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Inhibition of Spermatogonial Differentiation by Testosterone

From the Department of Experimental Radiation Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas.

Correspondence to: Marvin L. Meistrich PhD, M.D. Anderson Cancer Center, Department of Experimental Radiation Oncology–66, 1515 Holcombe Blvd, Houston, TX 77030 (e-mail: meistrich{at}mdanderson.org).

Received for publication July 11, 2002; accepted for publication September 19, 2002.

As will be described later, it is not clear whether the direct action of testosterone is to block an actual step of differentiation of the spermatogonia or to cause the apoptosis of the spermatogonia prior the step at which they would differentiate. However, throughout this review we will use the concept of "inhibition of spermatogonial differentiation" to encompass both possibilities.

Blocks of Spermatogonial Differentiation

The stem spermatogonia, designated A_s, maintain their numbers by self-renewal, and some differentiate to form by sequential divisions A_pr (A-paired), and A_al-4 and A_al-8 (A-aligned) spermatogonia, which go on to produce A₁ spermatogonia.

This differentiation may be blocked in 3 ways. In one way, which is the focus of this review, undifferentiated spermatogonia proliferate but their numbers remain relatively constant because of apoptosis (Figure 1) (Allard and Boekelheide, 1996; Shuttlesworth et al, 2000). We will call this the proliferation-apoptosis (PAp) block to distinguish it from the other 2 ways. The second type of block in spermatogonial differentiation, which is caused by vitamin A deprivation, is characterized by spermatogonial differentiation to the A_al stage, but then proliferation ceases and spermatogonia can remain at this stage for a period of only several weeks (van Pelt and de Rooij, 1990) and is designated Ar (arrest). In the third kind of block, which is observed in several types of transgenic mice, including bax-deficient, or bcl-2-overexpressing, or glial cell line-derived neurotropic factor (GDNF)-overexpressing mice (Knudson et al, 1995; Furuchi et al, 1996; Meng et al, 2000), type A spermatogonia proliferate and accumulate but produce few differentiated cells, and is designated proliferation-accumulation (PAc).

View larger version (11K): [in this window] [in a new window]   Figure 1. Outline of stem cell kinetics in (A) normal rodents and (B) rodents with a proliferation-apoptosis block in spermatogonial differentiation as described for toxicant-treated rats, cryptorchid mice, and some mutant mice. In normal rodents, no apoptosis is observed at these stages, and the Aal and some Apr spermatogonia are induced to undergo differentiation into A1 spermatogonia at stage VII–VIII of the cycle of the seminiferous epithelium. In the rodents with the PAp block, spermatogonia of all clonal sizes undergo apoptosis, with the probability of undergoing an apoptotic event, as opposed to a mitotic division, increasing with chain length.

The precise relationship between these 3 blocks in rodents and the clinical phenotype of spermatogonial arrest in humans, which is often the result of hypogonadotropism (Johnsen, 1970), is not known. However, the types of spermatogonia present in humans and their proliferative status have not been studied.

Conditions Causing the Proliferation-Apoptosis Block in Spermatogonial Differentiation

A variety of testicular toxicants produce similar testicular histology in rats consistent with the PAp type of block. These agents include hexanedione (Boekelheide and Hall, 1991), boric acid (Ku et al, 1993), radiation (Kangasniemi et al, 1996), procarbazine (Meistrich, 1999), dibromochloropropane (DBCP; Meistrich, unpublished results), and indenopyridines (Hild et al, 2001). The type A spermatogonia proliferate in atrophic tubules but they do not accumulate because they continue to be lost by apoptosis many months after the original acute or subchronic exposure. Atrophic tubules with actively dividing stem type A spermatogonia were also observed in testis cross-sections from 27-month-old Brown-Norway rats (Schoenfeld et al, 2001). The failure of these cells to differentiate is in part responsible for the decline in spermatogenesis with age in these rats.

In contrast to that of rats, brief exposures to such toxicants do not induce such a block in spermatogonial differentiation in mice. Whereas 3.5 Gy of irradiation was sufficient to induce this block in LBNF₁ rats, mouse spermatogonia maintain their ability to differentiate even after doses of 12 Gy (Meistrich et al, 1978). But a block in spermatogonial differentiation can be induced by continuous elevation of temperature. In cryptorchid C57BL/6 mouse testes, spermatogenesis fails to progress past the A_al spermatogonial stage (Haneji et al, 1983); these cells actively proliferate but die by apoptosis (de Rooij et al, 1999). Similar blocks in the differentiation of A spermatogonia were also observed in jsd (juvenile spermatogonial depletion) mice (Beamer et al, 1988) and in Sl^17H mice, which have an altered form of stem cell factor in Sertoli cells (Brannan et al, 1992). In these mice, an initial wave of spermatogenesis is not maintained, so that the adult testis tubules contain only Sertoli cells and type A spermatogonia; the latter proliferate but die by apoptosis (de Rooij et al, 1999). In addition, certain other mouse mutants, including XO-Sxr^b (Sutcliffe and Burgoyne, 1989) and Dazl (Schrans-Stassen et al, 1999), have PAp blocks in spermatogonial differentiation but they differ from the above 2 models in that this condition is apparent by postnatal day 10 and there is no initial wave of spermatogenesis.

It was indeed surprising that such a wide variety of toxicant exposures, conditions, and genetic mutations produced such a similar phenotype. For example, some of these toxicants, such as irradiation, are believed to act directly on germ cells (Lee et al, 1999), whereas other toxicants, such as hexanedione, are believed to act on Sertoli cells. Furthermore, at least in some instances, the block to spermatogonial differentiation does not begin to develop for almost 6 weeks after the insult. Both these observations imply that the block to spermatogonial differentiation is not a direct consequence of the initial event, but that different initiating events produce a common outcome, which in turn, leads to the block.

Characterization of the Proliferation-Apoptosis Block of Spermatogonial Differentiation

Although the PAp blocks in spermatogonial differentiation caused by different agents have much in common (Figure 1), they show some quantitative differences in terms of the stage to which spermatogonia differentiate before undergoing apoptosis.

The type A spermatogonia in atrophic testes were first identified following exposure of either Sprague-Dawley or Fischer F344 rats to hexanedione (Boekelheide and Hall, 1991). Stem cells (isolated type A spermatogonia), although reduced in number from controls, still constituted a substantial proportion of these remaining A spermatogonia (Allard et al, 1995). The A spermatogonia were in active proliferation, but their numbers remained constant because they underwent apoptosis (Allard and Boekelheide, 1996). Calculations based on numbers of stem cells and total spermatogonia indicated that the cells were progressing to the A₂ or A₃ spermatogonial stage (Allard and Boekelheide, 1996).

In contrast, direct, whole-mounted tubule analysis of mitotic clones of A spermatogonia in irradiated LBNF₁ rats revealed that most of the clones were isolated or paired A spermatogonia and few had a clone size greater than 4, indicating that they were early progeny from the stem cells (Shuttlesworth et al, 2000). Very few clones progressed to become A_al-8 and A_al-16, which are the clone sizes that most often undergo differentiation in normal rats (Figure 1A) because the probability of apoptosis increased as clone size increased (Figure 1B). Thus, failure of spermatogonia to differentiate appeared to be a consequence of their undergoing apoptosis first.

In jsd, Sl^17H, and cryptorchid mice, the clones of A spermatogonia in whole-mounted tubules were arranged as 1 to 16 cells (de Rooij et al, 1999). There were appreciable and similar numbers of clones of A_al-8 and A_al-16 in all 3 models. These undifferentiated A spermatogonia were proliferating, but they did not accumulate, and the larger clones in particular underwent apoptosis. Because the clone sizes indicate that spermatogonia develop to the point at which the A_al cells should differentiate into A₁ spermatogonia, the failure to do so indicates the lack of a signaling system rather than prior apoptosis.

The difference in numbers and stage of development of spermatogonia between the irradiated rat and the mouse models appears to be real because the same methodology was employed. It is not known whether these differences are due to how mice and rats respond to blocks at the spermatogonial level or whether differences in the cause of the blocks. The difference in stage at which the block was reported to occur in irradiated vs hexanedione-treated rats could be a result of the different analytical methods employed, in addition to the possible contributions of rat strain or the nature of the original toxic insult.

Hormone Levels During the Proliferation-Apoptosis Block to Spermatogonial Differentiation

As is typical in cases of testicular tubular atrophy, FSH and luteinizing hormone (LH) levels rise in most cases in which only type A spermatogonia remain in the tubules. FSH levels were elevated 1.5-fold to 2-fold and LH levels were elevated 2-fold to 4-fold after treatment of rats with hexanedione (Boekelheide and Hall, 1991), gamma radiation (Kangasniemi et al, 1996), procarbazine (Meistrich et al, 1999), indenopyridine (Hodel and Suter, 1978), boric acid (Ku et al, 1993), and DBCP (Meistrich, unpublished results) and in jsd mice (Shetty et al, 2001).

In all the cases studied, serum testosterone remained unchanged. It has been shown that when the germ cells in the testes are lost, testicular mass and, consequently, blood flow decline (Wang et al, 1983). The maintenance of serum testosterone levels is a result of the hypothalamic-pituitary axis acting to keep serum testosterone constant when there is a decline in testicular blood flow by adjusting LH levels accordingly. This results in a 2.5-fold to 3-fold increase in ITT concentrations, which was confirmed in irradiated, procarbazine-treated, and DBCP-treated rats and jsd mice. The greater proportion of Leydig cells (their numbers are not decreased) in the testis, the decreased clearance rate of newly produced testosterone from the testis, and the elevated LH levels are all responsible for the increase in ITT concentrations.

There were 2 exceptions to this pattern of hormone changes. First, in cryptorchid mice, FSH was elevated 1.5-fold, but LH was unchanged (Mendis-Handagama et al, 1990). Second, in aged rats, both serum and testicular interstitial fluid testosterone levels were depressed (Schoenfeld et al, 2001). This depression in testosterone levels may be a combined result of the general depression with aging in LH and Leydig cell function, which can no longer respond by increasing testicular testosterone production. Nevertheless, these results show that above normal levels of ITT are not necessarily required for inhibition of spermatogonial differentiation, which will be discussed later.

GnRH Analogue Treatment Reverses Proliferation-Apoptosis Blocks in Spermatogonial Differentiation

We first demonstrated the stimulation of recovery of spermatogenesis in rats using hormone treatment given after irradiation (Meistrich and Kangasniemi, 1997). All previous studies had focused on the possible protective effect of giving the suppressive hormones before the toxicant exposure (Ward et al, 1990). However, we ruled out many possible mechanisms (Meistrich et al, 1997) by which the hormone treatment could have protected the survival of the spermatogonia and concluded that the only explanation that fit the data was that the hormonal treatment given before the toxic insult helped somatic cells to support sustained recovery of spermatogenesis from surviving stem cells after the toxicant exposure (Meistrich et al, 2000). In all subsequent work we have focused on giving the GnRH analogue treatment after toxicant exposure, although others have given the hormones before and after the toxicant.

In our initial study (Meistrich and Kangasniemi, 1997), the tubule differentiation index (TDI; the percentage of tubule cross-sections containing differentiated cells) was only 37% at 10 weeks after 3.5 Gy irradiation in the absence of hormone treatment. When GnRH agonist treatment was started immediately after irradiation, the TDI at 10 weeks was dramatically increased to 91%. However, because GnRH analogue treatments suppress testosterone, which is required for spermatid differentiation, there is histological recovery to the round spermatid stage, but no sperm are produced. The production of sperm after cessation of a transient GnRH analogue block will be discussed below. We also showed that systemic exogenous administration of testosterone, which suppresses ITT concentrations, also maintains spermatogonial differentiation after irradiation.

In other cases involving a toxicant-induced PAp block to spermatogonial differentiation (Table 1) maintenance or recovery of spermatogenesis was enhanced by giving GnRH analogues after the toxicant treatment (Table 2). These include hexanedione, procarbazine, or DBCP.

In some other cases, GnRH analogue treatment has also proved beneficial to the maintenance or recovery of spermatogenesis after exposure to a toxicant for which the blocks in spermatogonial differentiation were not well characterized. Treatment with GnRH agonist for about 12 weeks after exposure to the anticancer agent busulfan significantly increased the TDI at week 18 (Udagawa et al, 2001). However, a 4-week hormone treatment prior to busulfan injection was ineffective. The irreversible loss of spermatogenic function that occurred after a single dose of heat to rat testes was likewise counteracted by GnRH analogue posttreatment (Setchell et al, 2001), and treatment with GnRH agonist before heating was also effective (Setchell et al, 2002). Finally, prevention of the indenopyridine-induced block to spermatogonial differentiation was achieved when GnRH analogues were given both before and after drug treatment (Hild et al, 2001). However, in a subsequent study using a GnRH antagonist, only prior, but not subsequent, treatment with the GnRH analogue was effective at restoring recovery of spermatogenesis following indenopyridine treatment (S.A. Hild, personal communication).

GnRH analogue treatment also enhanced the stimulation of recovery of spermatogenesis from stem cells following spermatogonial transplantation. When mouse testicular cells were transplanted into busulfan-treated mouse recipients, the efficiency of differentiated germ cell production from transplanted stem cells in the recipient tubules was enhanced with GnRH analogue treatment (Ogawa et al, 1998; Dobrinski et al, 2001). However, a significant benefit was derived only from pretreatment with GnRH analogue, indicating that the hormone treatment may be important for the stem cells to attach in their proper niche in the seminiferous tubules, but not for the initiation of differentiation. The importance of suppressing ITT levels with either GnRH agonist or exogenous testosterone treatment was also demonstrated in studies in which rat or mouse spermatogonia were transplanted into busulfan-treated rat hosts (Ogawa et al, 1999).

When GnRH treatment is given relative to the toxic exposure is important. Data from irradiated and hexanedione-treated rats showed that treating immediately after exposure to a toxicant was more effective than delayed treatments in the restoration of spermatogonial differentiation (Meistrich et al, 1999). However, there has not been a strict comparison between the effects of pretreatments and posttreatments in any of the models in which both treatments are effective.

Fertility can be restored in these pathological situations by GnRH analogue treatment. When a 10-week GnRH agonist or GnRH antagonist treatment was started immediately after 3.7-Gy irradiation, fertility was maintained at week 20 in the GnRH agonist and GnRH antagonist treated rats at normal and nearly normal levels, respectively, whereas none of the irradiated-only rats were fertile (Meistrich et al, 2001b). When treatment was initiated 10 weeks after 5 Gy irradiation, at which point spermatogenesis had completely declined, fertility was restored at week 30 to subnormal levels in 83% of GnRH agonist and 50% of GnRH antagonist treated rats. Thus we conclude that normal fertility can be restored by GnRH treatment after irradiation, although that may depend on initiation of the GnRH analogue treatment soon after a toxicant exposure that is not too severe. We have also demonstrated that GnRH analogue posttreatment significantly increases recovery of fertility in rats after procarbazine treatment (Meistrich et al, 1999). In contrast in the jsd mice, a transient increase in spermatogonial and spermatocyte differentiation was produced by the GnRH antagonist treatment; testicular sperm extraction and intracytoplasmic sperm injection (ICSI) were both required to produce offspring (Tohda et al, 2002).

Maintenance of Spermatogenesis After Reversal

Although the TDI in rats receiving 3.5 Gy of radiation and GnRH agonist for 10 weeks was 91%, testicular sperm head counts were only 0.1% of controls because the hormone treatment suppressed spermiogenesis. However, when additional time without further GnRH treatment was allowed before the rats were killed, the TDI recovered to 100%, and sperm counts reached about 50% of normal control levels at 6.5 weeks after stopping treatment and were maintained at this level for at least another 3.5 weeks.

The maintenance of spermatogenesis in irradiated rats after GnRH analogue treatment is stopped depends on the toxicant dose and time of initiation and duration of the hormone treatment. For example, when a 7-week GnRH analogue treatment was initiated at week 15 after 6 Gy of irradiation, the TDI was elevated from 0% in irradiated-only rats to 95% at week 24 (2 weeks after stopping the GnRH treatment), but then declined to 50% at week 36 (14 weeks after stopping GnRH; G.A. Shuttlesworth and M.L. Meistrich, unpublished data). Thus permanent progression and maintenance of spermatogenesis is not assured by this technique. Although no time course studies were done, extensive recovery of spermatogenesis in tubules after hexanedione treatment was observed 9 weeks after the end of a 10-week GnRH agonist treatment, and the degree of recovery was inversely correlated with the dose of hexanedione (Blanchard et al, 1998).

In contrast to the toxicant-treated rat models, spermatogenesis degenerated rapidly in jsd mice after withdrawal of the GnRH antagonist. Whereas a 6-week GnRH antagonist treatment increased the TDI from 11% in non-hormone treated mice to 95%, 5 weeks after cessation of the treatment the TDI progressively declined to 78% and to 8% after 13 weeks (Shetty et al, 2001). Although one wave of late spermatids was produced from the differentiating spermatogonia and spermatocytes that developed during the GnRH antagonist treatment, the maximum percentage of tubules that contained elongated spermatids was only 20% at week 4 after the hormone treatment was stopped (Tohda et al, 2002). However, these elongated spermatids were used in ICSI to effect a pregnancy.

The difference between the maintenance of spermatogenesis in the irradiated rat model and jsd mice is that the former likely involves an epigenetic change, whereas the latter is a genetic alteration. The epigenetic change caused by irradiation to render spermatogonial differentiation sensitive to inhibition by testosterone can be largely reversed by hormonal treatment. But the underlying defect in a genetic disorder manifests itself again as soon as the hormone treatment is stopped.

Role of Testosterone in Block of Spermatogonial Differentiation

Because the GnRH analogues that were used to stimulate or maintain spermatogonial differentiation in the various cases described above generally suppress LH, FSH, and testosterone, these hormones were implicated in the inhibition of spermatogonial differentiation. Using irradiated rat and jsd mouse models, we and others investigated the roles of these hormones in the regulation of spermatogonial differentiation.

One study involved the administration of exogenous LH to GnRH antagonist–treated jsd mice (Tohda et al, 2001). Whereas the GnRH antagonist restored spermatogonial differentiation, the addition of exogenous LH inhibited it. However, other experiments with jsd mice (Shetty et al, 2001; Tohda et al, 2001) and with irradiated rats indicated that it was the testosterone production stimulated by the LH, and not the LH itself, that inhibited spermatogonial differentiation. For example, GnRH agonist treatment of LBNF₁ rats did not suppress LH levels, but it did suppress ITT, serum testosterone, and FSH levels and stimulated spermatogonial differentiation (Meistrich and Kangasniemi, 1997; Meistrich et al, 1999). In another study, when irradiated rats treated with GnRH agonist were given exogenous testosterone, spermatogonial differentiation was inhibited despite a suppression of LH levels (Shetty et al, 2001). This led us to further investigate the precise roles of testosterone and FSH in the inhibition of spermatogonial differentiation after irradiation.

Various studies have indicated that testosterone had an inhibitory effect. Because there is a major increase in the ITT concentration in mice between 30 and 40 days of age (Jean-Faucher et al, 1978), the large decline in the numbers of B spermatogonia in jsd testes, which occurs between 6 and 7 weeks of age (Kojima et al, 1997), could very well be a consequence of the increase in ITT. In addition in these mice, the stimulation of spermatogonial differentiation by suppression of testosterone with GnRH antagonist was reversed by exogenous testosterone (Shetty et al, 2001). Furthermore, that inhibition by testosterone was reversed by treatment with the androgen-receptor antagonist flutamide.

In irradiated rats, we have shown that testosterone dose-dependently reduced the GnRH antagonist-stimulated spermatogonial differentiation. (Shetty et al, 2000, 2002). Further, the stimulatory action of low-dose testosterone alone, which reduces ITT concentrations, was also reduced with increasing doses of testosterone that increased both ITT and serum testosterone concentrations. The TDIs and the serum and ITT levels were similar for each given dose of testosterone, with or without the GnRH antagonist, showing that the testosterone levels in the testis or the serum, or both, limit the ability of spermatogonia to differentiate. The inhibition of spermatogonial differentiation by testosterone was further confirmed by showing that flutamide reversed the inhibition induced by exogenous testosterone in GnRH antagonist–treated, irradiated rats (Shetty et al, 2000). Further support for our hypothesis that it is indeed testosterone acting through the androgen receptor and not a nonandrogenic metabolite of testosterone that inhibits spermatogonial differentiation was obtained by showing that various androgens, including 5-dihydrotestosterone (a 5-reduced androgen), 7-methyl-19-nortestosterone (a non-5-reducible androgen but one that can be aromatized), and methyltrienolone (a nonmetabolizable androgen) also suppressed spermatogonial differentiation in GnRH antagonist–treated irradiated rats (Shetty et al, 2002). In the same study, we showed that estradiol (E₂) was not inhibitory.

When testicular testosterone levels in irradiated rats treated with various GnRH analogues and testosterone combinations were compared with the TDI, an excellent negative correlation was observed (Figure 2) with only 1 point deviating significantly from each of the fitted curves (Figure 2, B and D). Although a general negative trend was also noted for serum testosterone vs TDI, there was a very significant deviation (Figure 2, A and C, arrow and open circles) in which the irradiated rats with a moderate amount of serum testosterone showed no differentiation. However, irradiated rats treated with GnRH analogues and testosterone (Figure 2, A and C, open upward triangles and filled diamonds) showed higher serum testosterone, but a significantly higher percentage of the tubules contained differentiating cells. This led us to conclude that ITT is the major factor, as the irradiated-only rats (open circles) had much higher ITT concentrations than those also treated with GnRH analogues and testosterone (Figure 2, B and D, open upward triangle and filled diamond). However, there were some small but significant discrepancies in the correlation between ITT and TDI. For example, GnRH agonist–treated, irradiated rats (Figure 2B, filled square) showed a higher TDI but also higher ITT than a similar group of rats that also received testosterone implants (Fib. 2B, open triangle). Because the former group had much lower serum testosterone levels (Figure 2A), we suggested that although the ITT was the major factor inhibiting spermatogenic recovery, serum testosterone seemed to have a minor additive inhibitory role. The point that deviated from the curve in Figure 2D (filled triangle) was a result of treatment of irradiated rats with GnRH antagonist and daily injections of testosterone proportionate, which may result in varying levels of testosterone throughout the course of treatment.

View larger version (20K): [in this window] [in a new window]   Figure 2. Correlation between serum testosterone (A, C) and ITT (B, D) during hormone treatment and the levels of recovery of spermatogenesis at the end of experiment. (A, B) Data from combinations of testosterone with GnRH agonist, given during weeks 0–10 after 6 Gy irradiation. TDI analysis was performed on testicular histological sections prepared on week 20. (C, D) Data from combinations of testosterone with GnRH antagonist, given during weeks 3–7 after 5 Gy irradiation. TDI analysis was performed on testicular histological sections prepared on week 13. Equivalent symbols in (A through D) are from the same treatments. Regression curves were fitted to the data with the exception of the deviant points (open circles) in (A and C). Arrows indicate discrepancies from complete correlations. Data from 2 reports (Shetty et al, 2000, 2002) were combined.

In all these situations, ITT concentrations in the normal range (about 50 ng/g testis) seem to inhibit the differentiation of spermatogonia. Figure 2D shows that even ITT concentrations of 15–30 ng/g of testis inhibited spermatogonial differentiation. Further, the observed block in the spermatogonial differentiation in aged rats that had ITT concentrations below normal and spermatogonial differentiation was stimulated by further suppression of ITT with a GnRH agonist show that above normal levels of ITT are not necessarily required for the inhibition of spermatogonial differentiation. Rather, in these circumstances, spermatogonial differentiation becomes sensitive to physiological levels of testosterone.

Based on the concept that testosterone inhibited spermatogenesis in toxicant-treated rats, hexanedione-exposed rats were treated with ethane dimethane sulfonate (EDS), which specifically eliminates Leydig cells, followed by GnRH agonist, which prevented Leydig cell regeneration (Richburg et al, 2002). Even though EDS reduced testosterone levels to undetectable levels, the EDS treatment inhibited the recovery of spermatogonial differentiation that the GnRH agonist would normally induce. Although the results of this study seemed to contradict the hypothesis that testosterone inhibits spermatogonial differentiation, that hypothesis could still be valid if a Leydig cell factor is required for the stimulation of spermatogenic recovery in the atrophic testis and this factor were eliminated by EDS, but not by GnRH analogue treatment.

Role of FSH in Block of Spermatogonial Differentiation

The elevated FSH levels in these pathological models of testicular atrophy could contribute to the inhibition of spermatogonial differentiation. Although as shown above, testosterone appears to be an inhibitory factor, it is necessary to determine whether FSH also has a role.

The possible contribution of serum testosterone to inhibiting spermatogonial differentiation suggests that testosterone may act at an extratesticular site. One such likely site is the pituitary, where it could act by altering gonadotropin levels. We have already ruled out LH as having a significant contribution to the inhibition of spermatogonial differentiation, so we focused on a possible role for FSH. However, testosterone has a complex action on pituitary production of FSH. When testosterone is given to rats or mice that have normal GnRH production and action, it suppresses FSH levels by having a combined action on the hypothalamus and pituitary. However, when testosterone is given to GnRH antagonist–treated rats, but not mice (Shetty et al, 2001), it reverses the GnRH antagonist-induced reduction of FSH levels in these rats by direct up-regulation of FSHß gene transcription in the pituitary (Perheentupa et al, 1993). The levels of FSH in the presence of exogenous testosterone appear to be independent of whether or not a GnRH antagonist is also given (Shetty et al, 2000).

There was good inverse correlation between TDI and FSH levels (Figure 3), which could be due in part to the concomitant rise in FSH when testosterone was given. Several points did deviate from this correlation curve (Figure 3, arrow). Although TDI and ITT were even more closely correlated, (Figure 2), a role for FSH in inhibition of spermatogonial differentiation could not be ruled out. We directly tested the role of FSH by giving exogenous FSH to irradiated rats while suppressing levels and actions of androgens with GnRH antagonist and flutamide. Exogenous FSH significantly inhibited the tubule differentiation stimulated by GnRH antagonist-flutamide treatment, although not as drastically as did androgens (G. Shetty, unpublished data). From these data and the overall relationship between hormone levels and TDI (Figure 4), we conclude that primarily ITT, but also FSH, which is regulated by serum testosterone, inhibits spermatogonial differentiation in irradiated rats.

View larger version (13K): [in this window] [in a new window]   Figure 3. Correlation between serum FSH levels during hormone treatment and the subsequent levels of recovery of spermatogenesis in irradiated rats treated with various combinations of GnRH antagonist, 1 of the androgens, and the antiandrogen flutamide. The points indicated by arrows deviated from the curve fitted to the other points. The deviant points are from rats treated with GnRH antagonist, testosterone, and flutamide (open circle), or GnRH antagonist and daily injections of testosterone propionate (open square). Data from 2 reports (Shetty et al, 2000, 2002) were combined.

View larger version (40K): [in this window] [in a new window]   Figure 4. Schematic of observations in irradiated rats showing the different levels of FSH and testosterone in the serum and testis during various hormone treatments and the resulting changes in the differentiation of spermatogonia. Whereas no spermatogonial differentiation is observed in irradiated rats not treated with hormones (left panel), likely due to the high levels of ITT and FSH, differentiation is induced by suppression of testosterone and FSH (second panel). The fact that there is some differentiation in the third panel indicates that either ITT or FSH are inhibitory. The fourth panel indicates that testosterone is acting through the androgen receptor, although it could be acting at the pituitary or testis. The fifth panel (compare with the second panel) shows that FSH has an inhibitory role.

In contrast to the results with rats, administration of exogenous FSH in jsd mice during suppression of gonadotropins did not inhibit spermatogonial differentiation (Tohda et al, 2001). Further confirmation of the inability of FSH to inhibit spermatogonial differentiation in jsd mice was shown by the lack of correlation between FSH levels and the TDI (Shetty et al, 2001) and the lack of correlation in timing of the rise in FSH levels, which reaches near adult levels during the first 2 weeks after birth (Slegtenhorst-Eegdeman et al, 1998), and the major block in spermatogonial differentiation, which does not occur until between weeks 6 and 7 after birth (Kojima et al, 1997).

Relationship to Roles of Hormones in Normal Spermatogenesis

In the various pathological conditions we have described, it appears that testosterone and FSH may act additively to inhibit the differentiation of spermatogonia, whereas in normal spermatogenesis they act additively to support survival and differentiation of spermatocytes and spermatids. Thus the differences between the action of the hormones in the 2 situations involves not only the direction of action but also their targets during spermatogenesis (Figure 5). In spermatocyte and spermatid differentiation, the normal requirement for primarily testosterone, but also with some additive effects of FSH (O'Donnell et al, 1994; El Shennawy et al, 1998), appears not to be altered in the pathological situation, in which differentiation does not proceed past the spermatocyte or early spermatid stage during suppression of testosterone and FSH. However, in these pathological models the hormones act at an additional checkpoint. Spermatogonial survival and differentiation, which in normal rats can proceed in the absence of testosterone and FSH but is augmented by these hormones (Huang and Nieschlag, 1986; Meachem et al, 1999), becomes, in these pathological models, sensitive to inhibition by testosterone and in some cases, to inhibition by FSH as well. Possible mechanisms for the development of this checkpoint will be described in the next section.

View larger version (26K): [in this window] [in a new window]   Figure 5. Roles of testosterone and FSH in normal spermatogenesis and in pathological models involving a proliferation-apoptosis block in spermatogonial differentiation. Stimulatory and inhibitory roles are indicated by pluses and minuses, respectively, with the strength of the stimulatory or inhibitory action indicated by the numbers of pluses or minuses and the font size.

Possible Mechanisms for Block in Spermatogonial Differentiation

Although testosterone and FSH have effects on spermatogonial differentiation in these pathological models, spermatogonia are not known to have receptors for these hormones. According to currently accepted dogma, in normal animals, FSH receptors (FSHRs) are localized exclusively in the Sertoli cell (Kliesch et al, 1992) and androgen receptors (ARs) are localized in a variety of somatic cell types, including Sertoli, Leydig, peritubular myoid, and vascular smooth muscle cells (Bremner et al, 1994). Furthermore, normal development of germ cells that lack an AR is possible (Johnston et al, 2001). Because germ cells lack AR and FSHR, these hormones must act via paracrine or juxtacrine routes between the cells that contain the receptors for these hormones and the spermatogonia.

The model chosen to explain the apparent contradiction, that testosterone inhibits spermatogonial differentiation after some pathological insults or genetic defects but not in normal spermatogenesis, depends on whether the pathology directly alters the spermatogonia or the androgen-responsive somatic cell. In most cases the target is not known. Although certain toxicants are believed to act primarily on Sertoli cells (eg, hexanedione; Boekelheide, 1988) or germ cells (eg, radiation; Lee et al, 1999), it is not possible to prove that the long-term effects are due to action on these cells. Hence, Figure 6, which lays out our model, is divided into two parts, A and B, which assume the defect lies in the spermatogonia, whereas Figure 6C and D, assume it lies in the Sertoli cells. The Sertoli cell was used as the example of the androgen-responsive somatic cell because it is most likely, but we cannot rule out the possibility that the peritubular, Leydig, or vascular smooth muscle cells are instead involved in some cases. In Sl^17H mice the defective gene, stem cell factor, is indeed specifically produced by Sertoli cells. However, for the jsd mutation, transplantation experiments have shown conclusively that the defect is expressed in the spermatogonia, not in the somatic cells (Boettger-Tong et al, 2000; Ohta et al, 2001).

View larger version (35K): [in this window] [in a new window]   Figure 6. Models to explain testosterone-dependent inhibition of spermatogonial differentiation in pathological situations in mice and rats but not normal rodents. (A) It is assumed that the defect is in spermatogonia and they do not differentiate because a growth, survival, or differentiation factor is missing. Normal spermatogonia could have 2 pathways that support this step, but altered spermatogonia lack one receptor (square symbol) (or intracellular component of signal-transduction pathway) and therefore require the second receptor, the ligand (triangle), which is suppressed by the presence of testosterone. (B) It is assumed that the defect is in spermatogonia and they do not differentiate because they are killed by apoptosis. Sertoli cells could secrete an apoptotic effector (circle) in the presence of testosterone but normal spermatogonia lack the receptor or pathway for this ligand. The altered spermatogonia possess this receptor and therefore become sensitive to apoptosis in the presence of testosterone. (C) It is assumed that the defect is in somatic (Sertoli) cells and the reason spermatogonia do not differentiate is that a growth or differentiation factor is absent. Normal Sertoli cells make this growth factor constitutively, but in the altered Sertoli cells, it could be inhibited by testosterone. (D) It is assumed that the defect is in somatic (Sertoli) cells and the reason spermatogonia do not differentiate is that they are killed by apoptosis. Whereas normal Sertoli cells do not make an effector for this apoptotic process, the altered Sertoli cells could make this effector, but only in the presence of testosterone.

The appropriate choice of model also depends on whether the cause of the block is spermatogonial apoptosis (Figure 6, B and D), or the lack of a functional signal for the spermatogonia to differentiate (Figure 6, A and C). In the first case, the failure to differentiate is a secondary consequence of the failure of the cells to survive to an appropriate stage. In the second instance, the observed apoptosis would be a secondary consequence of cells remaining undifferentiated for too long.

Alterations in spermatogonia could make these cells either more sensitive to testosterone-induced proapoptotic factors from the Sertoli cell (Figure 6B) or more dependent on testosterone-suppressible growth and differentiation factors from their surroundings (Figure 6A). Alternatively, testosterone could act on somatic cells to induce proapoptotic factors (Figure 6D) or to inhibit normally secreted growth factors (Figure 6C). In any case, all of these models predict that there should be at least one gene or gene product specifically regulated by testosterone in the target somatic cell.

Possibilities for Clinical Application

The above animal models may be applicable to 4 areas of human infertility or fertility control: idiopathic male infertility involving spermatogenic arrest, infertility due to treatment of cancer and autoimmune diseases with chemotherapy or radiotherapy, infertility due to environmental or occupational exposures, and development of a male reversible contraceptive.

Many cases of male infertility involve testicular disorders with arrest at various stages of spermatogenesis, including arrest at the spermatogonial stage in 10% of such cases (Skakkebaek et al, 1973). If testosterone inhibits spermatogenesis as was the case with jsd mice (Tohda et al, 2002), the late spermatids might be produced with intermittent testosterone suppression and be used for ICSI.

Chemotherapy or radiotherapy induces prolonged or permanent azoospermia in 3000 men of reproductive age in the United States each year (Meistrich et al, 2001a). Azoospermia also results from cyclophosphamide treatment for autoimmune diseases (Watson et al, 1985). Although some of these treatments may kill all of the stem cells, sometimes stem spermatogonia do survive but they fail to differentiate, as was observed in the rodent models. This is evidenced by the spontaneous reinitiation of spermatogenesis in some patients after many years of azoospermia (Meistrich et al, 1992). There are also histological examples of failure to differentiate past the spermatogonial (Kreuser et al, 1989) or the spermatocyte (Meistrich and van Beek, 1990) stages during the azoospermic period. The trigger for the spontaneous recovery is not known. Although several earlier attempts to enhance recovery of spermatogenesis by treatment with GnRH analogues before and during chemotherapy or radiotherapy were unsuccessful (Morris and Shalet, 1990), low-dose systemic testosterone to suppress intratesticular testosterone levels did induce recovery of spermatogenesis in all men treated with cyclophosphamide (Masala et al, 1997). However, there has been only one trial of the use of hormonal suppression after the completion of chemotherapy, and in that trial, no recovery was observed (Thomson et al, 2002). It should be noted that all the patients had been treated before puberty with high doses of procarbazine or radiation, which likely led to a complete loss of stem cells. A study using GnRH analogues for adult patients whose azoospermia resulted from lower doses of cytotoxic agents should be conducted next.

Environmental and occupational exposures to toxicants that block spermatogonial differentiation in rats may also produce similar effects in men. Boric acid is in widespread commercial and consumer use. Hexanedione is the active metabolite of the widely used solvent n-hexane. As yet there are no reports of effects of these chemicals on human spermatogenesis. However, DBCP, which is now banned, produced azoospermia in all highly exposed workers involved in its production (Whorton et al, 1979), and many thousands of agricultural workers who were exposed to DBCP appear to have an increased incidence of azoospermia (Slutsky et al, 1999). That azoospermia in men following exposure to moderate doses of DBCP may be spontaneously reversible years later (Potashnik and Porath, 1995) indicates that the stem cells may have survived and that DBCP may cause azoospermia by producing a prolonged block in spermatogenic differentiation.

The ability to reversibly block the differentiation of spermatogonia has potential for use as a male contraceptive. Compounds such as indenopyridines, in a single dose produce apparently irreversible sterility in rats, mice, and dogs without other toxicity (Cook et al, 1995), but their development as contraceptives is limited by the irreversibility of the spermatogenic block. However, the presence of type A spermatogonia in the tubules of indenopyridine-treated rats suggests that it could be reversed. Although spermatogonial differentiation and recovery of spermatogenesis was enhanced by treating rats with GnRH analogues before and after indenopyridine treatment (Hild et al, 2001), further studies showed that only the treatment before indenopyridine was effective (S.A. Hild, personal communication). It is now important to determine whether spermatogonial differentiation can be reinitiated by hormonal or other forms of treatment given after the induction of a block to differentiation by the indenopyridine.

Although GnRH analogues and gonadal steroids have similar actions in humans and rodents, we do not know whether they will stimulate recovery of spermatogenesis in men with genetic or toxicant-induced blocks in spermatogonial differentiation because we do not know whether the mechanisms of the block are the same in the different species. Preliminary analyses of studies in irradiated monkeys show that GnRH antagonist treatment failed to prevent or reverse the reductions in spermatogenesis produced by radiation (A. Kamischke, personal communication; Richburg et al, 2002). Therefore, it is important to elucidate the mechanism by which testosterone inhibits spermatogonial differentiation in rodents to evaluate its application to men. Mechanistic knowledge can be used to find targets downstream from the initial action of androgen to develop restorative treatments that allow maintenance of androgen levels.

Footnotes

Supported by grants ES-08075 and HD-40397 from the National Institutes of Health.

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				J. Rohozinski and C. E. Bishop The mouse juvenile spermatogonial depletion (jsd) phenotype is due to a mutation in the X-derived retrogene, mUtp14b PNAS, August 10, 2004; 101(32): 11695 - 11700. [Abstract] [Full Text] [PDF]

				S. A. Hild, B. J. Attardi, and J. R. Reel The Ability of a Gonadotropin-Releasing Hormone Antagonist, Acyline, to Prevent Irreversible Infertility Induced by the Indenopyridine, CDB-4022, in Adult Male Rats: The Role of Testosterone Biol Reprod, July 1, 2004; 71(1): 348 - 358. [Abstract] [Full Text] [PDF]

				G. Shetty and C. C. Y. Weng Cryptorchidism Rescues Spermatogonial Differentiation in Juvenile Spermatogonial Depletion (Jsd) Mice Endocrinology, January 1, 2004; 145(1): 126 - 133. [Abstract] [Full Text] [PDF]

				M. L. Meistrich, G. Wilson, K. L. Porter, I. Huhtaniemi, G. Shetty, and G. A. Shuttlesworth Restoration of Spermatogenesis in Dibromochloropropane (DBCP)-Treated Rats by Hormone Suppression Toxicol. Sci., December 1, 2003; 76(2): 418 - 426. [Abstract] [Full Text] [PDF]

				F.-P. Zhang, T. Pakarainen, M. Poutanen, J. Toppari, and I. Huhtaniemi The low gonadotropin-independent constitutive production of testicular testosterone is sufficient to maintain spermatogenesis PNAS, November 11, 2003; 100(23): 13692 - 13697. [Abstract] [Full Text] [PDF]

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