**3. Age-dependent activity of HSDs**

In testis of mammals two morphologically and functionally different Leydig cell populations have been identified. One develops prenatally (fetal Leydig cells, FLCs) and the second arises postnatally (adult Leydig cells, ALCs), (Mendis-Handagama & Aryiaratne, 2001; Pinto et al., 2010). These two generations have different gene expression profiles, which indicate that they originate from separate types of stem cell (Dong et al., 2007; O'Shaugnessy et al., 2002b). Differentiation of FLCs is initiated by human chorionic gonadotropin (hCG), whereas development of ALCs is critically dependent on LH (O'Shaughnessy et al., 1998). After birth the population of FLCs decreases in size, although some fetal-type cells persist even in the adult testis. In the rat, FLCs that are arranged in large compact clusters and contain numerous lipid droplets. In ALCs the nuclei are large and cytoplasmic content is sparse with a few lipid droplets. The cytoplasm of Leydig cells of both populations contains abundant smooth endoplasmic reticulum (SER) and tubulovascular mitochondria, which are important organelles in biosynthesis of steroid hormones.

The FLCs are fully competent steroidogenically. It has been demonstrated that in rat, FLCs start to express LH receptors (LHR) and 3β-HSD I from fetal day (fd) 15.5 (Payne & Hales, 2004). However in mouse, 3β-HSD I expression has been detected shortly before fd 11 (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 been confirmed in FLCs of rat (Haider, 2004).

268 Dehydrogenases

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). 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, arrowheads-

In testis of mammals two morphologically and functionally different Leydig cell populations have been identified. One develops prenatally (fetal Leydig cells, FLCs) and the second arises postnatally (adult Leydig cells, ALCs), (Mendis-Handagama & Aryiaratne, 2001; Pinto et al., 2010). These two generations have different gene expression profiles, which indicate that they originate from separate types of stem cell (Dong et al., 2007; O'Shaugnessy et al., 2002b). Differentiation of FLCs is initiated by human chorionic gonadotropin (hCG), whereas development of ALCs is critically dependent on LH (O'Shaughnessy et al., 1998). After birth the population of FLCs decreases in size, although some fetal-type cells persist even in the adult testis. In the rat, FLCs that are arranged in large compact clusters and contain numerous lipid droplets. In ALCs the nuclei are large and cytoplasmic content is sparse with a few lipid droplets. The cytoplasm of Leydig cells of both populations contains abundant smooth endoplasmic reticulum (SER) and tubulovascular mitochondria, which are important

The FLCs are fully competent steroidogenically. It has been demonstrated that in rat, FLCs start to express LH receptors (LHR) and 3β-HSD I from fetal day (fd) 15.5 (Payne & Hales, 2004). However in mouse, 3β-HSD I expression has been detected shortly before fd 11

weak to moderate staining). Magnifications, x 320.

**3. Age-dependent activity of HSDs**

organelles in biosynthesis of steroid hormones.

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

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

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 & Mendis-Handagama, 2000; Haider, 2004).

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 described (Wu et al., 2007).

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).

Differentiation of progenitors to newly formed adult Leydig cell is associated with the cell cytoplasm shape change from spindle shaped to polygonal. Newly formed Leydig cells move toward the central interstitium and locate near blood capillaries although they do not exclusively arrange in clusters. These cells express LHR and the levels of 3β–HSD VI, P450scc and P450c17 increase with the further steps of Leydig cell differentiation (Ariyaratne & Mendis-Handagama, 2000; Shan et al., 1993). It has been demonstrated that in mice *Hsd3b3* and *Hsd3b6,* remain fairly stable after birth but show a pubertal rise in expression around pd 20 (O'Shaughnessy et al., 2002)

Hydroxysteroid Dehydrogenases – Localization, Function and Regulation in the Testis 271

aging Leydig cells adenyl cyclase is maintained that results in the defect of the cAMP-LH signaling cascade. In addition, protein and mRNA levels of StAR have been significantly reduced, suggesting deficits in the transport of cholesterol to the inner mitochondrial membrane of aged Leydig cells. Moreover, the activity, protein level, and mRNA level of P450scc, P450c17, 3β–HSD and 17β–HSD have been found markedly reduced in old Leydig cells (Ivell et al., 2003; Luo et al., 1996; Midzak et al., 2009; Zirkin & Chen, 2000). Interestingly, these authors have demonstrated that long-term suppression of steroidogenesis by administration of T prevents or delays the reduced steroidogenesis that accompanies Leydig cell aging due to suppressing the production of the reactive oxygen

Primary control of 3β-HSD expression occurs through the activation by LH its receptor and the induction of the cAMP second messenger system (Simard et al., 2005). Recent findings from our own laboratory have revealed that bank vole Leydig cells treated with LH have increased steroidogenic capacity and T secretion (Gancarczyk et al., 2003). A profound hypogonadal effect and suppression of T production has been demonstrated in boars treated with deslorelin, an agonist of gonadotropin-releasing hormone (GnRH), (Kopera et al., 2008). In Leydig cells of treated boars very weak or lack of LHR and 3β-HSD expression has been detected. In contrast, Lin et al. (2008) who treated mouse Leydig cells with GnRH agonists (I and II) have demonstrated that 3β-HSD has been stimulated directly resulting in

increase of T production. On the contrary, 17β-HSD was not induced in treated cells.

Formation of cAMP activates steroidogenesis by temporally distinct manners either, acutely (minutes) due to StAR action or chronically (hours) related on P450scc, P450c17, 3β-HSD and 17β-HSD activities. The delivery of cholesterol into the inner mitochondrial membrane is the rate-determining step in steroidogenesis. Also the differences in the way that cultured Leydig cells respond to cAMP have been reported. In mouse Leydig cells, cAMP has stimulated T production which then suppressed 3β-HSD at the mRNA level, whereas addition of cAMP to cultured rat Leydig cells increases 3β-HSD activity and expression at both the mRNA and protein level after 24–72 h (Keeney & Mason 1992; Payne & Sha 1991).

Interestingly, expression of 3β-HSD has been reported to be dependent upon steroidogenesis factor 1 (SF1). SF1 response element has been detected in the proximal promoter region of the human 3β-HSD type II gene. Recently Scott et al. (2009) have confirmed that the mouse 3β-HSD I gene promoter has three potential SF1 consensus binding sites. However, it is

A series of studies show that pituitary hormone-prolactin (PRL) and thyroid hormones regulate activity of HSDs. In Leydig cells of hypophysectomized rats treated with PRL, a significant increase in number of 3β-HSD immunopositive cells together with an increase in T and E2 concentrations was found (Dombrowicz et al., 1992; Manna et al., 2001). Also our

currently unknown whether SF1 regulates the expression of 17β–HSD.

species that are a by-product of steroidogenesis itself.

**4.1. Pituitary hormones and other peptides**

**4. Regulation of HSDs function**

5α-androstane-3α and 17β-diol is synthesized as the predominant androgen with the emergent increase in activity of 17β–HSD 3 and in a continuous presence of 5α-reductase and 3α-HSD (Hardy et al., 1990). It is worth noting, that in immature ALCs 11βhydroxysteroid dehydrogenase type 1 (11β-HSD 1) and 11β-hydroxysteroid dehydrogenase type 2 start to be expressed. In the rat testis, the presence of 11β-HSD 1 is in coincidence with the first appearance of elongated spermatids in the seminiferous tubules (Haider, 2004).

Adult Leydig cells are the dominant cell type of the Leydig cell lineage from pd 56 (Benton et al., 1995). Transformation of immature adult Leydig cells into mature adult Leydig cells is characterized by a significant increase in the average cell size and disappearance of cytoplasmic lipid droplets. The capacity to secrete T increases significantly in mature adult Leydig cells because of their enhanced responsiveness to circulatory LH due to the acquisition of higher numbers of LHR. During this time in the mouse testis, 3β-HSD VI becomes the predominant isoform of HSDs (Payne & Hales, 2004).

Additionally, the sharp decline in 5α-reductase activity overlaps. Shan et al. (1993) have reported that the mature Leydig cells by pd 90 produce 150 times more T than progenitors, and five times more than immature Leydig cells. Such high T levels are required for initiation, maintenance and regulation of the spermatogenesis. By day 90 the secretory capacity per ALC in rat has been estimated as 1.43 pg.

During puberty ALCs are particularly sensitive to androgens and expression of AR mRNA in this time is significant. Studies have shown that in the absence of AR, there is developmental failure of ALC maturation (O'Shaughnessy et al., 2010). However, there is well known phenomenon when ALCs destroyed by ethane dimethane sulphonate (EDS) administration can proliferate to regenerate the original population of Leydig cells (Teerds & Rijnities, 2007).

In aging human testis, both serum and intratesticular T concentrations progressively decline being in correlation to decreased LH level. In rat, these changes have been reported to be strain-dependent (Harman et al., 2001). In Brown Norway rats, the decrease in T level concomitantly with an increase in FSH level and unchanged LH level have been detected (Chen et al., 2002). Several studies have demonstrated that in men decrease in T level is associated with alterations in body composition, diminished energy, muscle strength and physical function, depressed mood and decreased cognitive function (Matsumoto, 2002). These age related changes result from the loss of steroidogenic capacity of the Leydig cells and/or reduction in their number (Chen et al., 2001, 2009). It has also been found that in aging Leydig cells adenyl cyclase is maintained that results in the defect of the cAMP-LH signaling cascade. In addition, protein and mRNA levels of StAR have been significantly reduced, suggesting deficits in the transport of cholesterol to the inner mitochondrial membrane of aged Leydig cells. Moreover, the activity, protein level, and mRNA level of P450scc, P450c17, 3β–HSD and 17β–HSD have been found markedly reduced in old Leydig cells (Ivell et al., 2003; Luo et al., 1996; Midzak et al., 2009; Zirkin & Chen, 2000). Interestingly, these authors have demonstrated that long-term suppression of steroidogenesis by administration of T prevents or delays the reduced steroidogenesis that accompanies Leydig cell aging due to suppressing the production of the reactive oxygen species that are a by-product of steroidogenesis itself.
