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 descended 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 joined to form the duct of the epididymis. This duct 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 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. Together with seminal vesicles, the prostate 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.

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.

SPERMATOGENESIS

Spermatogenesis represents the process by which precursors termed spermatogonia undergo a complex series of divisions to give rise to spermatozoa(4,5). This process takes place within the seminiferous epithelium which is a complex structure composed of germ cells and radially oriented supporting cells called Sertoli cells (Figure 5). 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. This photomicrograph illustrates the typical structure of the testis showing the seminiferous tubules containing the germ cells and Sertoli cells. The tubules are surrounded by thin plate-like contractile cells called peritubular myoid cells. The Leydig cells and blood vessels lie within the intertubular tissue.

Spermatogenesis can be divided into three major phases (i) proliferation and differentiation of spermatogonia, (ii) meiosis, (iii) spermiogenesis which represents a complex metamorphosis involved in the transformation of round spermatids arising from the final division of meiosis into the complex structure of the spermatozoon.

(i) Spermatogonial Renewal and Differentiation

These cells represent a population that divide by mitosis providing both a renewing stem cell population as well as spermatogonia that are committed to enter the meiotic process. The identification of different types of spermatogonia is complex due to a lack of definitive markers that can identify specific stages. To date, classification of these cells has depended on the features of their nuclei and, in particular, their chromatin patterns(5). The identification of the latter, which represents a crucial step in classification, is often obscured by poor fixation especially if the tissue is fixed in formalin. The ideal fixatives for testicular tissue are Bouin's or Cleland's solution. In general two main classes of spermatogonia can be identified in all mammals: Type A exhibiting fine pale staining nuclear chromatin and Type B with coarse chromatin collections found close to the nuclear membrane(8). In many mammals, the Type A spermatogonia can be divided into several subtypes that may represent different phases of proliferation and progression towards Type B spermatogonia. The Type B spermatogonia are generally agreed to represent the cells which differentiate and enter into the process of meiosis where they are called primary spermatocytes(9).

In the human and other primates, the Type A spermatogonia can be further divided into A dark (Ad) and A pale (Ap)(9) Some investigators have proposed that the Ad spermatogonia represent the reserve or non-proliferative spermatogonial population which can give rise to Ap(10-12) whereas others have suggested that the Ap spermatogonia are the true stem cell of the testis(13). Some of these differences arise from the difficulties in identifying spermatogonial cell types.

Spermatogonia do not separate completely after meiosis due to incomplete cytokinesis and remain joined by intercellular bridges(14). These intercellular bridges persist throughout all stages of spermatogenesis and are thought to facilitate biochemical interactions allowing synchrony of germ cell maturation.

Following damage to the seminiferous epithelium, some investigators have suggested new criteria which may facilitate the identification of the true spermatogonial stem cell within the epithelium. These criteria have emerged from studies of investigators engaged in transplantation of germ cells into the testis. Following the induction of cryptorchidism, the surviving spermatogonia, from which restoration of spermatogenesis is possible, show the presence of a6b1 integrin (15).

(ii) Meiosis

This process commences when Type B spermatogonia lose their contact with the basement membrane to form preleptotene primary spermatocytes. The preleptotene primary spermatocytes engage in DNA synthesis and condensation of individual chromosomes providing the appearance of thin filaments in the nucleus which identify the leptotene stage(15). 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. 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 repair(16).

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 synaptinemal 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 recognised 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(17,18).Meiotic maturation in the human takes about 24 days to proceed from the preleptotene stage to the formation of round spermatids. Several studies have identified key molecules that are necessary to allow meiosis to be completed. These include the synaptinemal complex protein (SCP1) and the chromosomal core protein (COR1)(19,20). Further, the heat shock protein, HSP 70-2, is required for desynapsis of the synaptinemal complexes and the completion of the first meiotic division(21).

(iii) Spermiogenesis

The transformation of a round spermatid into a spermatozoon represents a complex sequence of events that constitute the process of spermatogenesis. 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 are consistent between all species and consists (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 (Figure 7) (5, 21,22).

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 which migrates to come into contact with the nuclear membrane where it becomes applied as a cap-like structure over approximately 30-50% of the nuclear surface(22).

The nuclear changes involve a reorganisation of the nucleus and cytoplasm such that the nucleus comes into close apposition with the cell membrane in a region where it is covered by the acrosomal cap. Subsequently, there is a progressive condensation of chromatin to form progressively larger and more electron dense granules together with a change in the shape of the condensed nucleus. This change in shape varies significantly between species. The condensation of chromatin represents the morphological appearances of significant biochemical changes which result in the stabilization of DNA. These changes include the replacement of lysine-rich histones with transitional proteins which in turn are subsequently replaced by arginine-rich protamines(24,25). The resultant DNA becomes resistant to digestion by the enzyme DNase. Associated with these changes there is a marked decrease in nuclear volume and a cessation of gene transcription(26).

The formation of the tail commences early in spermiogenesis 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 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(27). As spermiogenesis proceeds, the outer dense fibres and fibrous sheath which characterize the regions of the sperm known as the mid and principle-piece are developed.

The formation of the mitochondrial sheath occurs at the time of the final reorganisation of the cytoplasm and organelles of the spermatid(5,22,23). 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). In the process of spermiation, the cytoplasm of the spermatid which has now migrated to a caudal position around the tail, is shed, most likely involving a process which requires the active participation of the Sertoli cell. 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(22). These cytoplasmic collections, termed residual bodies, which contain mitochondria, lipid and ribosomal particles are phagocytosed and moved to the base of the Sertoli cell where they are broken down by lysosomal mechanisms.

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.

(iv) 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 cycle of the seminiferous epithelium was defined by LeBlond and Clermont(28), 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 as identified by the periodic acid Schiff(PAS) stain (Figure 9). 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. This is 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 most mature spermatids are shed at stage VIII and this is reflected by the alteration in the transillumination patterns seen in the tubules (central representation of the tube). The stages at which proliferative events occur are shown as well as some key physiological events.

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 (17). 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 (29). Such observations amply confirmed the earlier studies of Perey and colleagues(30), that the stages of spermatogenesis were sequentially arranged along the length of the tubule (Figure 10). As the cycle progress, this arrangement resulted in a "wave of spermatogenesis" along the tubule. Regaud(30) interpreted his observations correctly by the statement "the wave is in space what the cycle is in time".

Figure 10. The pattern of the stages of spermatogenesis as they occur along the tubule are shown. Data based on Perey et. al. (30). Reproduced with permission from de Kretser and Kerr (1994) in "The Physiology of Reproduction" Ed E Knobil & J D Neill, Lippincott, Williams & Wilkins.

For many years, investigators believed that such a cycle did not occur in the human testis but the careful studies of Clermont(32) showed that human spermatogenesis could be subdivided into 6 stages. However unlike the rat, each stage often only occupied one quadrant of a tubule giving the disorganized appearance. By careful studies using tritiated thymidine injections into the testis, Clermont and Heller(18) 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(17).

The mechanisms that defined 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 recent demonstration that when rat germ cells were transplanted into the mouse testis, spermatogenesis proceeded at the normal rate for the rat, indicated that this rate is determined by intrinsic mechanisms within germ cells(33). Nevertheless, the Sertoli cell, because it extends from the base of the tubule to the lumen, restricts entry to the tubule because of the blood-testis barrier and is intimately associated with several generations of germ cells from spermatogonia to spermatids, is in a powerful position to influence the radially directed organization of cell associations. Given that the germ cells influence the metabolism of the Sertoli cells at each specific stage of the seminiferous cycle, the cell associations representing the stages of the seminiferous cycle may well be essential to allow successful completion of spermatogenesis.