**2. Steroidogenesis in the testis**

In the male, Leydig cells which are present in the interstitium of the testis express all of the enzymes essential for the conversion of cholesterol to androgens and estrogens. The major pathways of steroid hormone synthesis are well established, and the sequence of the responsible steroidogenic enzymes has been elucidated (Payne & Hales, 2004). There are two major classes of steroidogenic enzymes: the cytochrome P450 heme-containing proteins and the hydroxysteroid dehydrogenases (HSDs). The first class contains: cytochrome P450 side chain cleavage (P450scc), cytochrome P450 17α-hydroxylase C17-C20 lyase (P450c17) and cytochrome P450 aromatase (P450arom), whereas the second includes both 3βhydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3β-HSD) and 17β-hydroxysteroid dehydrogenase/ketosteroid reductase (17β-HSD/KSR), (Payne, 2007).

The initial step of T biosynthesis is the conversion of the C27 cholesterol to the C21 pregnenolone (Figure 1). This reaction is catalyzed by the cytochrome P450scc, which is located in the inner mitochondrial membrane. Pregnenolone diffuses across the mitochondrial membrane and is further metabolized by enzymes associated with the smooth

**Figure 1.** Steroid biosynthesis pathways in Leydig cell.

endoplasmic reticulum. P450c17 catalyzes the conversion of C21 pregnenolone or progesterone (P4) to the C19 dehydroepiandrosterone or androstenedione, respectively, while 3β-HSD catalyzes the conversion of Δ5-3β-hydroxysteroids (pregnenolone or dehydroxypregnenolone, and DHEA, respectively) to the Δ4-3-ketosteroids (P4, or 17α-hydroxyprogesterone, and androstenedione, respectively). Depending on the animal species, biosynthesis of sex hormones proceeds down either one or both of the Δ4 and Δ5 pathways. In rodents, the Δ4 pathway is primary whereas in primates, pigs and rabbits Δ5 pathway is dominant (Fluck et al., 2003; Mathieu et al., 2002). In the final step of sex hormones biosynthesis, conversion of androstenedione into T, 17β-HSD is involved. It was reported that the balance between these androgens depends on the type and activity of 17β-HSD present (Simard et al., 2005).

266 Dehydrogenases

**2. Steroidogenesis in the testis** 

particularly sensitive to androgen action, especially in four steps of germ cell development: spermatid adhesion and development, spermiation, progression through meiosis and spermatogonial differentiation (Verhoeven et al., 2010). Thus spermatogenesis is closely related and absolutely dependent on steroid hormone biosynthesis, action and control.

In the male, Leydig cells which are present in the interstitium of the testis express all of the enzymes essential for the conversion of cholesterol to androgens and estrogens. The major pathways of steroid hormone synthesis are well established, and the sequence of the responsible steroidogenic enzymes has been elucidated (Payne & Hales, 2004). There are two major classes of steroidogenic enzymes: the cytochrome P450 heme-containing proteins and the hydroxysteroid dehydrogenases (HSDs). The first class contains: cytochrome P450 side chain cleavage (P450scc), cytochrome P450 17α-hydroxylase C17-C20 lyase (P450c17) and cytochrome P450 aromatase (P450arom), whereas the second includes both 3βhydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3β-HSD) and 17β-hydroxysteroid

The initial step of T biosynthesis is the conversion of the C27 cholesterol to the C21 pregnenolone (Figure 1). This reaction is catalyzed by the cytochrome P450scc, which is located in the inner mitochondrial membrane. Pregnenolone diffuses across the mitochondrial membrane and is further metabolized by enzymes associated with the smooth

dehydrogenase/ketosteroid reductase (17β-HSD/KSR), (Payne, 2007).

**Figure 1.** Steroid biosynthesis pathways in Leydig cell.

3β-HSDs are membrane-bound enzymes that are distributed in both mitochondrial and microsomal membranes (Payne & Hales, 2004; Pelletier et al., 2001). The relevance of dual localization of these HSDs is related to substrate accessibility (Simard et al., 2005). Coprecipitation studies have shown that, in the inner mitochondrial membrane, 3β-HSD comprises a functional steroidogenic complex with P450scc, which immediately provides substrates converted from cholesterol to the 3β-HSD (Cherradi et al., 1995).

During the past decade, multiple isoforms of 3β-HSDs have been isolated and characterized in human, mouse and rat tissues. Six, highly homologous in their amino acid sequence isoforms have been identified in the mouse, but only two of them: 3β-HSD type I (3β-HSD I), and 3β-HSD type VI (17β–HSD VI) are expressed in the testis. In human testis only 3β-HSD I has been found (Payne & Hales, 2004).

Similarly to 3β-HSDs, 17β-HSDs are membrane-bound enzymes, and their soluble forms have also been reported. To date, 14 different types of 17β–HSDs have been identified (Blanchard & Luu-The, 2007). Unlike 3β-HSDs there is very little homology among the different 17β-HSD enzymes. Only three types, 17β–HSD type 3 (17β–HSD 3), 17β–HSD type 5 and 17β–HSD type 12 (17β–HSD 12), have been detected to be exclusively expressed in the testis. 17β-HSD 3 converts androstenedione to T as well as it is an important partner of P450arom involved in conversion of C18 steroid, estrone to E2 (Andersson et al., 1995). Recently, it has been confirmed for mice, humans and primates that 17β-HSD 12 shares high homology and function with 17β-HSD 3 (Blanchard & Luu-The, 2007; Liu et al., 2007).

The hydroxysteroid dehydrogenases belong to the same phylogenetic protein family, namely the short-chain alcohol dehydrogenase reductase superfamily. These enzymes are involved in the reduction and oxidation of steroid hormones requiring NAD+/NADP+ as acceptors and their reduced forms as donors of reducing equivalents. Studies have shown that mouse 3β-HSD has different cofactor preference: 3β-HSD I requires NAD+ while 3β-HSD type IV and V requires NADP+ as cofactors. Interestingly, 17β-HSD 3 prefers NADPH as a cofactor, and its primary activity is reductive. Studies have shown that a mutation in *HSD3B3* gene leads to decreased NADPH binding to tyrosine that has been identified as a critical residue for binding. Substitution of tyrosine with different amino acids resulted in alterations in cofactor preference switching from NADPH to NADH (Andersson et al., 1995; McKeever et al., 2002). In addition, Schäfers et al. (2001) have reported different cofactor preference for 17β-HSD 3 on different days of postnatal development in rat.

Both 3β-HSD and 17β-HSD are well known Leydig cell-specific markers in different mammals, at different times of development and under different perturbation regimes (Bilinska, 1994; Hejmej et al., 2011b; Mendis–Handagama & Ariyaratne, 2001; Teerds et al., 2007). In previous studies, activity of HSDs in testis of various mammals was mostly detected using histochemical techniques (Badrinarayanan et al., 2006; Bilinska 1979, 1983, 1994; Hutson, 1989), (Figure 2). Nowadays the resolution of their localization increased with applying specific antibodies (Kotula-Balak et al., 2011; Pelletier et al., 1999; Pinto et al., 2010).

Hydroxysteroid Dehydrogenases – Localization, Function and Regulation in the Testis 269

(Baker et al., 1999). Recently with the use both histochemical and immunohistochemical methods the presence of 3α-hydroxysteroid dehydrogenase (3α-HSD) and 17β-HSD has also

From fd 15.5 FLCs start actively producing T and its synthesis increases gradually (Habert & Picon, 1984). Expression of hormone receptors and enzymes in FLCs arise continuously during existence of this population in the testis. Interestingly, Ivell et al. (2003) have demonstrated that 17β-HSD type 10 (17β-HSD 10) starts to be expressed at the time when FLCs begin to involute. However, the pick of oxidative activity of these enzyme has been

Fetal population of Leydig cells is the primary source of T, androstenedione and DHT in both fetal and early postnatal testis (Ariyaratne & Mendis-Handagama, 2000; Huhtaniemi & Pelliniemi, 1992). Multiple studies have shown that T-producing capacity of FLC is significantly greater than that of ALC and is calculated to be even 87 pg per cell (Aryaratne

During the neonatal-prepubertal period T is required for differentiation and morphogenesis of the male genital tract, activation of the hypothalamo-hypophyseal-testicular axis, completion of the testicular descent, masculinization of the brain, control of Sertoli cell number, initiation of spermatogenesis and formation of ALC precursors (Ariyaratne &

Steroidogenic capacity of FLCs is still high through the first postnatal week, although concentrations of circulating T are much lower due to decrease in number of these cells. Moreover, the inhibitory effects of Müllerian Inhibiting Substance (MIS) and transforming growth factor-bs (TGF-bs) on FLCs steroidogenic activity in postnatal testis have been

Testosterone production gradually increases to high levels with the development of ALCs (Benton, 1995; Chen et al., 2010; Hardy et al., 1989). The proliferation and differentiation of the adult population is regulated by an interplay of multiple regulatory factors, that can simulate, as well as inhibit, Leydig cells at each developmental stage. The development of ALCs is initiated around day 14 after birth and finishes around day 60. This process consists of multiple steps of proliferation and differentiation such as: proliferation of precursor cells; differentiation of precursor cells to Leydig cell progenitors, progenitors into newly formed adult Leydig cells, newly formed adult Leydig cells into immature adult Leydig cells; and finally, maturation of the immature adult Leydig cells to mature adult Leydig cells. In the rat, it has been reported that stem cells and mesenchymal precursor cells do not express steroidogenic enzymes however precursor cells acquire 3β–HSD III and other setroidogenic enzymes like cytochromes: P450scc and P450c17 prior to gain LHR (Hardy et al., 1989; Teerds et al., 2007; Zirkin, 2010). These cells have negligible amounts of 17β-HSD 3 while expression of steroid metabolizing enzymes 5α-reductase and 3α-HSD is high. Thus precursor cells produce androsterone as their main androgen product (O'Shaughnessy et al., 2000). Also expression of AR by early developmental stages of ALCs lineage is required for

further transformations of these cells under androgen control (Ge & Hardy, 1997).

& Mendis-Handagama 2000; Huhtaniemi et al., 1982; Tapanainen et al., 1984).

been confirmed in FLCs of rat (Haider, 2004).

Mendis-Handagama, 2000; Haider, 2004).

described (Wu et al., 2007).

determined on postnatal day (pd) 16 (Schäfers et al., 2001).

It has been reported that 3β-HSD type III (3β-HSD III) as well as 17β-HSD 3 and 17β-HSD type 10 are useful markers also for germ cells in rat, mouse, equine and black bear testis (Almeida et al., 2011; Ivell et al., 2003; O'Shaugnessy et al., 2000). Recently Scott et al. (2009) have indicated 17β-HSD 3 as a good marker for Sertoli cells in fetal mouse testis.

**Figure 2.** (A-B) Histochemical localization of 3β-HSD (A) and 17β-HSD (B) in cultured mouse Leydig cells. Note various intensity of the staining in the individual cells (arrows-strong staining, arrowheadsweak to moderate staining). Magnifications, x 320.
