alone can restore or maintain qualitatively normal adult sperm production during gonadotropin suppression (327,332,333).

The amount of testosterone required to initiate spermatogenesis during puberty is higher than the concentration required to maintain it in adulthood (334). Because it is produced in the testis, testosterone concentrations in the adult testis are 25-125-fold higher than in serum (318). The very high levels of testicular testosterone are not necessary for spermatogenesis, as sperm production can be maintained at concentrations much lower than normal (318,327,332,333). In fact, LH-independent testosterone production, resulting in testicular levels of only 1-2% of normal, can promote qualitatively normal spermatogenesis in mice (335), which is consistent with the demonstration of focal areas of complete spermatogenesis in the testes of a patient with a mutated LHβ subunit that results in testicular testosterone levels 1-2% of normal (332). When testicular testosterone levels are low, such as in the pre-pubertal testis and during gonadotropin suppression in the adult, the 5α-reduction of testosterone to the more potent androgen DHT amplifies the androgenic signal to promote androgen-dependent effects (285). However, in the normal adult testis where testosterone levels are very high, testosterone likely acts directly on the AR, without conversion to DHT, to maintain androgen-dependent functions (336).

Within the testis, AR is expressed in Sertoli cells, PMC, Leydig cells and vascular endothelial cells ((337-339), whereas germ cells lack AR (340,341). Therefore, androgens act on AR within the testicular somatic cells to support spermatogenesis (318). Studies in mice show that androgen action on AR in each of the testicular somatic cell types is important for testis function. Sertoli cell AR is essential, as no sperm are produced in mice with targeted deletion of Sertoli cell AR expression (130,299) or in mice where the DNA binding domain of Sertoli cell AR has been deleted (342). AR expression in PMCs is also important for normal spermatogenesis (133) and in Leydig cells, for their development and function (201,318). The autocrine action of androgen on AR in Leydig cells is also required for normal steroidogenesis and hence optimal testosterone production (341), and AR in endothelial cells of the testicular arterioles is involved in maintaining normal fluid dynamics and vasomotion in the testis (339). In summary, androgens act on AR in various testicular somatic cells, but not germ cells, to support normal testicular function and sperm production (318).

Different phases of germ cell development rely on androgen action on somatic cells. In the absence of androgen signaling in Sertoli cells, spermatocytes cannot complete meiotic division, and no haploid round spermatids are produced (130,299,302,342). The progression of haploid spermatids through spermiogenesis also relies on androgen to prevent apoptosis and to ensure their ability to adhere to Sertoli cells (329,343,344). The release of mature spermatids at spermiation is also sensitive to androgen and/or gonadotropin inhibition (46). Many functions of Sertoli cells are androgen-dependent, such as the formation and maintenance of tight junction function at the blood-testis barrier (345-347), and are necessary to support germ cell development (318). The different androgen-dependent processes in germ cell development have different sensitivities to, or requirements for, androgens, reviewed in (348). For example, the completion of meiosis requires more androgen action than the completion of spermiogenesis (349). Individual variations in the sensitivities of different spermatogenic processes to androgens may explain why a correlation between sperm output and testicular testosterone levels has been so difficult to establish in gonadotropin-suppressed monkeys and men (350-353).

FSH and Sperm Production

Transgenic mouse models have provided important information regarding specific roles for FSH in sperm production (198,289,327,354). FSH receptors are found only on Sertoli cells and are expressed in a stage-dependent manner (355,356).

One of the most important functions of FSH is to establish a quantitatively normal adult Sertoli cell population, in terms of both number and function (289,332). FSH is a mitogen for postnatal Sertoli cell proliferation and is required for establishing normal Sertoli cell numbers in mice (198,288,289,332). Since Sertoli cell number determines spermatogenic output in adulthood (100), this function of FSH is important for quantitatively normal sperm production. FSH is also needed for normal Sertoli cell morphology, function and for their ability to support the maximal number of germ cells, (198,288,289,291,292,332). FSH stimulates the transcription of a large number of Sertoli cell genes during puberty, although many of these become less responsive to FSH in adulthood (357).

FSH action on Sertoli cells supports the number of spermatogonia, as revealed in studies in mice (302,317) and primates (321,322). Numbers of type B spermatogonia correlate more closely with circulating FSH than testicular testosterone levels in gonadotropin-suppressed monkeys and humans (331,358), indicating that these cells may be particularly supported by FSH. Transgenic human FSH expressed in hpg mice exerts stimulatory effects on spermatocyte numbers, indicating a permissive effect on meiosis, however FSH alone cannot support the completion of spermiogenesis (317). The acute suppression of FSH alone can also cause spermiation failure, presumably via effects on the Sertoli cell’s ability to release mature spermatids, which ultimately affects sperm output (303).

Transgenic mice lacking either the FSHβ subunit or FSHR remain fertile, however they exhibit small testes and reduced sperm counts due to the requirement for FSH to support the pre-pubertal proliferation of Sertoli cells, and to support quantitively normal numbers of germ cells in adulthood (291,292,302,359). A study in Finland identified 5 men homozygous for a mutation in the FSHR (360). While all 5 men had varying degrees of sperm in their ejaculates, ranging from oligospermia to normal numbers, none had normal semen parameters (360). This mutation caused the FSHR to have reduced ligand binding and signal transduction (360) however the receptor maintains residual activity (289). In contrast, five men with various mutations in the FSHβ subunit were all azoospermic, reviewed in (289,332).

The above studies suggest that FSH works in concert with androgens to promote normal sperm production. However, surprising observations in mice suggest that very high levels of FSHR activation can promote qualitatively normal sperm production in mice lacking LH receptors (361). In these studies, LHCGR null mice maintained low levels of testosterone as well as normal numbers of spermatogonia and spermatocytes but did not produce elongated spermatids. However, when this mouse line was crossed with a line expressing a highly constitutively active FSHR, sperm were produced (at levels ~50% of wildtype mice). This sperm production was not affected when the residual androgen action in the adult testis of these mice was inhibited by the AR antagonist flutamide. This study thus suggests that strong FSHR activation could compensate for the absence of androgen action in terms of the completion of spermiogenesis to produce elongated spermatids (289,361). The ability for high levels of FSHR activation to promote androgen-dependent spermiogenesis could perhaps be explained by the fact that androgens and FSH exhibit similar mechanisms of action in Sertoli cells (289).

In summary, the primary role of FSH in the testis is to support Sertoli cell proliferation and induce Sertoli cell gene expression that enables Sertoli cells to support the maximal number of germ cells. This action, along with androgen stimulation, is required for quantitatively normal sperm production.

Quantitatively Normal Spermatogenesis Requires Both Independent and Synergistic Actions of Androgens and FSH

Androgens activate nuclear AR to control androgen-dependent gene transcription and also promote rapid non-genomic effects involving AR-dependent activation of cAMP and mitogen-activated protein kinase (MAPK) pathways (296,297). FSH activates a cell-surface G protein-coupled receptor (FSHR) to activate calcium and cAMP-dependent signaling pathways (362). Sertoli cell expression of AR and FSHR peak at opposing stages of the spermatogenic cycle (363,364). Both hormones can activate intracellular calcium and cAMP-responsive element-binding protein (CREB) and MAPK signaling cascades in Sertoli cells (304). Testosterone also augments FSHR expression and FSHR-dependent gene transcription in pubertal monkey Sertoli cells (365). These properties of AR and FSH-mediated signaling in Sertoli cells highlight the fact that androgens and FSH can have independent and overlapping effects in Sertoli cells.

The data reviewed in the preceding sections have shown that androgens and FSH have distinct roles in spermatogenesis. However, they can also act co-operatively and synergistically to promote maximal spermatogenic output (198,285,288,304,318,332). Androgens and FSH co-operate by supporting different aspects of germ cell development, for example FSH contributes to the regulation of spermatogonia whereas androgen is required for the completion of meiosis and spermiogenesis. FSH establishes a quantitatively normal Sertoli cell population, whereas androgen initiates and maintains spermatogenesis; thus, both hormones co-operate to enable maximal spermatogenic output.

Androgens and FSH have certain overlapping functions in Sertoli cells, in that they can both influence Sertoli cell proliferation, morphology and function, albeit to different degrees, reviewed in (304). Both promote Sertoli cell functions that maintain germ cell survival, particularly of spermatocytes and round spermatids in the mid-spermatogenic stages in rodents (129,288). The fact that both hormones can prevent germ cell apoptosis explains why either hormone can maintain germ cell development, at least in the short term, following gonadotropin suppression in humans (293).

There are many examples of synergy between testosterone and FSH in terms of their regulation of spermatogenesis and ability to promote qualitatively or quantitatively normal sperm production (198,271,285,288,304,332). Testosterone and FSH support spermatogenesis at a lower dose when the other is present (285). Testosterone and FSH likely act synergistically in the control of signaling pathways and gene expression in Sertoli cells, which in turn are important for germ cell development (198,304). An example of such synergism is the demonstration that, after acute suppression of either androgen or FSH in rats, approximately 10% of mature spermatids fail to be released at spermiation, whereas suppression of both hormones resulted in 50% of spermatids failing to spermiate (303). Androgens and FSH both regulate Sertoli cell gene transcription, although some transcripts are preferentially affected by either androgens or FSH, while others are modulated by both in a synergistic manner (302,357,365).

It should be noted that there are species differences in the response of spermatogenesis to combined androgen and FSH suppression, reviewed in (271,366). In rodents, suppression of gonadotropins causes a decline in spermatogonial populations, but spermatogenesis is primarily arrested at the spermatocyte stage (367). In monkeys and humans however, spermatogenesis is primarily arrested at spermatogonial development, however meiosis and spermiogenesis can be maintained until they undergo a gradual attrition due to the lack of spermatogonia entering meiosis (271,351,358).

The requirement for both testosterone and FSH to support normal spermatogenesis in men was originally revealed in studies by Matsumoto and colleagues (368,369) whereby gonadotropins were suppressed by the administration of testosterone until suppression of spermatogenesis occurred. They then introduced injections of hCG to stimulate Leydig cell function and to restore intratesticular testosterone concentrations which increased sperm counts, but not to pre-treatment levels (Figure 13). These data suggested that, in association with undetectable FSH levels, increasing intratesticular androgen could partially restore sperm output (369). Using the same model, they initiated FSH treatment when sperm counts were suppressed and showed that, in the presence of low intratesticular testosterone concentrations, FSH alone could partially restore sperm output (370).

In summary, both androgen and FSH, acting via their receptors in Sertoli cells, co-operate and synergize to initiate and maintain quantitatively normal sperm output from the testis.

Figure 13.

The response in the sperm counts from normal volunteers to a suppression of FSH and LH by testosterone injections is shown. Note the recovery in sperm counts when human chorionic gonadotropin (hCG) and human FSH were introduced singly into the treatment regime. Data from Matsumoto et. al. (368,369) and Bremner et. al. (370).

Considerations for the Suppression of Sperm Production for Contraception

Because both androgens and FSH are required to maintain quantitatively normal spermatogenesis in adult males, the suppression of testosterone and FSH production to inhibit spermatogenesis is the principle of male hormonal contraception (MHC). The inhibition of sperm production in men by administering exogenous androgen to suppress LH, FSH and testosterone production is currently the leading approach to meet the requirement for a safe, reliable, reversible and acceptable male contraceptive (371).

MHC is based on the principle that the administration of exogenous androgen, alone or in combination with other agents (such as progestins or GnRH antagonists), suppresses GnRH and pituitary production of LH and FSH, reviewed in (371-374). The suppression of LH inhibits Leydig cell steroidogenesis, resulting in decreased testicular testosterone production to levels that are unable to fully support spermatogenesis. The suppression of FSH and testicular testosterone affects multiple aspects of spermatogenesis, particularly spermatogonia, the survival of germ cells and spermiation (293,331). Despite the inhibition of testicular steroidogenesis, the exogenous androgen provided by the MHC formulation maintains androgen bioactivity required to maintain androgen-dependent functions outside of the testis.

MHC reversibly suppresses sperm counts in men, and its efficacy has been evaluated in multiple phase II clinical trials, where the failure rate is defined by pregnancy in the female partner (371-373). The induction of azoospermia leads to extremely effective contraceptive efficacy (375) however not all men achieve azoospermia (371-374). Studies show that the suppression of sperm counts to <1million/mL provides high contraceptive efficacy (376) and this threshold of suppression should be achieved before efficacy of a particular formulation is tested (372).

A limitation of MHC is that around 5-20% of men do not achieve the levels of sperm count suppression that meet the target for desired efficacy, (371-374). The exact mechanisms underlying this heterogeneity are unclear but are likely due to individual variability in the sensitivity of their spermatogenesis to hormones (377). Various hypotheses have been proposed regarding the likely intra-testicular mechanisms that underly this variability (371-374).

The first is that, despite the fact that gonadotropins are suppressed to very low levels and do not appear to differ between so-called “responders” and non-responders”, it is possible that very low levels of gonadotropins could remain and act within the testis to support spermatogenesis in some men (373,374). Another possibility is that some men could maintain gonadotropin-independent testicular steroidogenesis, as has been observed in mice (378). Leydig cells continue to synthesize testicular androgens in men undergoing MHC-induced gonadotropin suppression, because administration of a CYP17A1 inhibitor causes further decreases in testicular testosterone (379). Another explanation could be differences in the production of the 5α-reduced androgen DHT in the testis, which is able to amplify the androgenic signal when testosterone levels are low (see 4.3). The administration of 5α-reductase inhibitors to suppress the conversion of testosterone to DHT did not improve sperm count suppression during MHC, however these inhibitors predominantly target the type 2 enzyme, whereas the type 1 enzyme is predominantly expressed in the testis (371). It is also worth mentioning that testicular DHT can also be produced via alternative pathways in many species including humans, see (159), and thus DHT derived from different pathways could also conceivably contribute to preserved androgen biosynthesis during MHC. Studies in mice have shown that, during conditions of Leydig cell steroidogenic insufficiency, the testis can up-regulate DHT production via alternate pathways and switch on other mechanisms to maintain androgen bioactivity (183,188). Taken together, these observations suggest that the extent of MHC-induced spermatogenic suppression could conceivably be improved by incorporating strategies that could further reduce androgen production and bioactivity within the testis.

Considerations for the Stimulation of Sperm Production for Fertility Treatment

Male infertility due to undetectable (azoospermia) or low (oligozoospermia) numbers of sperm in the ejaculate can occur in many clinical settings (380). Details of the approach to the treatment of men with reduced sperm counts are reviewed elsewhere (261,380,381). Gonadotropic stimulation of sperm production is appropriate in men with gonadotropin deficiency, such as hypogonadotropic hypogonadism (HH) or acquired androgen deficiency, may be of limited benefit in some men with oligospermia (381) but is of no or minimal benefit in men with non-obstructive azoospermia due to primary testicular failure (382) in whom gonadotropic drive is already high.

As androgens are essential for the initiation of sperm production (see section Androgens and Sperm Production), the induction of spermatogenesis in HH acquired after puberty is achieved by the administration of hCG (as an LH substitute) (381). Prolonged therapy is required to produce sperm in the ejaculate (261,381), given that human spermatogenesis takes more than 2 months to produce sperm from immature spermatogonia. Treatment with hCG alone may be sufficient for the induction of spermatogenesis in men with larger testes due to potential residual FSH action (261). However, for many men, and particularly for those with congenital HH, the co-administration of FSH (75–150 IU sc 3 times per week) is needed for maximal stimulation of sperm output (261,381). In men with congenital HH, FSH is needed to induce Sertoli cell maturation. Approximately 75-80% of men with congenital HH can show a return of sperm to the ejaculate following stimulation with combined hCG and FSH therapy or pulsatile GnRH therapy (383). Men with acquired HH and smaller testes also benefit from the co-administration of FSH with hCG, due to the well-known synergistic actions of FSH and androgens on spermatogenesis as described above. In some men, treatment may need to be particularly protracted (1-2 years) to enable pubertal maturation of the testis, for example the induction of spermatogenesis in Kallmann’s syndrome (381). Finally, it should be noted that because exogenous testosterone administration suppresses LH and intratesticular testosterone production (see section Considerations for the Suppression of Sperm Production for Contraception), exogenous testosterone therapy should not be given to men with HH who desire fertility (384).