Garlic has acquired a reputation as a formidable prophylactic and therapeutic medicinal agent over the centuries and many favorable experimental and clinical effects of the consumption of garlic, in different types of preparations (crude, powder...) have been reported (including cardiovascular diseases, stimulation of immune function, detoxification properties... for a review, see [3]). It has long been known, however, that the extraction process can increase potency compared with the crude plant. In the present study, the chemical analysis of the crude garlic used was not achieved, but Shukla and colleagues have quantified the concentration of the different compounds of garlic [9]. In this context, administration of garlic preparation to prevent hypercholesterolemia or arteriosclerosis might have side-effects on other organs. In terms of testicular functions, garlic or its metabolites have been studied as a protective adjuvant to different types of toxins [28]. Indeed, induction of testicular hypogonadism by heat is prevented in part by different types of garlic preparation (raw, heated garlic juices, dehydrated garlic powder of the more potent aged garlic extract [14]. Aqueous extract of garlic [29] or the metabolites diallyl sulfide [30] and diallyl tetrasulfide [31] offer a protection against cadmium-induced testicular damages. Garlic is also effective in restoring the testicular histology altered by EDTA [32]. The antioxidant activities of garlic extract were shown to decrease the toxic effects of free radicals induced by testicular torsion and detorsion [33]. While several studies show that As has a protective effect as an adjuvant, other studies show that it modifies spermatogenesis. Indeed, daily administration of 50 mg garlic powder over 70 days induced a spermatogenetic arrest at spermatocyte I stage [15]. Moreover, aqueous garlic extract [17] or the metabolite diallyl trisulfide [18] have spermaticidal effects. In contrast, a 90-day administration of 100 mg/kg aqueous extract of garlic has been shown to increase the number of spermatozoa [34]. In this context, to assess the positive or negative effect of garlic, it was important to analyze its action on the different testicular functions through the study of germ cell apoptosis, Leydig cell steroidogenesis and Sertoli cell markers (the present study). The present study has focused on the effects of As on testicular cells and its mechanisms of action. We showed here that oral administration of crude garlic induced germ cell death that targeted spermatocytes and spermatids, whereas spermatogonia and somatic Leydig and Sertoli cells were not affected. Given that CASP3 was immunodetected mainly in spermatocyte and spermatid cells (the TUNEL-positive germ cells) and that As treatment, at the same doses as observed for TUNEL, induced a dose-dependent increase in active CASP3 protein levels, the possibility exists that the germ cell apoptotic process could be related to CASP3 activation. The germ cell death might also, however, be related to another programmed cell death process since CASP3 activation has been associated with differentiation in some cell types [35]. In contrast, the active effector CASP6 seemed not to be involved in our model, while proCASP6 is expressed in normal germ cells [36]. While it was previously reported that chronic administration of 50 mg of garlic powder induced an arrest of spermatogenesis, our present study shows, for the first time, the involvement of an apoptotic process that targets testicular germ cells. Very few, if any, studies have reported such an apoptotic effect of As on non-tumoral cells, although it has been described in tumoral cells [37]. For example, administration of crude extract of garlic to a human colon cancer cell line induced apoptosis by increasing the levels of BAX, CYCS (previously known as cytochrome c) and CASP3 activity while it decreased the mitochondrial membrane potential [38]. More specifically, raw crushed garlic is high in allicin, a powerful bioactive compound of garlic that induced activation of CASP3, CASP8 and CASP9 and cleavage of poly (ADP-ribose) polymerase in several cancer cells [39].

Given that the balance between pro-apoptotic molecules (active effector caspase, IAPs inhibitors) and anti-apoptotic molecules (IAPs) levels determines the fate of the cells towards the executioner step of the death process [21], we have evaluated the expression of IAPs in our experimental model. BIRC2 and BIRC3 are highly expressed in rat testes [40]. BIRC3 was detected in the nucleus of B spermatogonia, spermatocytes at different stages and of somatic cells while BIRC2 was localized in the cytoplasmic compartment of spermatocytes (from stage VI pachytene onwards), spermatids (round and elongated) and Leydig cells [41]. While BIRC2 and BIRC3 play a role in the protection of germ cells from Fas-mediated apoptosis [41], their increased expression after treatment with crude garlic (the present study) suggested that germ cells are unable to inhibit CASP3 action. A potential explanation is that the high levels of DIABLO (in spermatocytes) or HTRA2 in spermatids by inhibiting the IAP action may favor the activity of CASP3 in the germ cells mainly affected by apoptosis after As treatment. The other IAPs studied here were expressed for BIRC5 in spermatocytes at first meiotic prophase and highly in Leydig cells [42] or exclusively in Leydig cells (our unpublished data) and for XIAP mainly in Sertoli cells (our unpublished data). BIRC5 and XIAP levels were unchanged after As treatment, suggesting that Leydig (BIRC5) and Sertoli (XIAP) cells are not affected in terms of apoptosis by crude garlic administration. These data are in accordance with the TUNEL approach used in the present study.

DNA degradation might also be triggered by a caspase-independent pathway through the action of AIFM1. During the apoptotic process, AIFM1 is released from mitochondria and translocated into the nucleus where its DNA binding activity mediates large-scale DNA fragmentation [26, 43]. Given that As treatment, at the doses used in the present study, had no effect on the expression of active AIFM1, we suggest that the caspase-dependent pathway is mainly involved in our experimental model. The next question to address was whether germ cell death induced by As was related to modifications in hormone profile and/or Leydig and/or Sertoli cell markers.

Crude garlic induced a dose-dependent decrease in plasma and intratesticular testosterone concentrations in treated rats and an increase in LH levels [16], suggesting that As targeted Leydig cells. In this context, we evaluated the different steps of testosterone biosynthesis. Conversion of cholesterol to biologically active testosterone is a multi-step enzymatic process, including Star, that controls the transport of cholesterol from the outer to the inner mitochondrial membrane [44], Cyp11a1, Hsd17b3 and Hsd3b5. Testosterone can be metabolized by Srd5a2 or Cyp19a1. We showed here that As alters testosterone production, since we found that Star, Cyp11a1, Hsd17b3 and Hsd3b5 mRNA levels were decreased in a dose-dependent manner. Given that testosterone protects germ cells, especially spermatocytes and spermatids, against apoptosis [45–47], its decrease induced by As treatment might be an explanation for the death of spermatocyte and spermatid cells via an apoptotic process. Interestingly, while garlic extract is known to reduce serum cholesterol levels (in humans and animals) and inhibit cholesterol biosynthesis [48] testosterone production was not related to cholesterol metabolism [16] but to steroidogenic enzyme modification (the present study).

In terms of Sertoli cells, we showed here that both hormones which regulate cellular functions are decreased, i.e. testosterone and plasma FSH levels. In addition, the germ cell number might be decreased since the number of empty seminiferous tubules increased in a dose-dependent manner [16] and it is well recognized that germ cell loss modifies Sertoli cell functions (for a review, see [49]). In this context, we evaluated several Sertoli cell markers such as TUBB3, a housekeeping gene involved in cytosqueleton network [50] and expressed exclusively in Sertoli cells (our unpublished data), or proteins known to be regulated by testosterone (GSTA2, RHOX5, AMH, CDKN1B) and FSH (AMH) or known to be involved in paracrine interactions (GATA4). TUBB3 expression was unchanged at all doses of As used, suggesting an absence of effect of crude garlic on Sertoli cell number (the present study). These data are in accordance with the absence of apoptosis in Sertoli cells discussed above. In terms of androgen-dependent genes, we showed here that two of them (GSTA2 and RHOX5) have their expression unchanged after treatment. In contrast, AMH and CDKN1B expression was decreased while GATA-4 protein expression was significantly increased after feeding with crude garlic (the present study). Given that RHOX5 and GSTA2 expression (genes that possess ARE or ARE-like sequences) was unchanged, the possibility exists that As effects on AMH and CDKN1B are not linked to testosterone modifications but rather linked to germ cell loss. Altogether, these data indicate that As modifies some Sertoli cell markers. In the present experimental model, we observed decreased levels of testosterone associated with decreased levels of FSH. As suggested, the decreased plasma FSH levels could not be accounted for a central alteration since LH plasma levels were increased. It is noteworthy that the increase in plasma LH levels observed here was higher (about double at 10% As and 3.3-fold at 15% As) than the decrease observed in FSH plasma levels (1.2-fold at 10 and 15% As). Two hypotheses, however, might explain the discrepancies observed in the plasma gonadotropin levels. First, increased estradiol production has been shown to be associated with decreased plasma FSH without effects on LH production [51]. It is likely that in our experimental model the estradiol production was not modified since in the rats fed with crude garlic, the aromatase expression was not different compared with control animals. Second, Sertoli cells produce inhibin B which inhibits FSH secretion. Inhibin B expression is stimulated by FSH or germ cells (pachytene spermatocytes, early spermatids) and inhibited by testosterone. In the present study, the possibility exists that the dramatic decrease in testosterone production induces an increase in inhibin production that in turn decreases FSH plasma levels.

Raw garlic consumption by humans ranges from one to two cloves (about 4 g) to 28 g per day (for a review, see [3]). The concentration used in the present study exceeds this amount of consumption, but various other types of garlic preparation are consumed (such as garlic powder, oil, extract or aged garlic) and the concentration in garlic active components is highly variable, particularly in powder and oil (for a review, see [3]). Moreover, the bioactive components of garlic are not fully characterized even if it is assumed that the sulfur-containing molecules are the active ones. Another point is that garlic consumption to reduce cardiovascular risk is a long-term consumption which is another potential negative effect with regard to spermatogenesis. In this context, and more widely, extrapolation from rat to human is a difficult task. It has long been known, however, that human spermatogenesis is more sensitive to stress than that of rats [52], suggesting that concentrations lower than those used in the present study might impair male spermatogenesis and, particularly, might induce azoospermia in men with low sperm count.