Androgen action on spermatogenesis

5.5.1 Testicular androgen production, metabolism and transport

Testosterone is produced by the interstitial Leydig cells. The details and mechanisms of testosterone synthesis and secretion are presented in chapter 1. Testicular concentrations of testosterone can exceed those found in circulating blood up to 100-fold or beyond. It was thought initially that spermatogenesis requires high local amounts of testosterone. This view could not be corroborated and spermatogenesis in the rat can proceed in the presence of 5-10% of normal intratesticular androgen levels as described in the hallmark paper by Cunningham and Huckins (1979). Interestingly, it has also been observed that testosterone can inhibit certain populations of A-type spermatogonia in the rat model (Huang and Nieschlag 1986) and these observations have been corroborated in rat gonadal protection models (Meistrich and Shetty 2003). A rather obvious need for high local androgen concentrations results from the fact that sufficient peripheral testosterone levels and pulses must be provided by rapid secretion of testosterone from testis into blood. This may well require a very high local availability of testosterone.

It is commonly believed that testosterone and other steroids freely diffuse throughout the testis. A percutaneous testicular aspiration study in fertile men revealed that the testosterone concentration exceeded that of SHBG/ABP by about 200-fold (Jarow et al. 2001). Hence, a substantial surplus of testosterone exists within the male gonad. On the other hand, in genetically engineered mice lacking ganglioside synthetase, spermatogenesis was altered and Sertoli cells showed vac-uolization (Takamiya et al. 1998). This enzyme deficiency prevented testosterone transport within the testis. It is conceivable that an active transport mechanism for androgens operates within the testis and plays a role in the regulation of the spermatogenic process.

Testosterone is metabolized to DHT by testicular 5a-reductase activity. It is unclear at present to what extent testicular 5a-reduction of testosterone is relevant for spermatogenesis. DHT can stimulate spermatogenesis even quantitatively in mice (Singh et al. 1995) and rat (Chen et al. 1994). The latter study suggested that DHT is more potent than testosterone for stimulation of spermatogenesis. On the other hand, finasteride treatment did not alter spermatogenesis in volunteers (Kinniburgh etal. 2001; Overstreet etal. 1999). Also, administration of finasteride to immature and adult rats had no effect on testes weights and spermatogenesis (Rhoden etal. 2002).

Testosterone within the testis is aromatized to estradiol. Whether estradiol directly regulates spermatogenesis is unknown. The occurrence of testicular atrophy in estradiol receptor-deficient mice has been attributed to back-pressure effects of lack of fluid resorption in the efferent ducts (Hess etal. 1997). Aromatase activity is present in Sertoli cells but has also been found in germ cells (spermatocytes, spermatids and sperm) (Carreau et al. 2001). The use of aromatase inhibitors has not resulted in clear suppression of spermatogenesis (Turner etal. 2000) except in the studies by Shetty et al. (1997; 1998) in bonnet monkeys. This group reported impaired spermiogenesis and altered sperm chromatin condensation on the basis of flow cytometric analysis. Mice bearing lack of estrogen receptor expression or aromatase expression or overexpression of aromatase (Murata et al. 2002) display various degrees of spermatogenic disturbance. Clinically, isolated cases with estrogen receptor deficiency or aromatase deficiency did not provide clues as to whether spermatogenesis is causally affected by estradiol (O'Donnell etal. 2001b).

5.5.2 Testicular androgen concentrations and spermatogenesis

The precise quantitative relationship between local testosterone concentrations and spermatogenesis has been the subject of numerous studies and debates (Rom-merts 1988; Sharpe et al. 1988a; 1988b). It has been suggested that under testosterone alone, approximately 30% of testicular androgen concentrations are needed to quantitatively support spermatogenesis in the rat. In this study testosterone was administered to Leydig cell-depleted rats, thus also demonstrating that testosterone is the only Leydig cell factor relevant for maintenance of spermatogenesis. In a specific experimental setting in which FSH secretion was retained, quantitative maintenance and restoration of spermatogenesis could be achieved with only 10% of normal testicular testosterone levels in the rat model (Rea et al. 1986a; 1986b). Generally, however, it has been difficult to establish a linear relationship between testosterone concentrations and the number of germ cells being produced. Several studies related to the issues of testicular androgen concentration and spermatoge-nesis are available for primates.

In nonhuman primate studies, experimental hypogonadotropism and hypogo-nadism were induced by administration of supraphysiological amounts of testosterone (Narula etal. 2002; Weinbauer etal. 2001a; 2001b) or treatment with a GnRH antagonist and testosterone substitution (Weinbauer et al. 1988). A clinical contraceptive study is also available in which volunteers were exposed to testosterone alone or testosterone plus DMPA (McLachlan et al. 2002b). Study durations were 15-26 weeks in nonhuman primates and 12 weeks in the clinical study. Animals

Testosterone

Dihydrotestosterone (DHT)

] T before

DHT before DHT after

Testosterone

Dihydrotestosterone (DHT)

] T before

DHT before DHT after

GnRH GnRH + 40 mg T GnRH + 200 mg T GnRH GnRH + 40 mg T GnRH + 200 mg T

Fig. 5.4 Testicular concentrations of testosterone and DHT in biopsates of cynomolgus monkeys before and after 15 weeks of treatment with an GnRH antagonist (GnRH) alone or in combination with either 40 mg or 200 mg testosterone buciclate (T). Asterisk denotes significant differences compared to baseline (before). Note that DHT levels did not differ significantly before and after treatments although animals treated with GnRH antagonist alone were azoospermic and testosterone-supplemented animals were severely oligozoospermic. Data modified from Weinbauer etal. (1988). The observations on DHT have been confirmed in a recent study in gonadotropin-suppressed men (McLachlan etal. 2002b).

GnRH GnRH + 40 mg T GnRH + 200 mg T GnRH GnRH + 40 mg T GnRH + 200 mg T

Fig. 5.4 Testicular concentrations of testosterone and DHT in biopsates of cynomolgus monkeys before and after 15 weeks of treatment with an GnRH antagonist (GnRH) alone or in combination with either 40 mg or 200 mg testosterone buciclate (T). Asterisk denotes significant differences compared to baseline (before). Note that DHT levels did not differ significantly before and after treatments although animals treated with GnRH antagonist alone were azoospermic and testosterone-supplemented animals were severely oligozoospermic. Data modified from Weinbauer etal. (1988). The observations on DHT have been confirmed in a recent study in gonadotropin-suppressed men (McLachlan etal. 2002b).

and subjects had markedly suppressed (bioactive) LH levels and were rendered severely oligozoospermic or azoospermic. All studies yielded a very similar finding, e.g. testicular levels of testosterone/androgen lacked any correlation to either testicular germ-cell numbers or numbers of sperm in the ejaculates. Equally surprising are those observations that the testicular levels of DHT did not differ significantly from control or baseline levels study (Fig. 5.4) (McLachlan et al. 2002b; Narula et al. 2002; Weinbauer and Nieschlag 1998; Weinbauer et al. 1988) nor did the levels of 5a-androstane-3a. In a shorter-term study of 16 and 25 days exposure to GnRH antagonist in cynomolgus monkeys (Zhengwei etal. 1998a; 1998b), no significant change of testicular testosterone was seen but a clear reduction of germ cell numbers.

Given the need for testosterone in spermatogenesis, it is unclear at present how severe spermatogenic suppression could be achieved in the above studies albeit having significant amounts of testicular testosterone and unchanged amounts of testicular DHT remaining. It is theoretically possible that sampling of testis tissue and the time elapsed until snap-freeze was a confounding factor (Maddocks and Sharpe 1989). However, if so, this confounding factor is surprisingly reproducible between different laboratories and experiments. It can also be considered that in those studies with testosterone administration, some testosterone diffused into the testis although this explanation does not hold for the study using GnRH antagonist (Weinbauer et al. 1988; 1998). Since the primate testis has a lobular architecture with much connective tissue and several layers of myoid cells surrounding the seminiferous tubule, it might also be that androgens are trapped in the involuting gonad and are locally bound and retained but are biologically inactive.

Certain experimental paradigms of testicular damage exist for rodents in which "high" intratesticular testosterone exerts an inhibitory effect on spermatogonial development and repopulation of seminiferous tubules. 2,5-hexanedione damages Sertoli cells in rats and interrupts spermatogenesis at the spermatogonial level in the majority of tubules. However, suppression of testicular testosterone concentrations using GnRH agonist treatment was associated with spermatogonial activation and repopulation in a large proportion of damaged seminiferous tubules (Boekelheide and Schoenfeld 2001). Studies using gonadal protection approaches in rats also indicated that the suppression of Leydig cell function can advance spermatogenic recovery in the rat model (Meistrich and Shetty 2003). In the juvenile spermatogonial depletion (jsd) mouse in which only one wave of spermatogenesis takes place, followed by sterility, suppression of testicular testosterone initiated spermatogonial activation (Matsumiya etal. 1999). In this study administration of a GnRH antagonist further reduced testis size but clearly increased the number of differentiating seminiferous tubules (spermatogonia ^ spermatocytes).

5.5.3 Testicular androgen receptor and sites of androgen action

The testicular androgen receptor is only expressed in somatic cells (Fig. 5.5). Sertoli cells, Leydig cells and peritubular cells have been shown to express the androgen receptor (van Roijen et al. 1995). Whether androgen receptor is really present in some rat elongating spermatids has remained enigmatic (Vornberger etal. 1994). It is assumed that the stimulatory effects of testosterone upon spermatogenesis are indirect and mediated via somatic cells. Alternatively it has also been suggested that testosterone can act directly upon germ cells and is transported into these cells by androgen binding protein (ABP). Observations that rats made transgenic for ABP and expressing high testicular androgen levels, have disturbed spermatogenesis (Joseph et al. 1997a; 1997b; Larriba et al. 1995) would suggest active testosterone could be bound and inactivated by high ABP levels (Fig. 5.5).

Studies in nonhuman primates revealed that testosterone induced the appearance of a-smooth muscle actin in the peritubular cells of the testis (Schlatt et al. 1993). This observation strongly suggests that testosterone initiates the contractile function of these cells. These data suggest a very specific effect of testosterone on the differentiation of testicular primate cells. In these studies it was also observed that testosterone but not FSH induced an "adult-type" actin distribution in the Sertoli cells (Schlatt et al. 1995). Generally, testosterone appears to be a differentiation

Fig. 5.5 Testicular production, distribution and action of testosterone (T). Testosterone is produced in Leydig cells and acts on specific receptors on Leydig cells, peritubular cells and Sertoli cells. Germ cells lack androgen receptors. Once inside the seminiferous tubule, testosterone can be bound to androgen binding protein (ABP) for further transport. The Testosterone/ ABP complex has been reported to be internalized by germ cells. Within the seminiferous tubules testosterone is metabolised into DHT and estradiol. Whether these conversions of testosterone are essential for spermatogenesis is not entirely clear. Although it is reasonable to assume that testosterone induced the formation and secretion of essential pro-spermatogenic factors in peritubular cells and Sertoli cells, the nature of these factors is still to be discovered.

Fig. 5.5 Testicular production, distribution and action of testosterone (T). Testosterone is produced in Leydig cells and acts on specific receptors on Leydig cells, peritubular cells and Sertoli cells. Germ cells lack androgen receptors. Once inside the seminiferous tubule, testosterone can be bound to androgen binding protein (ABP) for further transport. The Testosterone/ ABP complex has been reported to be internalized by germ cells. Within the seminiferous tubules testosterone is metabolised into DHT and estradiol. Whether these conversions of testosterone are essential for spermatogenesis is not entirely clear. Although it is reasonable to assume that testosterone induced the formation and secretion of essential pro-spermatogenic factors in peritubular cells and Sertoli cells, the nature of these factors is still to be discovered.

factor for somatic testicular cells. Differentiation of Leydig cells from fibroblast precursors is also under LH/androgen control (Teerds etal. 1989). In the rat model, testosterone is also involved in Sertoli cell-spermatid adhesion (McLachlan et al. 2002b for review).

The expression of the androgen receptor varies in relation to the spermatogenic stage of spermatogenesis. In the rat, highest expression and also highest levels of this androgenic steroid were observed in spermatogenic stages VII and VIII (Bremner et al. 1994). In these stages the spermatids finally elongate. Cytometric data for human testis suggest a peak of androgen protein expression immediately after the final stage of spermatid maturation (stage III) (Suarez-Quian et al. 1999). The expression of testicular androgen receptor is under the control of both testosterone and FSH.

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