**4.5. Endocrine disrupting chemicals**

A large body of information concerning the effects of endocrine disrupting compounds (EDCs) on Leydig cells steroidogenesis during fetal development and in adult mammals has been accumulated in the past decades. EDCs can disturb morphology and normal endocrine functions of the Leydig cells or oppose the actions of androgen through their estrogenic or anti-androgenic properties (Hejmej et al., 2011a). A number of compounds act directly on Leydig cells to diminish T production by interfering with the expression of steroidogenic enzymes, at the protein and/or mRNA level (Skakkebaek, et al., 2001).

Our recent results have shown that administration of the estrogenic compound, 4-*tert*octylphenol (OP), to adult bank voles has caused the significant decrease of 3β-HSD and increase of P450arom expression concominantly with the alteration of the androgen/estrogen balance within the tesis of sexually active animals (Hejmej et al,. 2011b). Similar results have been reported by Victor-Costa et al. (2010) on rats treated with atriazine. These authors concluded that inhibition of 3β-HSD function is one of the possible mechanism through which xenoestrogens disturb spermatogenesis. *In vitro* studies on Leydig cells obtained from various mammals have revealed decrease in the activity and expression of 3β-HSD after OP, bisphenol A (BPA) and genistein administration (Hu et al., 2010; Kotula-Balak et al., 2011; Ye et al., 2011). Our study demonstrated that OP markedly disturbes morphology and steroidogenic function of the Leydig cells through direct effect on 3β-HSD expression and localization (Kotula-Balak et al., 2011). In detail, treatment with high doses of OP (10-4–10-6 M) resulted in a reduced staining intensity and the staining was usually located near the nucleus, whereas in the low OP doses (10-7 and 10-8 M) it was manly dispersed throughout the cytoplasm (Figure 3).

Hydroxysteroid Dehydrogenases – Localization, Function and Regulation in the Testis 275

In recent years, disorders of human male reproductive development increased in incidence (Sharpe & Skakkebaek, 1993; Toppariet al., 1996). Cryptorchidism and hypospadias are the two most common congenital malformations that comprise a testicular dysgenesis syndrome (TDS), which arises during fetal development and manifests in adulthood

Several studies have shown that increased intratesticular temperature in cryptorchid testes affects spermatogenesis, resulting in either reduced fertility or infertility. Other evidences collected over the years have indicated that increased testicular temperature negatively influences the development and differentiation of Leydig cells causing impairments in sex

Our recent studies have shown that in cryptorchid horses disturbances in differentiation and/or maturation of Leydig cells may be related to altered intracellular communication. In these animals decreased immunoexpression of gap junction protein, connexin 43, in testicular cells were accompanied with reduced expression of LHR, 3β-HSD and disturbed androgen/estrogen balance (Hejmej et al., 2005, 2007; Hejmej & Bilinska, 2008), (Figure 4). Altered expression of these proteins and imbalance in sex hormones level detected in cryptorchid horses suggested their additional influence on morphology and function of undescended testis. Markedly reduced expression of 3β-HSD has been also reported in rats with experimentally induced cryptorchidism (Wisner & Gomes, 1978). No significant changes in T levels have been detected in patients with cryptorchidism as well as other mammalian species (Bilinska et al., 2003; Farrer et al., 1985; Illera et al., 2003; Kawakami et

**Figure 4.** Immunohistochemical localization of 3β-HSD in testis of normal (A) and cryptorchid stallion (B). Counterstaining with Mayer's haematoxylin. The presence of 3β-HSD is confined to Leydig cells (arrows). Note a clearly weaker staining in the cryptorchid horse (A) than in the healthy stallion (B). LC-

hormones biosynthesis (Huff et al., 2001; Kotula-Balak et al., 2001; Pinart et al., 2000).

**5. HSDs in pathological conditions**

**5.1. Temperature and 3β-HSD activity**

al., 1999; Ren et al., 2006; Ryan et al., 1986).

Leydig cells, ST-seminiferous tubules. Bars 20 µm.

(Skakkebaek et al., 2001).

**Figure 3.** (A-C) Immunostaining for 3β-HSD. Positive staining of various intensity is confined to the cytoplasm of Leydig cells (arrows). Note, clearly reduced staining for 3β-HSD in Leydig cells treated with high OP dose (B). In many cells weak to moderate staining in the perinuclear region is visible (arrowheads). In Leydig cells treated with low OP dose (C) the intensity of immunostaining is similar to that of the control (A), (arrows). Only in a few cells staining in the perinuclear region is visible (arrowhead). Bars 20 µm.

It is worth noting that the effect of EDCs on HSDs function can be diverse depending on the choice of animal species, age, routes of administration and dose levels. Studies of Pogrmic-Majkic et al. (2010) have shown that in rat Leydig cells atriazine stimulated 17β-HSD, whereas other authors reported inhibition of these enzyme in rat and human microsomes treated with various xenoestrogens (Hu et al., 2010; Vaithinathan et al., 2008; Ye et al., 2011). Recently, it has also been found that antiandrogens such as tributylin, triclosan and flutamide modified HSDs expression in Leydig cells and microsomes of various mammals (Kim et al., 2008; Kumar et al., 2009; McVey & Cooke, 2003; Ohno et al., 2005; Ohsako et al., 2003).
