ABSTRACT

The testes synthesize two important products: testosterone, needed for the development and maintenance of many physiological functions; and sperm, needed for male fertility. The synthesis of both products is regulated by endocrine hormones produced in the hypothalamus and pituitary, as well as locally within the testis. Testosterone is indispensable for sperm production, however both testosterone and Follicle Stimulating Hormone (FSH) are needed for optimal testicular development and maximal sperm production. Sperm are produced via the extraordinarily complex and dynamic process of spermatogenesis that requires co-operation between multiple testicular cell types. While it has long been known that testosterone and FSH regulate spermatogenesis, years of research has shed light on many of the intricate mechanisms by which spermatogonial stem cells develop into highly specialized, motile spermatozoa. Spermatogenesis involves the concerted interactions of endocrine hormones, but also many paracrine and growth factors, tightly co-ordinated gene and protein expression programs as well as epigenetic modifiers of the genome and different non-coding RNA species. This chapter provides a comprehensive overview of the fascinating process of spermatogenesis and of its regulation, and emphasises the endocrine regulation of testicular somatic cells and germ cells. The chapter also provides a summary of the clinically significant aspects of the endocrine regulation of spermatogenesis. For complete coverage of all related areas of Endocrinology, please see our online FREE web-book, www.endotext.org.

CLINICAL SUMMARY

The testes synthesize two essential products: testosterone, needed for the development and maintenance of many physiological functions including normal testis function; and sperm, needed for male fertility. The synthesis of both products is regulated by endocrine hormones produced in the hypothalamus and pituitary, as well as locally within the testis.

The secretion of hypothalamic gonadotropin-releasing hormone (GnRH) stimulates production of luteinizing hormone (LH) and follicle stimulating hormone (FSH) by the pituitary. LH is transported in the blood stream to the testes, where it stimulates Leydig cells to produce testosterone: this can act as an androgen (via interaction with androgen receptors) but can also be aromatized to produce estrogens. The testes, in turn, feedback on the hypothalamus and the pituitary via testosterone and inhibin secretion, in a negative feedback loop to limit GnRH and gonodotropin production. Both androgens and FSH act on receptors within the supporting somatic cells, the Sertoli cells, to stimulate various functions needed for optimal sperm production. Spermatogenesis is the process by which immature male germ cells divide, undergo meiosis and differentiate into highly specialized haploid spermatozoa. Optimal spermatogenesis requires the action of both testosterone (via androgen receptors) and FSH.

Spermatogenesis takes place within the seminiferous tubules of the testis. These tubules form long convoluted loops that pass into the mediastinum of the testis and join an anastomosing network of tubules called the rete testis. Spermatozoa exit the testes via the rete and enter the efferent ductules prior to their passage through, and final maturation in, the epididymis. The seminiferous tubules are comprised of the seminiferous epithelium: the somatic Sertoli cells and the developing male germ cells at various stages of development. Surrounding the seminiferous epithelium is a layer of basement membrane and layers of modified myofibroblastic cells termed peritubular myoid cells. Between the tubules is the interstitial space that contains blood and lymphatic vessels, immune cells including macrophages and lymphocytes, and the steroidogenic Leydig cells.

Male germ cell development relies absolutely on the structural and nutritional support of the somatic Sertoli cells. Sertoli cells are large columnar cells, with their base residing on basement membrane on the outside of the seminiferous tubules, and their apical processes surrounding germ cells as they develop into spermatozoa. Androgens (and estrogens) and FSH act on receptors within Sertoli cells: germ cells lack both androgen and FSH receptors, therefore these hormones act directly on Sertoli cells to support spermatogenesis. Sertoli cells regulate the internal environment of the seminiferous tubule by secreting paracrine factors and expressing cell surface receptors needed for germ cell development. Sertoli cells form intercellular tight junctions at their base: these occluding junctions prevent the diffusion of substances from the interstitium into the tubules and create a specialized milieu required for germ cell development. These junctions are a major component of the so-called ‘blood-testis-barrier’, wherein the passage of substances from the circulation is prevented from entering the inner part of the seminiferous tubules. The most immature germ cells, including germline stem cells, reside near the basement membrane of the seminiferous tubules and thus have free access to factors from the interstitium, however germ cells undergoing meiosis and haploid cell differentiation develop “above” the blood-testis-barrier and thus are entirely reliant on the Sertoli cell microenvironment. The seminiferous tubules are also an immune-privileged environment. Meiotic and post-meiotic germ cells develop after the establishment of immune tolerance, and could thus be recognized as “foreign” by the immune system, therefore the seminiferous tubules, via a number of different mechanisms including the blood-testis-barrier, actively exclude immune cells and factors from entering the seminiferous tubules and being exposed to meiotic and haploid germ cells.

The number of Sertoli cells determines the ultimate spermatogenic output of the testes. In humans, Sertoli cells proliferate during the fetal and early neonatal period and again prior to puberty. At puberty, Sertoli cells cease proliferation and attain a mature, terminally differentiated phenotype that is able to support spermatogenesis. Disturbances to Sertoli cell proliferation during these times can result in smaller testes with lower sperm production. Conversely, disturbances to the cessation of proliferation can result in larger testes with more Sertoli cells and a greater sperm output. It seems likely that the failure of many men with congenital hypogonadotropic hypogonadism (HH) to achieve normal testicular size and sperm output, when treated by gonadotropic stimulation, may result from deficient Sertoli cell proliferation during fetal and prepubertal life. The action of both androgens and FSH on Sertoli cells is necessary for the ability of Sertoli cells to support full spermatogenesis. In addition, the expression of many genes and paracrine factors within Sertoli cells is necessary for spermatogenesis.

Spermatogenesis relies on the ability of Leydig cells to produce testosterone under the influence of LH. Fetal Leydig cells appear following gonadal sex differentiation (gestational weeks 7-8 in humans) and, under the stimulation of placental human chorionic gonadotropin (hCG), results in the production of testosterone during gestation. In humans, fetal cells decrease in number towards term and are lost from the interstitium at about twelve months of age. The adult population of Leydig cells in the human arises from the division and differentiation of mesenchymal precursor cells under the influence of LH at puberty. Factors secreted by Sertoli cells and peritubular myoid cells are also necessary for Leydig cell development and steroidogenesis. Optimal Leydig cell steroidogenesis also relies on a normal complement of macrophages within the testicular interstitium as well as on the presence of androgen receptors in peritubular myoid cells, presumably because these cells secrete factors necessary for Leydig cell development and function.

The process of spermatogenesis begins in the fetal testis, when the Sertoli cell population is specified in the embryonic testis under the influence of male sex determining factors, such as SRY and SOX9. Newly-specified Sertoli cells enclose and form seminiferous cord structures and direct primordial germ cells to commit to the male pathway of gene expression. Fetal Sertoli cells proliferate and drive seminiferous cord elongation; this process is also dependent on factors secreted by Leydig cells. In the neonatal testis, primordial germ cells undergo further maturation and migrate to the basement of the seminiferous tubules where they provide a pool of precursor germ cells for postnatal spermatogenesis.

Spermatogonia are the most immature germ cell type. This heterogeneous population includes spermatogonial stem cells, which self-renew throughout life to provide a pool of stem cells available for spermatogenesis, as well as proliferating cells that differentiate and become committed to entry into meiosis. Spermatogonial development is hormonally independent and as such they are present even in the absence of GnRH. Spermatogonia eventually differentiate into spermatocytes that proceed through the process of meiosis that begins with DNA synthesis resulting in a tetraploid gamete. During the long meiotic prophase, which lasts ~2 weeks, homologous chromosomes pair and meiotic recombination occurs; this involves the induction and repair of DNA double-strand breaks allowing the exchange of genetic information between paired chromosomes, thereby creating genetic diversity between gametes. At the end of prophase, the meiotic cells proceed through two rapid and successive reductive divisions to yield haploid spermatids. The completion of meiosis depends absolutely on androgen action in Sertoli cells; in the absence of androgen, no haploid spermatids will be produced.

Newly formed haploid round spermatids differentiate, with no further division, into the highly specialized spermatozoan during the process of spermiogenesis. This involves many complex processes, including development of the acrosome (an organelle on the surface of the sperm head that contains enzymes required to penetrate the zona pellucida of the oocyte and thus facilitate fertilization), the flagella (the motile microtubule-based structure required for sperm motility) and the remodelling of the spermatid’s DNA into a tightly coiled structure within a small, streamlined nucleus that will not hinder motility. This remodelling of the DNA involves the cessation of gene transcription up to 2 weeks prior to the final maturation of the sperm; therefore spermiogenesis involves the translational delay of many mRNA species which must then be translated at precise times throughout their final development. Spermatogenesis ends with the process of spermiation. This involves removal of the spermatid’s large cytoplasm, revealing the streamlined mature spermatozoa, and the final disengagement of sperm from the Sertoli cells into the tubule lumen, prior to their passage to the epididymis. Both the survival of spermatids during spermiogenesis and their release at the end of spermiation is dependent on optimal levels of androgen and FSH.

Spermatogenesis is a long process, taking up to 64 days in the human, and its inherent complexity demands precise timing and spatial organization. Within the seminiferous tubules, Sertoli cells and surrounding germ cells in various phases of development are highly organized into a series of cell associations, known as stages. These stages result from the fact that a particular spermatogonial cell type, when it appears in the epithelium, is always associated with a specific stage of meiosis and spermatid development. The stages follow one another along the length of the seminiferous tubule, and the completion of a series of stages is termed a “cycle”. This cycle along the length of tubule is obvious in rodents, however in humans several cycles are intertwined in a helical pattern; thus a human seminiferous tubule viewed in cross section will contain up to three stages. The completion of one cycle results in the release of mature spermatozoa into the tubule lumen; the cycles are repeated along the tubules, resulting in constant “pulses” of sperm production. These pulses of sperm release allow the testes to continually produce millions of sperm, with the average normospermic man able to produce approximately 1000 sperm per heartbeat.

The precise timing and co-ordination of spermatogenesis is achieved by many factors. Emerging evidence suggests that retinoic acid, metabolized within the testis from circulating retinol (a product of vitamin A) is a major driver of spermatogenesis. A precise pulse of retinoic acid action is delivered to a particular stage of the spermatogenic cycle; this pulse is achieved by the constrained expression of enzymes involved in retinoic acid synthesis, degradation and storage, as well as the local expression of retinoic acid receptors. This pulse of retinoic acid acts directly on spermatogonia to stimulate their entry into the pathway committed to meiosis. It also acts directly on Sertoli cells to regulate its cyclic functions. Sertoli cells contain an internal “clock” that allows them to express genes and proteins at precise times. This clock appears to be set by retinoic acid, however the timing of the clock can be influenced by the germ cells themselves.

The timing of spermatogenesis also relies on an extraordinarily complex program of gene transcription and protein translation. Alternative splicing of mRNA is highly prevalent in the testis, and generates many germ cell-specific transcripts that are important for the ordered procession of germ cell development. Noncoding RNAs, including microRNAs, small interfering RNAs, piRNAs and long noncoding RNAs, are highly expressed in the testis, particularly by the germ cells. Indeed, studies on male germ cells have revealed much of what is known about the biology and function of non-coding RNAs. These non-coding RNAs have many and varied roles and are particularly required for the transcriptional program executed during meiosis and spermiogenesis.

The male germ cell transmits both genetic and epigenetic information to the offspring. Epigenetic modifications of the genome are heritable; epigenetic processes such as DNA methylation and histone modifications regulate chromatin structure and modulate gene transcription and silencing. The male germ cell undergoes major epigenetic programming in the fetal testis, during the genome wide de-methylation and re-methylation to establish the germline-specific epigenetic pattern that is eventually transmitted to the offspring. The sperm epigenome is then further remodelled during postnatal spermatogenesis by various mechanisms. It is now known that a man’s sperm epigenome can be altered by environmental factors (including diet and lifestyle as well as exposure to environmental factors) throughout his lifetime and this altered sperm epigenome can influence both his fertility and the health of his future children.

It is clear from the above summary that spermatogenesis relies on many intrinsic and extrinsic factors. However spermatogenesis is absolutely dependent on androgen-secretion by the Leydig cells; androgens stimulate and maintain germ cell development throughout life. Testicular testosterone levels are very high, by virtue of its local production, however they are considerably higher than those required for the initiation and maintenance of spermatogenesis. Androgen action on receptors within Leydig cells, peritubular myoid cells and Sertoli cells is essential for normal steroidogenesis and spermatogenesis. While testosterone is essential for spermatogenesis, it is also important to note that exogenous testosterone administration resulting in even slightly supraphysiological serum levels suppresses gonadotropin secretion via negative feedback effects on the hypothalamus and pituitary, leading to the cessation of sperm production.

In contrast to androgens, spermatogenesis can proceed in the absence of FSH; however, testes are smaller and sperm output is reduced. This is due to FSH’s role in the peri-pubertal proliferation and differentiation of Sertoli cells and in the maintenance of germ cell survival. While FSH is thus not essential for spermatogenesis, it is generally considered that optimal spermatogenesis requires the combined actions of both androgen and FSH, with both hormones having independent, co-operative and synergistic effects to promote maximal sperm output.

These factors are an important consideration in the stimulation of spermatogenesis in the setting of HH. As androgens are essential for the initiation of sperm production, the induction of spermatogenesis in HH acquired after puberty is achieved by the administration of hCG (as an LH substitute). Prolonged therapy is required to produce sperm in the ejaculate, given that human spermatogenesis takes more than 2 months to produce sperm from spermatogonia. Treatment with hCG alone may be sufficient for the induction of spermatogenesis in men with larger testes due to potential residual FSH action, however, for many men, and particularly for those with congenital HH, the co-administration of FSH is needed for maximal stimulation of sperm output. In men with congenital HH, FSH is needed to induce Sertoli cell maturation, whereas men with acquired HH and smaller testes benefit from the co-administration of FSH due to the synergistic actions of FSH and androgens on spermatogenesis.

In summary, the testes, under the influence of gonadotropins, produce testosterone and sperm. These processes require the co-ordinated actions of multiple cell types and the secretion of paracrine factors. Spermatogenesis is a long and complex process that relies on multiple somatic cells as well as on the co-ordinated expression of genes, proteins and non-coding RNAs. Inherent vulnerabilities exist in spermatogenesis meaning that lifestyle and environmental factors can potentially influence a man’s sperm epigenome, his fertility and the health of his future children.

GENERAL ANATOMY OF THE MALE REPRODUCTIVE SYSTEM

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 (1).

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. 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 (1). 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 (2). 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 (3).

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 and 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.

AN OVERVIEW OF SPERMATOGENESIS

Spermatogenesis is the process by which precursor germ cells termed spermatogonia undergo a complex series of divisions to give rise to spermatozoa (4-5). This process takes place within the seminiferous epithelium (Figure 5), a complex structure composed of germ cells and radially-oriented supporting somatic cells called Sertoli cells. The latter cells extend from the basement membrane of the seminiferous tubules to reach the lumen. The cytoplasmic profiles of the Sertoli cells are extremely complex as this cell extends a series of processes that surround the adjacent germ cells in an arboreal pattern (5-7).

Figure 5

The top panel illustrates the typical structure of the human seminiferous epithelium containing the germ cells and Sertoli cells. The position of Sertoli cell nuclei within the epithelium is indicated, as is the tubule lumen. The tubules are surrounded by thin plate-like contractile cells called peritubular myoid cells. The Leydig cells and blood vessels lie within the interstitium. The bottom panel illustrates the nuclear morphology of the major cell types found within the human seminiferous epithelium, showing the progress of spermatogenesis from immature spermatogonia through meiosis and spermiogenesis to produce mature elongated spermatids. Abbreviations: Ad: A dark spermatogonia, Ap: A pale spermatogonia, B: type B spermatogonia, Pl: preleptotene spermatocyte, L-Z: leptotene to zygotene spermatocyte, PS: pachytene spermatocyte, M: meiotic division, rST: round spermatid, elST: elongating spermatid, eST: elongated spermatid. All germ cell micrographs were taken at the same magnification to indicate relative size. Micrograph of seminiferous epithelium was provided by Dr Sarah Meachem.

Spermatogenesis can be divided into three major phases (i) proliferation and differentiation of spermatogonia, (ii) meiosis, and (iii) spermiogenesis which represents a complex metamorphosis of round haploid germ cells into the highly specialized structure of the spermatozoon (Figure 5). It is important to note that, as germ cells divide and differentiate through these phases, they do not separate completely after mitosis but remain joined by intercellular bridges (8). These intercellular bridges persist throughout all stages of spermatogenesis and are thought to facilitate biochemical interactions allowing synchrony of germ cell maturation.

Meiosis

Meiosis is the process by which gametes undergo reductive division to provide a haploid spermatid, and in which genetic diversity of the gamete is assured via the exchange of genetic material. 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 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. The 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 (29). 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 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 (30).

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.

The diplotene stage is recognized by partial separation of the homologous pairs of chromosomes that still 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 (12, 31). 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, however whether this phenomenon occurs in males has been the subject of debate. A recent study in mice showed that advanced age was associated with increased defects in chromosome pairing, however no increase in anueploidy was observed at Metaphase II, suggesting that such errors were corrected during metaphase checkpoints in males (32). Therefore advanced age, at least in mice, has more of an impact on gamete aneuploidy in females compared to males.

Spermiogenesis and Spermiation

The transformation of a round spermatid into a spermatozoon represents a complex sequence of events that constitute the process of spermiogenesis. No cell division occurs, but a conventional round cell becomes 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 reorganisation of the cytoplasm and cell organelles and (e) the process of release from the Sertoli cell termed spermiation (5, 33-37).

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.

The formation of the acrosome commences by the coalescence of a series of granules from the Golgi complex. These migrate to come into contact with the nuclear membrane where they form a cap-like structure which becomes applied over approximately 30-50% of the nuclear surface (33). Acrosome biogenesis begins early in round spermatid development, and progressively extends as a “cap” over the nucleus as round spermatids differentiate further.

Once the acrosome is fully extended, round spermatids begin what is known as the elongation phase of spermiogenesis. As spermatid elongation commences, 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 this polarization, 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 subsequently replaced by arginine-rich protamines (38-39). The spermatid chromatin subsequently 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 (40). Therefore, the subsequent spermatid elongation phase proceeds in the absence of active gene transcription (see (36)).

At the commencement of spermatid elongation, a complex, microtubule-based structure known as the manchette is formed. The microtubule network 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 is thought to participate in nuclear head shaping, perhaps by exerting a force on the nucleus as it progressively moves distally towards the posterior portion of the nucleus (41-43).

The formation of the tail commences early in spermiogenesis in the round spermatid phase, when a filamentous structure emerges from one of the pair of centrioles which lie close to the Golgi complex. Associated with the changing nuclear-cytoplasmic relationships, 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. This basic structure is modified at the region of its articulation with the nucleus through the formation of a complex structure known as the connecting piece (44).

Metamorphosis of the flagella proceeds during the elongation phase, as it acquires its characteristic neck region, mid-, principal- and end-pieces (37). The development of the flagella is thought to involve a mechanism known as Intra-Manchette Transport (IMT), which is proposed to be similar to the Intra-Flagellar Transport (IFT) systems 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 (42-43, 45).

The middle and principal pieces contain the mitochondrial and fibrous sheath components, respectively, and include the outer dense fibers. The biochemical characteristics of these components of the sperm tail are emerging (46-51), reviewed in (37). While these components provide some structural stability to the tail, evidence suggests that they may serve as a molecular scaffold to position key enzymes critical to successful sperm motility. For instance, CatSper 1, an ion channel plasma membrane-associated protein present in the principal-piece, has been shown to regulate calcium ion fluxes critical for the process of hyperactivation of sperm motility associated with capacitation (52). Studies demonstrate that CatSper, or a directly associated protein, is a non-genomic progesterone receptor that mediates the effects of progesterone on sperm hyperactivation and acrosome reaction (53-54). Further studies have shown that plasma membrane calcium-ATPase 1 is also located in the principal-piece and has been shown to be critical for the process of hyperactivation of sperm motility (55). While these are plasma membrane-located complexes, TPX1 (also called CRISP2), a protein localized to the outer dense fibers of the tail and acrosome (56) has been shown to regulate ryanodine receptor calcium signalling (57).

The formation of the mitochondrial sheath occurs at the time of the final reorganization of the cytoplasm and organelles of the spermatid (5, 33, 58). The mitochondria that had remained around the periphery of the spermatid aggregate around the proximal part of the flagellum to form a complex helical structure (Figure 8).

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 Sertoli cells prior to their passage to the epididymis, see (35) for review. 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 cytoplasm of the spermatid migrates to a caudal position around the tail and is markedly reduced in volume. Some observations suggest that prolongations of Sertoli cell cytoplasm send finger-like projections which invaginate the cell membrane of the spermatid cytoplasm and literally 'pull' the residual cytoplasm off the spermatid (33). The remnants of the spermatid cytoplasm form what is termed the residual body. The residual bodies contain mitochondria, lipid and ribosomal particles, and are phagocytosed and moved to the base of the Sertoli cell where they are broken down by lysosomal mechanisms. The final release of sperm 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 (35), culminating in the Sertoli cell “letting go” of the mature spermatid (59). The morphological features of spermiation are relatively conserved between species, particularly among mammals (60). Spermiation is highly susceptible to perturbation by pharmacological modulators and by agents that suppress gonadotropins, reviewed in (35), and failure of spermiation can be recognized by the presence of mature elongated spermatid nuclei being phagocytosed by the Sertoli cells (61).

Figure 8. A

cross-section through the developing mid-piece of the sperm tail shows the aggregation of mitochondria (arrows) surrounding the outer dense fibres (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.

The Cycle of the Seminiferous Epithelium

Within the seminiferous epithelium, the cell types that constitute the process of spermatogenesis are highly organized to form a series of cell associations or stages. These cell associations, or stages of spermatogenesis, result from the fact that a particular spermatogonial cell type, when it appears in the epithelium, is always associated with a specific stage of meiosis and spermatid development. The stages follow one another along the length of the seminiferous tubule, and the completion of a series of stages is termed a “cycle” (see Figure 9). This cycle along the length of tubule is obvious in rodents, however in humans the situation is more complex (see below). The completion of one cycle results in the release of mature spermatozoa into the tubule lumen; the cycles are repeated along the length of the tubules (Figure 9), resulting in constant “pulses” of sperm production along the tubules. Thus the cyclic nature of spermatogenesis enables continual sperm production within the testis. These pulses of sperm release along the length of the seminiferous tubules allow the testes to continually produce millions of sperm, with the average normospermic man able to produce approximately 1000 sperm per heartbeat.

The cycle of the seminiferous epithelium was defined by LeBlond and Clermont (62), as the series of changes in a given area of the seminiferous tubule between two appearances of the same developmental stage or cell association. They defined 14 stages in the rat cycle based on the 19 phases of spermiogenesis (Figure 9) as identified by the periodic acid Schiff (PAS) stain. In effect, if it was possible to observe the same region of the seminiferous epithelium by phase contrast microscopy over time, the appearance would progress through the 14 stages before stage I reappeared. They also demonstrated that the duration of any one stage was proportional to the frequency with which it was observed in the testis. As type A spermatogonia in any one area of the epithelium progress through meiosis and spermiogenesis to become spermatozoa, the specific area of the tubule would pass through the 14 stages four times. In each progression, the progeny of the spermatogonia progressively move toward the lumen of the tubule.

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 in a cyclic manner along the length of the seminiferous tubule, as illustrated in the diagram in the middle panel. Examples of the histology of the seminiferous epithelium at two different stages are given in the bottom panel.

Studies in many mammalian species demonstrated that the cycle of spermatogenesis could be identified for each species but showed that the duration of the cycle varied for each species (12). In many species, especially the rat, the same stage of spermatogenesis extends over several millimetres of the adjacent tubule and it is possible, by observation under transillumination, to dissect lengths of seminiferous tubules at the same phase of spermatogenesis (63). Such observations amply confirmed the earlier studies of Perey and colleagues (64), that the stages of spermatogenesis were sequentially arranged along the length of the tubule (Figure 9). As the cycle progress, this arrangement resulted in a "wave of spermatogenesis" along the tubule. Regaud (65) interpreted his observations correctly by the statement "the wave is in space what the cycle is in time".

For many years, investigators believed that such a cycle did not occur in the human testis but the careful studies of Clermont (66) showed that human spermatogenesis could be subdivided into 6 stages. However unlike the rat, each stage often only occupied one quadrant of any given tubule cross section giving the disorganized appearance. By careful studies using tritiated thymidine injections into the testis, Clermont and Heller (31) demonstrated that the duration of the cycle in the human took 16 days and the progression from spermatogonia to sperm took 70 days or four and a half cycles of the seminiferous cycle. Other studies showed that the cycle length was specific for each species (eg rat 49 days) and the progression of each cell type in spermatogenesis involved a defined duration (12). It is likely that the relatively poor definition of stages in human seminiferous tubules, compared to the rat, is due to a greater number of spermatogonia entering each phase of the cycle in the rat, their cell progeny therefore occupying a greater length of the tubule.

Transcriptional profiling studies described the changing patterns of gene expression across the rat spermatogenic cycle, and demonstrated that Sertoli cells and germ cells showed highly co-ordinated stage-dependent changes in gene expression (67). The mechanisms underlying these temporal constraints on spermatogenesis have been the subject of speculation as to whether these were intrinsic or were imposed by the Sertoli cells. The latter proposition is supported by the demonstration that when rat germ cells were transplanted into the mouse testis, spermatogenesis proceeded at the normal rate for the rat, indicating that the kinetics of the spermatogenic cycle are determined by intrinsic mechanisms within germ cells (68). In contrast however, Sertoli cells demonstrate cyclic expression of certain proteins in the embryonic and pre-pubertal period, even in the absence of germ cells (69). Recent studies demonstrate that retinoic acid “sets the clock” within post-pubertal Sertoli cells, however differentiating germ cells are required to “fine tune” the clock (70) (see below for further information). Taken together, these observations demonstrate that the Sertoli cell contains a “clock” that modulates cyclic gene and protein expression, and that the precise timing of this clock is modulated by germ cells.

THE ROLE OF SERTOLI CELLS IN SPERMATOGENESIS

Sertoli cells have an intimate physical relationship with the germ cells (Figure 10) during the process of spermatogenesis (5, 7, 71). The cytoplasmic extensions that pass between the germ cell populations surrounding the Sertoli cell provides structural support through a microfilament and microtubular network present in the cytoplasm of the Sertoli cell (72). This architecture is not static but changes in the tubule depending on the stage of the spermatogenic process.

Sertoli cells regulate the internal environment of the seminiferous tubule. This regulation is facilitated by specialized inter-Sertoli cell occluding-type junctions which are formed at the sites where processes of Sertoli cell cytoplasm from adjacent cells meet (73). These junctions contribute to the blood-testis barrier that regulates the entry of a variety of substances into the seminiferous tubule (74). These occluding junctions towards the base of Sertoli cells prevent the diffusion of substances from the interstitium into the inner part of the seminiferous tubule (see 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 their access to factors from the interstitium restricted by the blood-testis-barrier. This effectively divides the seminiferous epithelium into a basal compartment containing spermatogonia, and an adluminal compartment containing meiotic and post-meiotic germ cells. As preleptotene spermatocytes migrate from the basement membrane of the tubule into the adluminal compartment, these tight junctions open up to allow this cellular migration to take place (Figure 11) and reform beneath the preleptotene spermatocytes which have now left the basement membrane to form leptotene spermatocytes. The formation and dissolution of these junctional specializations are under the control of numerous physiological regulators including endocrine (75-76) and paracrine (77) factors, see for (78) recent review.

The Sertoli cell junctions and the blood-testis barrier are required for fertility (79). These junctions allow the environment of meiotic and post-meiotic germ cells to be precisely controlled by the Sertoli cell, enabling the precisely timed delivery of factors uniquely required for germ cell development. For example, the Sertoli cell provides substrates for germ cell glycolysis (80-82); lactate rather than glucose is the preferred substrate for glycolysis in primary spermatocytes and Sertoli cells generate lactate from glucose.

The blood-testis barrier has long been thought to contribute to the immune-privileged environment within the seminiferous epithelium. Meiotic and post-meiotic germ cells develop after the establishment of immune tolerance, and could thus be recognized as “foreign” by the immune system, therefore this barrier protects the developing germ cells from immune cell attack (83). However some studies show that seminiferous tubules continue to exclude immune cells when Sertoli cell junctions are absent (79) or even when Sertoli cells are ablated (84), raising questions as to the precise role of these junctions in immune privilege. It seems likely that many factors, including the production of anti-inflammatory cytokines, regulate the immune privileged environment of the testis.

In the adult rat testis, activin A protein peaks at the time of blood-testis-barrier remodeling and migration of leptotene spermatocytes into the adluminal compartment, suggesting that activin A could regulate blood-testis barrier function (for review see (85)). More recently it has been shown that elevated activin A action in vivo and in vitro suppresses the Sertoli cell tight junctions that form a major component of the blood-testis barrier (86), suggesting that activin A could facilitate blood-testis-barrier remodeling.

Recent studies have revealed that the blood-testis-barrier shows differential permeability and can exclude different sized molecules depending on its functional status (87). Tracer studies showed that the barrier can exclude all molecules between 0.6-150kDa in size when it is “fully sealed”, however in some situations and stages it can exclude large (150kDa+ molecules) but remain permeable to smaller molecules. These studies reveal that the barrier is more selective in its function than previously thought, and highlight the complexity of this structure and its important role in spermatogenesis.

Sertoli cells are indispensible for germ cell development, as they provide physical, metabolic and nutritional support at precisely timed intervals as dictated by the spermatogenic process. Transgenic mouse models have revealed many Sertoli cell genes that are required for all aspects of spermatogenesis, reviewed in (88). For example, the Etv5 transcription factor within Sertoli cells is essential for the maintenance of the stem cell niche (89), reviewed in (90). Sertoli cells respond to the changing needs of the developing germ cells as evidenced by the remarkable stage-specificity in the expression patterns of many Sertoli cell genes (67).

The differentiation status of Sertoli cells is related to their capacity to support spermatogenesis. For example, perinatal hypothyroidism extends the duration of Sertoli cell proliferation but also delays their maturation; this is also associated with a delay in the onset of spermatogenesis (91-92). It was widely believed that once Sertoli cells ceased pre-pubertal proliferation, they attained a so-called “terminally differentiated” phenotype. However it is now clear that Sertoli cells can de-differentiate in certain conditions of impaired spermatogenesis, reviewed in (93). For example, a loss of claudin 11 (a protein involved in Sertoli cell occluding junctions) causes Sertoli cells to remain proliferative during development and to lose their epithelial phenotype (94). De-differentiated Sertoli cells in cell cycle are not observed in normospermic men, but are present in men after 12 weeks of gonadotropin suppression (95). 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 (96). Therefore the maintenance of an adult Sertoli cell phenotype is essential for normal spermatogenesis.

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. Recent studies using a mouse model of acute and specific ablation of Sertoli cells have revealed they are essential for the maintenance of peritubular myoid cell fate and function, and are required for Leydig cell development and normal steroidogenesis (84, 97). Therefore Sertoli cells are required for both sperm and androgen production within the testis.

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.

Figure 11

The position of the blood testis barrier in the seminiferous epithelium, which is formed by tight, occluding and adhesion junctions between adjacent Sertoli cells. This barrier restricts the diffusion of substances from the interstitum and blood vessels, and thus allows the Sertoli cell to determine the microenvironment above the junctions. This barrier effectively divides the seminiferous epithelium into two compartments, the basal compartment with free access to substances from outside the tubule, and the adluminal compartment, the environment of which is controlled by the Sertoli cell. Meiosis and the differentiation of spermatids occurs in the adluminal compartment. The inter-Sertoli cell junctions transiently remodel to allow germ cells to move from the basal to the adluminal compartments, whilst protecting the functionality of the barrier. Diagram provided by Jenna Haverfield.

The number of Sertoli cells determines the ultimate spermatogenic potential of the testis. In rodents, Sertoli cells proliferate in fetal and early postnatal life and even into adulthood, reviewed in (93), whereas 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 (98), reviewed in (93, 99). Studies in mice show that apoptosis of Sertoli cells during fetal life results in abnormal cord development, smaller testes and reduced seminiferous tubule size (100), suggesting the proliferation of Sertoli cells during the fetal period is an important driver of seminiferous tubule formation. That Sertoli cell number determines the total sperm output of the testis, reviewed in (93, 101), is emphasized by studies showing that perinatal induction of hypothyroidism extends the duration of Sertoli cell proliferation, which in turn leads to increased Sertoli cell numbers and increased sperm output of the adult testis (91, 102). Other Sertoli cell mitogens such as FSH and activin (103-104), together with thyroxine, can also exert significant changes in the number of Sertoli cells in the testis, depending on the temporal pattern of their secretion. The latter must occur before the cessation of Sertoli cell proliferation. In the rat, this occurs at about 20 days whereas in the human, Sertoli cells cease to divide during the pubertal process (98). It is possible that the failure of many men with hypogonadotropic hypogonadism to achieve normal testicular size and normal sperm counts, when treated by gonadotropic stimulation, may result from abnormal Sertoli cell proliferation during fetal and prepubertal life resulting in a decreased Sertoli cell complement (105).

LEYDIG CELLS AND STEROIDOGENESIS

The Leydig cells lie within the intertubular regions of the testis and are found adjacent to blood vessels and the seminiferous tubules (5, 106). They are the cell type responsible for testosterone production which is essential for the maintenance of spermatogenesis. There are very significant organizational differences in the intertubular tissue betweens species reflecting the number of Leydig cells and differing architecture involving blood vessels and lymphatic sinusoids (107). Additionally, fibroblasts, macrophages, lymphocytes and small numbers of mast cells are found in the intertubular regions of the testis (108-109), reviewed in (110-111).

In most species there are two populations of Leydig cells, fetal and adult (112-113), that differ in terms of morphology, androgen synthesis, and regulation by paracrine and autocrine factors, reviewed in (110, 114-115). The fetal population appears following gonadal sex differentiation (gestational weeks 7-8 in humans) and, under the stimulation of hCG, results in the production of testosterone during gestation (116). In the human, these cells decrease in number towards term and degenerate and are lost from the intertubular region at about twelve months of age (117), although recent lineage-tracing experiments have indicated that fetal Leydig cells persist in the postnatal rodent testis (118). The adult population of Leydig cells in the human results from LH stimulation commencing at the time of puberty. This generation arises by division and differentiation of mesenchymal precursors under the influence of LH (119). Evidence in humans also supports a third neonatal Leydig cell population that peaks at 2-4 months after birth although their function is poorly understood (120), for review see (110). Whether or not the various Leydig cell populations share a common stem cell precursor also remains unclear (111).

Much of the data investigating gene regulatory systems that control fetal and adult Leydig cell differentiation is derived from rodent models, and differences may exist in the human. For example, placental hCG action via the LH/hCG receptor is required for human fetal Leydig cell development but not for mouse fetal Leydig cells (121). However, both species have in common the two main factors that influence fetal Leydig cell differentiation; Desert hedgehog (Dhh) and Platelet-derived growth factor A (Pdgfa). Interestingly both of 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 ((122-124), also see (110) and references therein). Dhh and Pdgfra also play an important role in adult Leydig cell development (124-126). Targeted deletion of Sertoli cell Dhh in mice causes major reductions in fetal Leydig cell number and androgen synthesis and results in undescended testes and feminized external genitalia (124, 127). A similar phenotype, termed complete pure gonadal dysgenesis, is observed in 46,XY patients with mutations in the DHH gene (128). A number of other important regulatory genes are also recognized to influence fetal and adult Leydig cell differentiation [e.g. Wt1, (129), Nrg1 (130), Inhba (125), for review see (110).

Leydig cells have the capacity to synthesize cholesterol from acetate or to take up this substrate for steroidogenesis from lipoproteins (106, 131). Typical of any steroid secreting cell, the Leydig cell contains 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 requiring intracellular transport of substrates between these organelles to achieve successful androgen production.

Leydig cells also produce the peptide hormone, insulin-like factor 3 (INSL3), which is structurally related to the insulin, IGF1 and IGF2 family (132-133), for review see (134). Targeted disruption of the Insl3 gene in mice causes bilateral cryptorchidism due to failure of gubernaculum development during embryogenesis (133). In the adult testis, INSL3 acts via its receptor, RXFP2 (formerly known as LGR8) found both on meiotic and post-meiotic germ cells, and on Leydig cells themselves (135-136). In gubernacular tissue, RXFP2 expression is up-regulated by androgen and abolished by an androgen receptor antagonist, suggesting a link between INSL3 and androgen signaling pathways (137). INSL3 has an anti-apoptotic function in the germ cell compartment (136), and could form part of an autocrine feedback loop in Leydig cells (135) which respond in vitro by increasing cyclic AMP and testosterone (138). In the human testis, INSL3 is a constitutive biomarker of both Leydig cell differentiation status and cell number, otherwise known as Leydig cell ‘functional capacity’ (134). This functionality has been useful to follow pubertal onset and increasing testicular volume (139) or to evaluate treatment for hypogonadism (134, 140), but does not have predictive value for sperm retrieval in patients with Klinefelter’s syndrome (141).

ROLE OF PERITUBULAR MYOID CELLS

External to the basement membrane of the seminiferous tubule, are several layers of modified myofibroblastic cells termed peritubular myoid cells (PMCs) (169-170). PMCs are contractile and are responsible for the irregular contractions of the seminiferous tubules which propel seminiferous tubule fluid and released spermatozoa through the tubular network to the rete testis (171). PMC contractility is stimulated by various factors, reviewed in (171) including endothelin, prostaglandin F2 alpha and angiotensin (172-174). These contractions are associated with dramatic changes in PMC shape and their cytoskeletal actin networks (175). PMCs and Sertoli cells both contribute to the composition of the basement membrane that surrounds the seminiferous tubules, reviewed in (171). PMCs also produce various growth factors such as activin A and platelet derived growth factors (176-177), that may influence the function of other testicular cells, reviewed in (171).

PMCs have long been known to influence Sertoli cell function and protein expression, reviewed in (178) and the presence of Sertoli cells is required for normal PMC development and function (84, 97). PMCs influence Sertoli cell number, function and ability to support germ cell development, as revealed by studies in mice lacking androgen receptor expression in PMCs (179). This model also revealed that PMCs influence Leydig cell development and steroidogenesis (168). Further studies in transgenic mice reveal that an R-spondin receptor, LGR4, is selectively expressed in PMCs, participates in Wnt/β-catenin signaling and is necessary for germ cell development during meiosis (180). PMCs, under the influence of androgen, secrete the growth factor glial cell line-derived neurotrophic factor (GDNF), which is necessary for the maintenance of the spermatogonial stem cell niche (181-182). Therefore it is clear that PMCs modulate spermatogenesis via the regulation of Leydig, Sertoli and germ cell development and function.

THE REGULATION OF SPERMATOGENESIS

Many studies in the past 30 years have focused on the endocrine regulation of spermatogenesis. It is clear that the gonadotropins LH and FSH are required for the initiation and maintenance of quantitatively normal spermatogenesis. LH targets the Leydig cells to stimulate androgen biosynthesis, and the resulting androgens (testosterone and its androgen metabolites) act on receptors within the seminiferous epithelium to stimulate and support spermatogenesis. FSH targets receptors in the Sertoli cells directly to support spermatogenesis. However the roles of other endocrine factors, such as vitamin A and its metabolite retinoic acid, are emerging. While both androgens and FSH are required for optimal spermatogenesis (see below), spermatogenesis relies on the local production of growth factors, signaling molecules and other intrinsic mechanisms.

The following sections consider key aspects of the regulation of Sertoli cells and germ cell development and function, with the roles of the “traditional” endocrine regulators, androgen and FSH, briefly discussed at the end of each section. The role of estrogens in spermatogenesis is considered elsewhere in Endotext (Endocrinology of Male Reproduction Section, Chapter 17, Estrogens and Male Reproduction (144)).

THE HYPOTHALAMIC-PITUITARY-TESTIS AXIS

The successful initiation of testicular function is dependent on the hypothalamic secretion of GnRH which in turn stimulates FSH and LH to act on the testis. These actions initiate spermatogenesis and testosterone production.It is well recognised that the testis in turn, through the secretion of hormones produced in the Sertoli and Leydig cells, exerts a negative feedback control on the production of gonadotropins.

The presence of such a negative feedback control by the testis on pituitary FSH and LH secretion is best demonstrated by the rapid rise of FSH and LH after castration. The mechanisms by which the secretion of FSH and LH increases in response to castration involves a rise in the hypothalamic secretion of GnRH and also involves direct actions at the pituitary level which allow an increase in pulse amplitude. Further, the fact that LH and FSH are co-secreted by the majority of gonadotroph cells in the anterior pituitary raises a number of unresolved questions as to how GnRH and the inhibitory signals act on the pituitary to result in the differential regulation of FSH and LH secretion.

The secretion of the gonadotropins FSH and LH are regulated by the episodic secretion of gonadotropin releasing hormone (GnRH) produced in the hypothalamus (also see Endotext, Endocrinology of Male Reproduction section, Chapter 5,Hypogonadotropic Hypgonadism (HH) and Gonadotropin Therapy (346)). There is now a substantial body of evidence that indicates that the kisspeptins, a family of neuropeptides localized to the arcuate nucleus of the brain are upstream regulators of GnRH secretion (for reviews see (347) (348). For instance, arcuate kisspeptin-neurokinin B-dynorphin expressing hypothalamic neurons are critically involved in the increase in gonadotropin secretion that occurs after gonadectomy (349). The regulation is further complicated by the isolation and characterization of gonadotropin-inhibitory hormone (GnIH), which acts both upstream of GnRH and also may operate at the levels of the gonads as an autocrine/paracrine regulator of steroidogenesis (350-351).

The pituitary secretion of FSH and LH by the gonadotrophs is also controlled by the feedback inhibition that occurs via the steroids, testosterone and estradiol (for an extensive review on the role of estradiol in the hypothalamic-pituitary-testis axis, see Endotext, Endocrinology of Male Reproduction section, Chapter 17, Estrogens and Male Reproduction (144)). The secretion of FSH and LH is also regulated by protein inhibitors, inhibin, secreted by the gonads, and follistatin, produced locally within the pituitary by the follicular-stellate cells (352), reviewed in (353). Follistatin exerts its inhibition of FSH secretion by its capacity to bind and block the actions of the activins A and B, the latter locally produced by the pituitary gland (354).

SUMMARY OF THE ENDOCRINE REGULATION OF SPERM PRODUCTION: CLINICAL CONSIDERATIONS

Optimal Spermatogenesis Requires Synergistic Actions of Androgens and FSH

The data reviewed above indicate that androgens and FSH have distinct roles in spermatogenesis but that these hormones also act co-operatively and synergistically to promote maximal spermatogenic output (156, 203-204, 219).

Androgens and FSH co-operate by supporting different aspects of germ cell development, for example FSH stimulation of spermatogonial populations and androgen stimulation of spermiogenesis. FSH establishes a quantitatively normal Sertoli cell population, whereas androgen initiates and maintains sperm production, thus both hormones co-operate via independent functions to enable maximal spermatogenic output.

Both androgens and FSH facilitate normal Sertoli cell morphology and function, which are likely essential for the ability of Sertoli cells to support the maximum number of germ cells. Both hormones also promote germ cell survival, particularly of spermatocytes and round spermatids in the mid-spermatogenic stages in rodents (264), reviewed in (204). 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 (209).

There are many examples of synergy between testosterone and FSH, reviewed in (156, 203-204). It has been demonstrated in many experimental settings that testosterone and FSH can support spermatogenesis at a lower dose when the other is present, reviewed in (203). 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 (156, 219). An example of such synergism is the demonstration that, after acute suppression of either androgen or FSH in rats, approximately 10% of mature spermatids failed to be released at spermiation, whereas suppression of both hormones resulted in 50% of spermatids failing to spermiate (218). Both testosterone and FSH modulate the expression of many miRNA species in Sertoli cells, which likely mediate a large spectrum of proteomic changes important for Sertoli and germ cell function (220).

It should be noted that there are species differences in the response of spermatogenesis to combined androgen and FSH suppression, reviewed in (241, 443). In rodents, suppression of gonadotropins causes a decline in spermatogonial populations but spermatogenesis is primarily arrested at the spermatocyte stage (444). 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 (241, 435, 442).

The requirement for both testosterone and FSH to support normal spermatogenesis in men was revealed in studies by Matsumoto and colleagues (445-446) 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 12). These data suggested that, in association with undetectable FSH levels, increasing intratesticular androgen could partially restore sperm output (446). Using the same model, they initiated hFSH treatment when sperm counts were suppressed and showed that, in the presence of low intratesticular testosterone concentrations, FSH alone could partially restore sperm output (447). The latter study strongly suggests a role for FSH which appears to be able to synergise with low testosterone to stimulate sperm production in men.

Figure 12

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 hCG and hFSH were introduced singly into the treatment regime. Data from Matsumoto et. al. (reference 171, 172) and Bremner et. al. (reference 172).

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