II. Origin of the Spermatozoon

Table of Contents

• I. Introduction
• II. Origin of the Spermatozoon
• III. Epididymal Transit and Maturation
• IV. Spermatozoal Structure: Form to Function
• V. Physiological Considerations
• VI. Clinical Considerations: Semen Evaluation
• VII. Concluding Remarks

The life of a spermatozoon begins within the testes, unless, of course, one wishes to consider the embryonic origin of the primordial germ cells. The testis, an elaborately designed organ, is classically considered to possess two functions: (1) exocrine-spermatogenesis, and (2) endocrine-production of hormones important to spermatogenesis, sexual differentiation, development of secondary sex characteristics, and libido. Although this simplistic description provides one with the general concept of testicular function, it does not portray the extremely complex nature and elegant interplay of these two processes. Conventional descriptions convey the role of hypothalamic-and pituitary-derived hormones on regulation of testicular function, as well as feedback mechanisms required for homeostasis. Although such pathways are undoubtedly the key orchestrators of testicular function, emerging information is revealing a multitude of subcellular, molecular-mediated events that "cloud" our understanding of the events that actually occur within the testes. As with any area of study, the more learned we become about a topic, the more queries surface that require additional clarification. Such is the case with testicular function. Without question, a thorough understanding of testicular function will require a keen appreciation of the mechanisms by which genes and gene products are expressed and repressed [2-8]. As an example of the genetic complexity surrounding control of testicular function, new information has revealed that single nucleotide polymorphisms (SNPs) have been identified in the follicle-stimulating hormone (FSH) receptor gene of humans, resulting in a mutation of the FSH receptor (as is found on the surface of the Sertoli cell) that can influence its activity [9]. As another example, use of transgenic mice deficient in estrogen receptor genes has shown that estrogens are likely to play a more important role in testicular function than was once thought to be the case [10]. Similarly, use of mice with a selective androgen receptor knockout in Sertoli cells revealed that the androgen receptor in the Sertoli cell is an absolute requirement for normal spermatogenesis [11,12]. Furthermore, experimentation with germ cell-specific androgen receptor knockout mice revealed normal spermatogenesis, suggesting that germ cell androgen receptors may play different roles as the germ cells progress through spermatogenesis [13]. As seen here, to more fully understand what makes the testes tick, we must capitalize on the powerful molecular tools that have been developed in this capacity. Most of these types of studies are conducted in human and laboratory specimens, so it will be important to test the relevance to the horse.

Organization of the Testis

The testes are composed of testicular parenchyma encapsulated by the thick fibrous tunica albuginea. Extensions of the tunica albuginea penetrate the underlying testicular parenchyma, dividing it into numerous lobules (Fig. 1 and Fig. 2) [14]. The tunica albuginea is composed of collagen and elastic fibers, myoid cells, and a network of blood vessels (Fig. 3) [15-17]. Testicular parenchyma occupies nearly 90% of the total testicular mass in adult horses [16], and >70% of the testicular parenchyma is occupied by seminiferous tubules [18]. The interstitial component of the testis is located between seminiferous tubules and consists primarily of the Leydig cells intermingled with fibroblasts, lymphocytes, mast cells, blood vessels, lymphatic vessels, and extracellular matrix (Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9 and Fig. 10) [17-19]. Nerves are rarely seen in the testicular interstitium []17, but they are well established in the area of the spermatic cord leading to the testis.

Spermatogenesis

Spermatozoal production, i.e., spermatogenesis, occurs within the seminiferous tubules. Both ends of these highly coiled tubules open directly into the rete testis, such that the products (both cellular and non-cellular) of the seminiferous tubules are excreted into the rete testis and delivered to the excurrent duct system (e.g., the epididymis and ductus deferens). The numerous and tortuous seminiferous tubules, with a combined average length of 2419 m per testis in the stallion [20], are comprised of an epithelial wall, termed the seminiferous or germinal epithelium, and a lumen. The seminiferous epithelium consists of germ cells in various steps of development intermingled with Sertoli cells that serve to provide structural support and a nurturing source to the germ cells [17]. The Sertoli cells are anchored to the basement membrane, and extend to the lumen, of the seminiferous tubules. The seminiferous tubules are bordered by peritubular myoid cells (myofibroblasts) that, through peristaltic contractions, may aid in evacuation of luminal contents into the rete testis. These myoid cells are also considered to be involved in paracrine signaling events [21-23].

Figure 1. Drawings of the left equine testis with attached epididymis and spermatic cord in lateral view (A) and medial view (B). The vascular pattern over the surface of the testis is shown. The testis is normally oriented in the scrotum with the long axis directed horizontally. The epididymis courses over the dorsolateral aspect of the testis. (a) Cranial pole of testis (extremitas capitata). (b) Caudal pole of testis (extremitas caudata). (c) Appendix testis (vestigial remnant of the embryologic Müllerian [paramesonephric] duct). (d) Epididymal border of the testis, showing the entrance to the testicular bursa. (e) Proper ligament of the testis. (f) Ligament of the tail of the epididymis. (g) Reflected parietal tunic (tunica vaginalis parietalis). (h) Ductus deferens [14].

Figure 2. Schematic representation of the testis and associated epididymis. The thick tunica albuginea (a) covers the surface of the testis and also extends into the testicular parenchyma, thereby separating the parenchyma into lobules that contain both seminiferous tubules (b) and interstitial tissue. The luminae of the seminiferous tubules empty into the interconnecting tubules of the rete testis (f). The tubules of the extra-testicular portion of the rete testis connect to the epididymis through several efferent ducts (c) on the dorsocranial aspect of the testis. The epididymis forms a single duct that spans up to 45 m in length and consists of an initial segment (d), a caput (or head; e), a corpus (or body; g), and a cauda (or tail; h), which, in turn, connects with the ductus deferens (i).

A critical feature of the seminiferous epithelium is the formation of tight junctional complexes that develop between Sertoli cells, thereby physically dividing the seminiferous epithelium into basal and adluminal compartments (Fig. 11) [17]. This structural complex is termed the blood-testis barrier because it restricts direct access of blood-borne substances into the adluminal compartment. The described actions of the blood-testis barrier are (1) to segregate meiotic (except the earliest preleptotene spermatocytes) and post-meiotic germ cells from immunologic attack, because these germ cells are considered to be in an immunologically privileged site, and (2) to provide a unique microenvironment for the final stages of germ cell development within the adluminal compartment [24]. Although several blood barriers exist in the general body, there is no counterpart to the blood-testis barrier in the female. In the female, development of primary oocytes occurs before the immune system recognizes self, and the female does develop the counterpart to spermatids (i.e., ootids). In the male, germ cell development to spermatocytes, and spermatids does not occur until puberty. This is well after the immune system recognizes self and considers these cells as foreign. Especially intriguing is the molecular control over the transient disassembly of the blood-testis barrier to facilitate migration of germ cells from the basal into the adluminal compartment [24,25].

Spermatogenesis is an extremely complex process that involves germ cell proliferation, germ cell differentiation, and, paradoxically, programmed germ cell death (termed apoptosis). This lengthy process, 57 days in the stallion [20,26], is controlled by a vast array of messengers acting through endocrine, paracrine, and autocrine pathways [27-31].

Spermatogenesis not only involves transformation of undifferentiated diploid germ cells into highly differentiated and specialized haploid spermatozoa (Fig. 12 and Fig. 13) [17,26], but it also involves profound transcriptional modifications within the cells [3,32]. As a partial list, the finished product of spermatogenesis has (1) a one time chromosomal complement that is profoundly repackaged, (2) a newly formed and intricately designed flagellum, (3) the biogenesis of a highly complex secretory vesicle, the acrosome [33], and (4) retention of some mRNA (in a largely depleted cytoplasmic package) that likely has implications in fertilization and post-fertilization events [32,34,35].

Figure 3. The equine testis viewed in a gross cross-section (A), in a low-magnification histologic section (B), and a higher-magnification histologic section (C). (A) The testis is compromised of the tunica albuginea (capsule; TA) and testicular parenchyma (TP). The central vein (CV) has associated connective tissue. (B) The testicular parenchyma contains numerous tightly coiled seminiferous tubules (STs). (C) The interstitium between seminiferous tubules (STs) contains numerous Leydig cells (LCs), blood vessels (BVs), and lymphatic vessels (LVs). Scale bars = 10 mm in A,1 mm in B, and 30 µm in C [15-17].

Figure 4. Micrograph of cross-sections of equine seminiferous tubules and interstitial tissue, after toluidine blue staining of 0.5-µm Epon section. Stages I, III, VI, and VII of the seminiferous epithelial cycle are depicted, and show respective groupings of spermatogonia (A and B), primary spermatocytes (L, Z, and P), and spermatids (Sa, Sb1, Sc, Sd1, and Sd2) that are intimately associated with Sertoli cells (SCs) and contained by myoid cells (MCs) marking the outer limits of the seminiferous tubules. The interstitium between tubules is characterized by robust Leydig cells (LCs), lymphatic ducts (LDs), and blood capillaries (BCs). Scale bar = 10 µm.

Spermatogenesis is initiated by the differentiation of spermatogonia from a stem cell pool that is continually replenished for most of a stallion’s adult life. Under normal circumstances, this is a highly productive process, yielding on the order of 5 - 6 billion spermatozoa per day for an adult stallion. This translates into 30 - 40 trillion spermatozoa produced over the course of a stallion’s life. From an equally impressive view, an average healthy stallion produces 60,000 - 70,000 spermatozoa per second. These newly formed spermatogonia enter a proliferative phase, whereby continuous mitotic amplifications yield a dramatic increase in spermatogonial numbers. Interestingly, the cytoplasmic component of mitotic divisions is often incomplete, resulting in daughter cells that remain connected by intracytoplasmic bridges (Fig. 14) [36]. Such an arrangement permits direct communication within this syncytium of developing germ cells, thereby assisting in their synchronous development. After completion of spermatocytogenesis, the spermatozoa enter a meiotic phase, characterized by duplication and exchange of genetic information (i.e., genetic recombination) and two meiotic divisions that reduce the chromosome complement to form haploid round spermatids. It is during the meiotic stage that germ cells pass through the blood-testis barrier to enter the adluminal compartment. During the final phase of development, spermiogenesis, the round spermatids undergo a dramatic transformation that includes nuclear reshaping through chromatin compaction, creation of a flagellum, development of the acrosome, and considerable loss of cytoplasm (Fig. 13 and Fig. 14). The fully developed spermatids are released as spermatozoa into the lumen of the seminiferous tubules by a process termed spermiation (Fig. 15) [19,37]. It is during spermiogenesis that the germ cells may be most vulnerable to both structural and genetic defects [34].

Figure 5. Scanning electron micrograph of the equine testis revealing seminiferous tubules (STs) and Leydig cells (LCs). Tails of developing spermatids (T) can be seen projecting into the lumen of seminiferous tubules. Scale bar = 50 µm [19].

Figure 6. Transmission electron micrograph of equine Leydig cells found in testicular interstitium. Equine Leydig cells have an abundance of cytoplasm containing smooth endoplasmic reticulum (SER) and a large number of mitochondria (M). Gap junctions (GJs) for intercellular communication are located in the plasma membranes of two adjacent cells. The nucleus (N) is spherical and largely euchromatic, and has a distinct nucleolus (No). Scale bar = 3 µm [18].

Figure 7. The effect of stallion age on pigmentation of unfixed equine testicular parenchyma, as observed grossly. (A) Light parenchyma is from a post-pubertal stallion (2 - 3 yr). (B) Moderately dark parenchyma is characteristic of adult (4 - 5 yr) stallions. (C) Dark parenchyma characterizes aged stallions (13 - 20 yr) [18].

Cellular Associations During Spermatogenesis

The spermatogenic cycle, or cycle of the seminiferous epithelium, represents the series of changes in a given region of a seminiferous tubule between two appearances of the same developmental stages [38-41]. For instance, if spermiation were used as a reference point, the cycle would consist of all the cellular associations occurring within a given cross-section of a seminiferous tubule between two consecutive spermiations (Fig. 16). These cross-sectional cellular associations can be divided into eight distinct stages (Fig. 16, Fig. 17, Fig. 18 and Fig. 19) [19,42,43]. The spermatogenic cycle length is constant at 12.2 days in the stallion [20,26]. The spermatogenic wave, on the other hand, refers to the spatial, sequential order of stages along the length of a seminiferous tubule at a given point in time (Fig. 20) [42]. These two characteristics of the seminiferous epithelium can be defined because of the synchronous nature of development for cohorts of differentiating germ cells along the length of the seminiferous tubules. Histologic evaluation of the stages of the seminiferous epithelial cycle permits one to assess the efficiency of spermatogenesis [39,44].

Germ Cell Degeneration

Germ cell degeneration can be amplified in stallions with abnormal testicular function, but it is also a normal phenomenon in spermatogenesis [45]. In fact, "normal" spermatogenesis is a relatively inefficient process and has been reported to result in an estimated loss of 25 - 75% of the potential number of spermatozoa produced by spermiation in the rat [46,47]. In the stallion, germ cell degeneration is more profound during the physiologic breeding season than during the non-breeding season [41]. Developing spermatogonia are the most vulnerable to apoptotic degeneration (Fig. 21) [43]. It is possible that the "physiologic" form of germ cell degeneration is a homeostatic mechanism that prevents overloading the Sertoli cells, i.e., to maintain a fine balance in the germ cell:Sertoli cell ratio. Apoptosis is a well-defined physiological process of cell elimination [48], and the apoptotic process is required for normal spermatogenesis in mammals [49,50]. A protective role of luteinizing hormone (LH) against germ cell apoptosis has been reported in rats, based on expression of various apoptotic genes following immunoneutralization of LH in germ cells [51]. Apoptosis of spermatogonia and spermatocytes has been reported in normal stallion testes, a finding that is consistent with the expected time that germ cells may be susceptible to removal from the system [43]. Of interest, formation of the developing seminiferous tubules and the Sertoli cell (i.e., blood- testis) barrier coincides with increased germ cell apoptotic rates in stallions, providing evidence that apoptosis may play an intricate role in initiation of spermatogenesis [52]. As expected, these changes are coincident with gene expression patterns [53]. Studies involving rats indicate that FSH is an important regulator of this event [54,55].

Figure 8. Composite of the interstitium between seminiferous tubules (STs) and Leydig cells (LCs) in stallions at different ages in the middle (A, C, and E) or onset (B, D, and F) of the breeding season. (A and B) The post-pubertal (2 - 3 yr) stallion has large Leydig cell clusters in the interstitium. (C and D) The young adult stallion (4 - 5 yr) has larger clusters of Leydig cells that increase the density. (E and F) The aged stallion testis (13 - 20 yr) has mostly Leydig cells in its interstitium; however, some Leydig cells may contain a large accumulation of lipofuscin (Lf). No difference in Leydig cell structure is noted between the onset and middle of the breeding season. Scale bars = 75 µm in A, C, and Ea nd 25 µm in B, D, and F [18].