J Endocrinol. 2005 Oct;187(1):117-24.
Influence of long-term dietary administration of procymidone, a fungicide with anti-androgenic effects, or the phytoestrogen genistein to rats on the pituitary-gonadal axis and Leydig cell steroidogenesis.
Svechnikov K, Supornsilchai V, Strand ML, Wahlgren A, Seidlova-Wuttke D, Wuttke W, Söder O.
Department of Woman and Child Health, Pediatric Endocrinology Unit, Q2:08, Karolinska Institute and University Hospital, Stockholm, Sweden.
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Procymidone is a fungicide with anti-androgenic properties, widely used to protect fruits from fungal infection. Thereby it contaminates fruit products prepared for human consumption. Genistein-containing soy products are increasingly used as food additives with health-promoting properties. Therefore we examined the effects of long-term dietary administration (3 months) of the anti-androgen procymidone (26.4 mg/animal per day) or the phytoestrogen genistein (21.1 mg/animal per day) to rats on the pituitary-gonadal axis in vivo, as well as on Leydig cell steroidogenesis and on spermatogenesis ex vivo. The procymidone-containing diet elevated serum levels of LH and testosterone and, furthermore, Leydig cells isolated from procymidone-treated animals displayed an enhanced capacity for producing testosterone in response to stimulation by hCG or dibutyryl cAMP, as well as elevated expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450 scc) and cytochrome P450 17alpha (P450c17). In contrast, the rate of DNA synthesis during stages VIII and IX of spermatogenesis in segments of seminiferous tubules isolated from genistein-treated rats was decreased without accompanying changes in the serum level of either LH or testosterone. Nonetheless, genistein did suppress the ex vivo steroidogenic response of Leydig cells to hCG or dibutyryl cAMP by down-regulating their expression of P450 scc. Considered together, our present findings demonstrate that long-term dietary administration of procymidone or genistein to rats exerts different effects on the pituitary-gonadal axis in vivo and on Leydig cell steroidogenesis ex vivo. Possibly as a result of disruption of hormonal feedback control due to its anti-androgenic action, procymidone activates this endocrine axis, thereby causing hyper-gonadotropic activation of testicular steroidogenesis. In contrast, genistein influences spermatogenesis and significantly inhibits Leydig cell steroidogenesis ex vivo without altering the serum level of either LH or testosterone.

From the full text article:

The present investigation demonstrates that long-term dietary administration of the phytoestrogen genistein or of procymidone, a fungicide with anti-androgen action, to rats exerts different effects on their pituitary-gonadal axis in vivo and on ex vivo steroidogenesis by their Leydig cells. In another experiment with an identical treatment regime serum concentrations of genistein were found to be 8 µM, concentrations at which uterine weight is slightly increased as a sign of estrogenic effect (results not shown). Concentrations of between 1 and 10 µg/ml (corresponding to high-nanomolar or low-micromolar concentrations) were reported in women under commercial soy isoflavone supplementation (Setchell et al. 2001). It can be speculated that the soy intake of some males is similar to that of females. Similarly, low-micromolar concentrations of genistein were found in infants who consumed large amounts of food products derived from soy beans (Setchell et al. 1997). Therefore, the effects to be discussed below may be of specific concern for male fertility. ... At the same time, genistein suppressed the steroidogenic response of Leydig cells to hCG and (Bu)2cAMP by down-regulating the expression of P450 scc.

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The present study also revealed that long-term dietary administration of the non-steroidal phytoestrogen genistein to rats affected their pituitary-gonadal axis in vivo and ex vivo steroidogenesis by their Leydig cells in a fashion different from procymidone. Despite the lack of any changes in their serum levels of LH or testosterone, Leydig cells isolated from genistein-treated rats lost their responsiveness to hCG and (Bu)2cAMP almost completely, suggesting that genistein and/or its metabolites have direct effects on Leydig cell function. This reduced steroidogenesis is presumably due to a decrease in the expression and activity of P450 scc, although there were no apparent decreases in serum testosterone levels. Similarly, a decrease in the activity of the steroidogenic enzymes without apparent reduction in serum testosterone levels after treatment by phthalate has been reported earlier (Akingbemi et al. 2001). It might be suggested that the inhibition of P450 scc was not profound enough to affect testosterone production by Leydig cells at the time of serum testosterone measurement or that compensatory mechanisms were activated in vivo to counteract the effect of decreased enzyme activity. Another possible explanation for this finding is that phytoestrogens can inhibit the activities of 5'-reductase and aromatase in Leydig cells, as has been shown in peripheral tissues (Kellis & Vickery 1984, Evans et al. 1995), thereby decreasing the rate of testosterone metabolism and supporting the level of circulating androgen despite suppression of upstream steroidogenic enzyme.

To date, relatively little is known concerning the effects of genistein on steroidogenesis by Leydig cells. It has been reported that long-term dietary administration of this substance reduces serum levels of testosterone and androstenedione without affecting the expression of StAR in rats (Weber et al. 2001) and, furthermore, suppresses both basal and LH-stimulated androgen production by rooster Leydig cells in vitro (Opalka et al. 2004). Moreover, genistein has been shown to inhibit follicle-stimulating hormone- and forskolin-stimulated production of progesterone by primary cultures of rat ovarian cells (Whitehead & Lacey 2000). In contrast to the study by Weber et al.(2001), we and others (Makela et al. 1995, Ohno et al. 2003) could not find significant changes in plasma androgen levels in longer-term studies of phytoestrogen effects on reproductive function. This discrepancy may be due to a difference in the composition of the phytoestrogens used to treat the animals. In our study, rats received a chow containing genistein only, whereas in the report by Weber and co-workers (2001) animals were fed with a phytoestrogen-rich diet containing seven different derivatives of genistein, with genistein being a minor component. It can be speculated that these phytoestrogens, due to their synergistic or additive effects on Leydig cells, could have a more potent suppressive effect on steroidogenesis than genistein alone.

In the present study we have demonstrated that dietary genistein down-regulates the expression of mitochondrial P450 scc, which catalyses the conversion of cholesterol to pregnenolone. The mechanism remains unknown, but may involve activation of the estrogen receptor by genistein. This suggestion is supported by the recent report that the xenoestrogen methoxychlor (a derivative of DDT) alters steroidogenesis by immature rat Leydig cells by suppressing the expression of P450 scc and that the antiestrogen ICI 182 780 prevents this effect (Akingbemi et al. 2000). In addition, genistein and its derivatives were found to be potent inhibitors of P450c21 and human adrenocortical 3'-hydroxysteroid dehydrogenase in vitro (Ohno et al. 2002). It should be also noted that the mechanisms underlying the biological actions of genistein are complex, since in addition to its estrogen-like effects (Murkies 1998), this compound is also a tyrosine kinase inhibitor (Akiyama et al. 1987). Therefore, the observed biological responses to genistein will be determined by both activation of the expression of specific estrogen-dependent genes and alterations in signalling pathways in the target cells.

The present investigation also reveals that chronic dietary exposure of rats to genistein significantly reduces the rate of meiotic DNA synthesis in preleptotene spermatocytes during stages VIII and IX of spermatogenesis, without affecting the other stages examined. This observation is consistent with the previous finding that genistein inhibits both proliferation and differentiation of spermatogonia and primary spermatocytes in primary testis cell cultures, isolated from the medaka (Song & Gutzeit 2003). Similarly, pre- and postnatal dietary high-dose exposure of rats to this phytoestrogen has been shown to cause aberrant and delayed spermatogenesis, including degeneration of spermatocytes and depletion of spermatids, resulting in a deficit of sperm in the epididymis (Delclos et al. 2001).

Categories: 2005, Soy, Phytoestrogens, Endocrine, Estrogen, Hormone disruptors, DNA synthesis, Steroid hormones, Progesterone, Testes, Sexual development, Nutrition and diet