Organisation and kinetics of spermatogenesis

5.2.1 Basic and common features

Spermatogenesis comprises the development of sperm from stem spermatogonia. This process encompasses the multiplication and differentiation of stem cells into differentiated and proliferating germ cells, the redistribution of genetic information during meiosis and the maturation and differentiation of haploid germ cells. Following proliferation of A-type spermatogonia into B-type spermatogonia, these cells enter meiosis and are termed spermatocytes and after completion of reduction divisions, the emerging haploid germ cells are denoted as spermatids. These sper-matids undergo a major and complex morphological, structural and functional maturation and development process resulting in the production of spermatozoa. Terminally elongated spermatids (testicular spermatozoa) do not exhibit progressive motility but are capable of fertilization as evidenced by in vitro fertilization techniques.

Among mammals, the entire process of spermatogenesis is topographically determined andduring the various developmental phases, only specific germ cell associations assemble. The specific germ cell associations are known as stages of spermatogenesis. Every stage is thought to derive from one stem cell and hence represents a cell clone with intercellular bridges remaining that allow continued cellular communication (Alastalo etal. 1998;Ren and Russell 1991).The commonlyused staging system is based upon the morphology of the developing acrosome in spermatids (Clermont 1972). It is obvious that the subdivision of the spermatogenic process into various stages is somewhat arbitrary and, hence, that the number of spermatogenic stages varies between species. Dividing the spermatogenic process into stages is critical since many processes and actions occur physiologically in a stage-specific manner. Conversely, disturbances of spermatogenesis imposed by endocrine deficiencies or exposure to toxicants - at least initially-become frequently manifest in a stage-specific response pattern.

Germ cell development is tightly coupled to intratubular somatic cells, the Sertoli cells. These cells possess highly specialised cytological and structural features enabling them to functionally and also physically support germ cell development and movement from the basement to the tubular lumen. Sertoli cells divide during the prepubertal period until the establishment of the blood testis barrier. Two major functions are assigned to the Sertoli cell: to determine adult testis size and sperm production and to enable and coordinate germ cell proliferation and development. Evidence for the former is compelling since it has been demonstrated under normal and pathological conditions that the number of Sertoli cells correlates precisely with the number of sperm produced (de Franca etal. 1995).

Whereas previously it was believed that the Sertoli cells govern the spermatogenic process it is now thought that this ability may well reside in the germ cell itself. Modulation and elimination of specific germ cell types by administration of specific toxins provoked stage-specific alterations of Sertoli cell inhibin secretion (Jegou 1993; Sharpe 1994). This view is further corroborated by xenogeneic germ cell transplantation studies in mice testes being injected with germ cells derived from rats (Clouthier etal. 1996). In these testes - although only mouse Sertoli cells could be found (Russell and Brinster 1996) - mouse and rat spermatozoa were produced simultaneously and the mouse-specific and rat-specific timing of spermatogenesis was retained (Franca etal. 1998). The latter is particularly interesting given the fact that the entire spermatogenic process requires approx. 35 days in mice but approx. 50 days in rats.

During spermatogenesis the developing germ cells are relocated from the basement towards the lumen of the seminiferous tubule, and during spermiation, the spermatozoa are released into the lumen. In order to be able to propel the sperm within the testis and into the epididymis via the excurrent testicular ducts, the seminiferous tubules contract in a peristaltic manner (Assinder et al. 2002; Santiemma et al. 2001; Tripiciano et al. 1999). These contractions are believed to proceed along the length of the seminiferous tubules and are induced by the peritubular cells. These cells exhibit the features of myoid cells and contain a-smooth muscle actin, panactin, smooth muscle myosin and desmin (Holstein et al. 1996) and their contractions are controlled by oxytocin and endothelin. Isolated rat spermatogenic stages VII-VIII segments were most responsive to oxytocin (Harris and Nicholson 1998). Oxytocin and endothelin have also been found in human testes (Ergun etal. 1998).

Description and evaluation of the spermatogenic process can be qualitative and quantitative. Qualitatively normal spermatogenesis refers to the presence of all germ cell types and spermatogenic stages. Quantitatively normal spermatogenesis implies that the numbers of all germ cell types are produced and present in normal quantity. This distinction is very important for the discussion of the relative role of testosterone and FSH in spermatogenesis and for the assessment of toxic actions on spermatogenesis.

5.2.2 Species-specific features

Although the spermatogenic process has common and universal features, substantial differences must also be kept in mind. For the purpose of this chapter, the discussion of species-specific aspects is largely confined to a comparison between rodents (mouse, rat, hamster) and primates (nonhuman primates and man).

The system of spermatogonial renewal is quite different between rodents and primates. Rodent stem spermatogonial development is well described and several generations of differentiating and dividing A-type spermatogonia exist prior to formation of B-type spermatogonia (de Rooij and Grootegoed 1998). In the primate, stem spermatogonia (Ad(ark)-type spermatogonia) are easily recognized and only one generation of renewing spermatogonia, i.e. (Ap(a]e)-type spermatogonia), has been described (Meistrich and van Beek 1993). The precise relationship between Ad-type and Ap-type spermatogonia and their kinetics are still under investigation. Currently the view predominates that the Ap-type spermatogonia - following division - provide one daughter cell to enter the spermatogenic process and the other daughter cell to replenish Ad-type population if needed. Conversely, Ad-type sper-matogonia - which rarely divide in the intact testis - are considered to replenish Ap-type spermatogonia in case of severe spermatogonial depletion, e.g. following testicular irradiation (van Alphen etal. 1989).

Among rodents, a tubular cross-section is occupied by a single spermatogenic stage (single-stage arrangement), whereas in primates a full range of arrangements comprising predominantly single-stage tubules, predominantly multi-stage tubules (>1 spermatogenic stage/tubular cross-section) and intermediate arrangements have been described (Wistuba etal. 2003). In New World monkeys, hominoids and man, tubules are predominantly multi-stage but are predominantly single-stage in macaques and intermediate in baboon. The human multi-stage arrangement has been suggested to derive from a helical arrangement of spermatogenic stages (Schulze and Rehder 1984; Zannini et al. 1999) but this view has also been challenged (Johnson et al. 1996). Alternatively, it might merely be the clonal size that determines whether a particular spermatogenic stage entirely occupies a tubule cross-section or not, i.e. whether the tubule is single- or multi-stage. The observation that neotropical primate testes - unlike those from Old World monkeys but similar to man - have predominantly multi-stage tubules implies that this feature has been developed several times during evolution and does not represent a selection criterion for spermatogenesis.

Interestingly, and contrary to previous beliefs, the single-stage vs multi-stage arrangement is not related at all to spermatogenic efficacy, i.e. germ cell loss during meiosis and spermatid maturation (Wistuba etal. 2003). For cynomolgus monkeys and man this has also been shown earlier by the use of unbiased stereological techniques for cell enumeration (Zhengwei et al. 1998). Hence the differences in testicular germ cell production and sperm output are now believed to be determined by the number of spematogonia entering meiosis and this aspect can be species-specific.

In terms of the number of spermatogenic stages, 14 stages are used for the rat, 12 for the hamster, 8 for the mouse, 6 or 12 for marmoset, 12 for macaques and 6 for chimpanzee and man (Clermont 1972; Millar et al. 2000; Smithwick et al. 1996; Weinbauer et al. 2001a). Originally 12 spermatid development stages were described for man (Clermont and Leblond 1955) and this has been reduced to 6 spermatogenic stages for practical reasons (Clermont, 1969; Fig 5.1). The human 6-stage classification has been applied to other primates and is useful for comparative studies (Dietrich etal. 1986; Wistuba etal. 2003).

The succession of all given stages is denoted as the cycle of spermatogenesis and the duration of a spermatogenic cycle has been determined using 3H-thymidine, 5-bromodeoxyuridine or the depopulation/repopulation of germ cells following testicular irradiation. In terms of the duration of one spermatogenic cycle, it is 12-14 days for the rat, 10 days for the hamster, 7-9 days for the mouse, 17 days in the Chinese hamster, 10 days for marmosets, 9-11 days for macaques, 14 days for chimpanzees and 16 days for man (Clermont 1972; Millar et al. 2000: Smithwick et al. 1996; Weinbauer and Korte 1999). For the completion of the entire spermatogenic process, i.e. formation of sperm from stem cell, between 4 and 4.5 spermatogenic cycles are needed.

Reproductive hormones do not influence the frequency of spermatogenic stages and the duration of the spermatogenic cycle (Aslam et al. 1999) whereas

Fig. 5.1 Schematic representation of the spermatogenic process and the six spermatogenic stages in men. The succession of all six stages requires approx. 16 days. Spermiation takes place during stage II^III transition. If stage III is taken as the starting point for the next spermatogenic cycle, the duration of the entire spermatogenic process from renewing spermatogonium (Ap, hatched box) to fully elongated spermatid (Sd2) would require four spermatogenic cycles, i.e. approx. 64 days. If the renewing spermatogonia in stage I are taken as starting point, the duration of the spermatogenic process would require 4.4 - 4.6 spermatogenic cycles. The authors favour the former approach. The human stage classification system has been used for other nonhuman primates (Aslam etal. 1999; Dietrich etal. 1986; Wistuba etal. 2003).

Fig. 5.1 Schematic representation of the spermatogenic process and the six spermatogenic stages in men. The succession of all six stages requires approx. 16 days. Spermiation takes place during stage II^III transition. If stage III is taken as the starting point for the next spermatogenic cycle, the duration of the entire spermatogenic process from renewing spermatogonium (Ap, hatched box) to fully elongated spermatid (Sd2) would require four spermatogenic cycles, i.e. approx. 64 days. If the renewing spermatogonia in stage I are taken as starting point, the duration of the spermatogenic process would require 4.4 - 4.6 spermatogenic cycles. The authors favour the former approach. The human stage classification system has been used for other nonhuman primates (Aslam etal. 1999; Dietrich etal. 1986; Wistuba etal. 2003).

toxicants (Rosiepen et al. 1995) or vitamin A deficiency/replenishment (Bartlett etal. 1990b; Siiteri etal. 1992) can do so. Forexample, 2,5-hexandione, a neurotoxin and Sertoli cell toxicant disrupting microtubule arrangements (Boekelheide et al. 2003), prolonged the duration of one spermatogenic cycle in the rat by one day. In the vitamin A depleted model, germ cell progression is arrested at the level of preleptotene spermatocytes but is restarted in most tubules simultaneously during vitamin A replacement. As a consequence, at a given later time point, most of the seminiferous tubules are in the same spermatogenic stage quite different from the normal stage distribution. However, this synchrony is lost over approximately 10 spermatogenic cycles and frequency distribution of spermatogenic stages returned to normal, strongly suggesting a change in the timing of the relative duration.

5.3 The hypothalamo-hypophyseal-testicular circuit

The hypothalamus-pituitary-testis circuit represents the core unit for the maintenance of the endocrine balance and fertility. Testicular functions, i.e. production of testosterone and of spermatozoa, are entirely subject to regulation by endocrine factors derived from the brain. Gonadotropin-releasing hormone (GnRH) is secreted from the hypothalamus and stimulates the synthesis and release of the gonadotropic hormones luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary gland (Fig. 5.2). LH acts on testicular Leydig cells and governs the production and secretion of testosterone by these cells. Within the testis, testosterone acts on peritubular cells that surround the seminiferous tubules and on the somatic Sertoli cells within the seminiferous epithelium. Beyond that, testosterone exerts a variety of physiological effects in the periphery and, in fact, androgen receptors have been detected in about 40 organs of the cynomolgus monkey (Dankbar etal. 1995).

FSH acts directly within the seminiferous tubules. In the immature testis FSH can also stimulate Leydig cell production. These stimulatory effects have been observed in the absence of endogenous LH (Haywood et al. 2003) and are mediated via the FSH receptor. It seems that the FSH receptor is involved in Leydig cell functional maturation and reduced peripheral testosterone levels, but increased tes-ticular testosterone concentrations were observed in FSH receptor-deficient mice (Krishnamurthy et al. 2001). In combination with LH/hCG activity, FSH potentiates Leydig cell testosterone production in the immature primate testis (Schlatt etal. 1995). The factor(s) that mediate the effects of FSH on immature Leydig cells are yet unknown.

The secretion of GnRH and gonadotropic hormones is controlled by testicular steroid and protein factors. Testosterone is the major steroid eliciting a negative feedback effect on LH and FSH secretion in the male. An additional feedback loop

Hypothalamus

GnRH

Pituitary

Inhibin B FSH / \ LH

f Seminiferous^ ^

( Leydig V

Testis

V cf

;li J

Peripheral effects

Fig. 5.2 Hypothalamic-hypophyseal-testicular communication in primates. Gonadotropin-releasing hormone (GnRH) stimulates the synthesis and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the pituitary. LH acts on Leydig cells and induces the synthesis and release of testosterone (T). Testosterone acts within the testis and -in the periphery - exerts an inhibitory feedback effect on hypothalamic GnRH secretion. Hence testosterone is the main regulator of LH secretion and also of FSH secretion. In part, the inhibitory effects of testosterone are mediated via aromatization of testosterone into estradiol (E) in the brain. For FSH an additional inhibitory feedback loop operates via testicular inhibin B at the level of the pituitary. Unlike LH, FSH acts on spermatogenesis directly. In the immature testis, FSH activity also stimulates Leydig cell differentiation and testosterone production via yet unknown factor(s) (denoted by dashed line and question mark).

has been described for FSH that is mediated by the effects of inhibins (de Kretser and Phillips 1998). Activin and follistatin are also involved in FSH feedback regulation but act more as local regulators rather than endocrine factors. The actions of testosterone can follow 5a-reduction to dihydrotestosterone (DHT) or aromatization to estradiol. In primates, negative feedback actions can be exerted at the hypothalamic and at the pituitary levels. It would appear, however, that testosterone predominantly acts via hypothalamic action (Fingscheidt etal. 1998; Veldhuis etal. 1997) whereas inhibin directly influences gonadotropins at the hypophyseal level in vivo and in vitro (Fingscheidt et al. 1998; Ramaswamy et al. 1998). Activins selectively stimulate FSH secretion and follistatin binds to activin and presumably determines and regulates activin-associated effects through this mechanism (McConnell etal. 1998). The physiological relevance of activin for spermatogenesis is strongly indicated by the observation that over-expression of follistatin is associated with spermatogenic defects, reduced testis size and reduced fertility in mice (Guo etal. 1998).

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