**5. 11-hydroxysteroid dehydrogenase in developing testis- marker for differentiation of the Leydig cells**

The enzyme 11-hydroxysteroid dehydrogenase (11-HSD) is hypothesized to modulate LCs steroidogenesis by controlling the intracellular concentration of glucocorticoids. By doing so, 11-HSD can protect the LCs against the suppressive effect of glucocorticoids [109- 112]. Glucocorticoids have been found to directly inhibit the transcription of genes encoding the key enzymes of testosterone biosynthesis [113,114]. Excessive glucocorticoid exposure suppress androgen synthesis and thus decrease serum testosterone (T) levels by inducing LC apoptosis and reducing the number of LCs per testis [115,116]. The effects of glucocorticoids on LCs are not only associated with the classic glucocorticoid receptormediated mechanism but possibly through the plasma membrane receptor or prereceptormediated action by the glucocorticoid metabolizing enzyme 11β-HSD1 [117]. Both isoforms of 11-HSD are localized in testicular LCs [118-121]. Recent studies showed that reductase activity predominates in both human and rat type 1 11-HSD [109]. In contrast, the other 11-HSD isoform, type 2, has been found to be exclusively oxidative [118,110,131]. Predominance of oxidative activity results in glucocorticoid inactivation, whereas the reductive activity of the enzyme has an opposite effect [109]. Hu et al. [122] postulated that inhibition of 11β-HSD1 in rats *in vivo*, increases intracellular active glucocorticoid concentration and thereby affects serum T concentration and steroidogenic enzyme expression in the LCs. The above mentioned data suggest an important role of 11β-HSD1 in modulating intracellular corticosterone concentrations and, in turn, for a direct effect of glucocorticoids on LCs. On the other hand, 11β-HSD type 1 mRNA and its activity was decreased corticosterone deficiency, and it seems that LCs need to maintain their intracellular concentration of corticosterone for normal function [123].

Several authors have demonstrated that 11-HSD in LCs is predominantly an oxidase [109- 111] and the enzyme has been suggested as a marker for the functional maturity of rat adult LCs [111,112,124,125]. The appearance of 11-HSD correlates with the postnatal increase in testicular weight, LCs number, total surface area of the intracellular membranes and T production by LCs [112]. Neumann *et al.* [126] reported a temporal coincidence of the first appearance of elongated spermatids in the seminiferous epithelium and the first histochemical demonstration of 11-HSD in the rat LCs on 35 pnd. The developmental pathway of ALCs population is accompanied with an increase in the 11-HSD activity and thus the enzyme can be used as a marker for steroidogenic differentiation of LCs [112,124,126,127]. Examination of 11-HSD in the LCs revealed that both oxidative and reductive activities were barely detectable in the progenitors (PLCs), intermediate in immature type (ILCs), and highest in ALCs. The ratio of the two activities favored reduction in PLCs and ILCs and oxidation in ALCs [109]. Clear recognizable oxidative activity of 11- HSD is present from 31 pnd onward, first in single ALCs and later in majority of these cells [127]. ALCs population expresses high levels of 11-HSD oxidative activity [109,125] and enzymatic behavior of 11-HDS in LCs is not consistent with the presence of type 1 alone [127,128]. Developmental analysis of 11-HSD in rat LCs revealed that 11-HSD reductive activity predominated in LCs precursors, whereas in adult LCs, the enzyme was primarily oxidative [118]. This switch, observed in the predominant direction of catalysis of 11-HSD from reduction to oxidation in adult LCs, may protect this cell type from glucocorticoidmediated inhibition of steroidogenesis. It was demonstrated that the adult LCs expressed not only 11-HSD type 1, an oxidoreductase, but also type 2, an unidirectional oxidase [129, 130]. Due to its high affinity for glucocorticoid substrates and exclusively oxidative activity, 11-HSD type 2 may also play a protective role in blunting the suppressive effects of glucocorticoids on LCs steroidogenesis. The inhibition of 11-HSD1 predominantly lowered reductase activity whereas by inhibition of 11-HSD2 alone, the oxidase activity was more prominently suppressed [131]. Recently, it has been reported that products such 7αhydroxytestosterone significantly switched 11β-HSD1 oxidoreductase activities toward reductase in developing rat testis and thus regulates the direction of 11β-HSD1 activity in LCs [132]. It seems that the switch of 11-HDS activity from reduction to oxidation during the transition from PLCs to ALCs [109] can be associated with the presence of 11-HSD2.

128 Dehydrogenases

metabolite syndrome.

**differentiation of the Leydig cells** 

understood. The enzymatic inactivation of cortisol and corticosterone by 11β-HSD enzymes appears to be of central importance for protection of gonadal steroidogenesis, prevention of

This review focuses on the importance of 11β-HSD isoenzymes in the developing and aging testis, ovary, adrenal gland, placenta and adipose tissue. The current work aims to provide recent understanding of the biological roles played by 11β-HSD in different processes and diseases including reproduction, adrenal gland function, cystic ovarian disease, and the metabolite syndrome. In addition, this review summarizes recent knowledge based on human data and genetic models on the clinical importance of 11β-HSD in relation to

**5. 11-hydroxysteroid dehydrogenase in developing testis- marker for** 

intracellular concentration of corticosterone for normal function [123].

Several authors have demonstrated that 11-HSD in LCs is predominantly an oxidase [109- 111] and the enzyme has been suggested as a marker for the functional maturity of rat adult LCs [111,112,124,125]. The appearance of 11-HSD correlates with the postnatal increase in testicular weight, LCs number, total surface area of the intracellular membranes and T production by LCs [112]. Neumann *et al.* [126] reported a temporal coincidence of the first appearance of elongated spermatids in the seminiferous epithelium and the first

The enzyme 11-hydroxysteroid dehydrogenase (11-HSD) is hypothesized to modulate LCs steroidogenesis by controlling the intracellular concentration of glucocorticoids. By doing so, 11-HSD can protect the LCs against the suppressive effect of glucocorticoids [109- 112]. Glucocorticoids have been found to directly inhibit the transcription of genes encoding the key enzymes of testosterone biosynthesis [113,114]. Excessive glucocorticoid exposure suppress androgen synthesis and thus decrease serum testosterone (T) levels by inducing LC apoptosis and reducing the number of LCs per testis [115,116]. The effects of glucocorticoids on LCs are not only associated with the classic glucocorticoid receptormediated mechanism but possibly through the plasma membrane receptor or prereceptormediated action by the glucocorticoid metabolizing enzyme 11β-HSD1 [117]. Both isoforms of 11-HSD are localized in testicular LCs [118-121]. Recent studies showed that reductase activity predominates in both human and rat type 1 11-HSD [109]. In contrast, the other 11-HSD isoform, type 2, has been found to be exclusively oxidative [118,110,131]. Predominance of oxidative activity results in glucocorticoid inactivation, whereas the reductive activity of the enzyme has an opposite effect [109]. Hu et al. [122] postulated that inhibition of 11β-HSD1 in rats *in vivo*, increases intracellular active glucocorticoid concentration and thereby affects serum T concentration and steroidogenic enzyme expression in the LCs. The above mentioned data suggest an important role of 11β-HSD1 in modulating intracellular corticosterone concentrations and, in turn, for a direct effect of glucocorticoids on LCs. On the other hand, 11β-HSD type 1 mRNA and its activity was decreased corticosterone deficiency, and it seems that LCs need to maintain their

intra-uterine growth retardation and metabolite syndrome.

As mentioned above the main function of glucocorticoids in adult LCs is inhibition of T biosynthesis [111]. Glucocorticoids directly regulate T production in LCs through glucocorticoid receptor (GR)-mediated repression of the genes that encode T biosynthetic enzymes [143,109]. The response of LCs to glucocorticoids depends not only on the number of GR and the circulating concentration of glucocorticoids, but also on the ratio of 11-HSD oxidative and reductive activities [144]. When oxidation predominates over reduction, 11- HSD decreases the intracellular availability to active glucocorticoid, attenuating GRmediated responses [118]. In this way, T production is maintained in the presence of normal serum concentrations of corticosterone and it is inhibited only if 11-HSD oxidative capacity in LCs is reduced.

By using experimental model for treatment with ethane-dimethnesulphonate (EDS) of mature rats our studies provided new data about expression pattern of 11-HSD during renewal of LCs population [133]. The quantitative immunohistochemical analysis of 11 HSD2 pattern after EDS treatment revealed progressive increases in the reaction intensity during postnatal development (on d 21after EDS) and reached a maximum on d35 and that is a turning point in the development from immature to mature LCs [133]. These changes in 11-HSD2 expression are consistent with previous data about structural and functional

maturation of the new population of LCs after EDS [134,135]. Therefore, 11-HSD2 can be a useful marker for ALCs differentiation and the reaction intensity might be associated with increased 11-HSD oxidative activity that occurred during the transition from PLCs to ALCs in postnatal rat testis [109,127]. Moreover, the gene profiling of rat PLCs, immature LCs and ALCs showed increased expression of 11-HSD2 gene that is in parallel with enhanced 11- HSD2 enzyme activity during postnatal development [136]. Together with previous studies [126] the data from EDS model suggest the relationship between 11-HSD and kinetics of spermatid differentiation and restoration of T production by new LCs population.

Hydrohysteroid Dehydrogenases – Biological Role and Clinical Importance – Review 131

**Figure 6.** 11-HSD type 2 in developing Leydig cells (LC)- 7, 21 and 35 days after EDS; and aging

**7. 11-hydroxysteroid dehydrogenase in the adrenal gland - expression** 

As mentioned above, the enzyme11-HSD catalyzes the interconversion of glucocorticoids to inert metabolites in man and rodents and plays a crucial role in regulating the action of corticosteroids. Inhibition of 11-HSD allows access of cortisol or corticosterone to the mineralcorticoid receptors where they act as mineralcorticoids [148]. Northern blot analyses revealed expression of mRNAs encoding both 11-HSD1 and 11-HSD2 in the whole rat

**profile under conditions of testosterone withdrawal** 

Leydig cells- 3, 18 and 24-months of age. x 400.
