Several signals, such as hormones and signaling molecules, have been identified as important regulators of Leydig cell differentiation and function. Conveying these signals and translating them into a genomic response to ensure an accurate physiological output requires the action of a network of transcription factors, including those belonging to the nuclear receptor superfamily. Nuclear receptors regulate expression of genes important for growth, differentiation, development, and homeostasis. Several nuclear receptors, such as steroid hormone receptors (NR3A and NR3C families), are activated upon ligand binding, whereas others, including members of the NR2C, NR2F, and NR4A families, either do not require a ligand or ligands have yet to be identified. Several nuclear receptors (e.g., NR2F2 and NR5A1) have been shown to play essential roles in Leydig cells, whereas for others (e.g., NR2B1 and NR4A1), the assessment of their function has been precluded by the early embryonic lethality associated with null mice or by redundancy mechanisms by other family members. This is now being overcome with the generation of novel approaches, including Leydig cell-specific knockout models. This review provides an overview of the nuclear receptor family of transcription factors as they relate to Leydig cell gene expression and function.

Introduction

The mammalian testis is composed of two main compartments: the seminiferous tubules, where germ cells are surrounded by Sertoli cells, and the region between the tubules that contains predominantly Leydig cells. Although Leydig cells were first described in 1850, their endocrine role in the control of male secondary sexual characteristics was only suggested in 1903 [1]. Today, the importance of these cells for male sex differentiation and fertility is indisputable. Leydig cells produce insulin-like 3 (INSL3) and testosterone, which are critical hormones for normal male sex differentiation and reproductive function. INSL3 regulates testis descent during fetal life and germ cell survival in adults [24]. Testosterone controls several critical processes, such as virilization of the male fetus, male sexual behavior, male sex accessory gland development and function, and initiation and maintenance of spermatogenesis (reviewed in Walker [5] and Zirkin [6]). Although INSL3 and androgens are essential for the development and function of the male reproductive system during both fetal and postnatal life, two distinct populations of Leydig cells are responsible for the production of these hormones during these periods: fetal Leydig cells (FLCs) and adult Leydig cells (ALCs). Fetal Leydig cells differentiate from stem progenitor cells that gradually acquire the potential to synthesize androgens (reviewed in Barsoum and Yao [7] and Griswold and Behringer [8]). These cells atrophy shortly after birth and are essentially absent from the adult testis. As such, they do not contribute to the ALC population. Adult Leydig cells derive from undifferentiated precursors that are present after birth and start to differentiate prior to puberty [9].

Cell differentiation creates the diversity of cell types that arise during development. This process is essential for proper functioning of every living organism because each differentiated cell type is responsible for a highly specialized function. As for all cellular processes, cell differentiation and function are tightly regulated by several hormones, signaling molecules, and paracrine factors. Conveying these signals and translating them into a genomic response to ensure a proper physiological output requires a network of transcription factors. Identifying these factors and elucidating their mechanism of action are of paramount importance for our understanding of cell differentiation and function in all tissues. This includes testicular Leydig cells.

Nuclear receptors are a family of transcription factors known to regulate several physiological and pathological processes. Several hundred nuclear receptors have been isolated so far from several species [10], including 47 in the rat, 48 in humans, and 49 in the mouse [11]. As depicted in Figure 1, they share a well-conserved modular structure composed of six domains: a ligand-independent activation domain (AF-1), a zinc finger DNA-binding domain (DBD), a hinge to allow for conformational change, a ligand-binding domain (LBD) and ligand-dependent activation domain (AF-2), and a C-terminal domain of unknown function (reviewed in Bain et al. [12]). All nuclear receptors bind to an element called the hormone-response element (HRE) with a core consensus sequence AGGTCA or AGAACA found in the promoter of target genes (reviewed in Glass [13]). The core sequence AGAACA is predominantly recognized by the glucocorticoid, mineralocorticoid, progesterone, and androgen receptors, whereas the sequence AGGTCA is mainly recognized by the estrogen, thyroid hormone, retinoic acid, and vitamin D receptors, as well as most other members of the nuclear receptor superfamily (Fig. 2). Some nuclear receptors bind as monomers to an HRE that contains a three-nucleotide 5′ extension, whereas others bind as homodimers or heterodimers to two HREs that are organized as direct (DR; → n →), inverted (IR; → n ←), or everted (ER; ← n →) repeats separated by a variable number of nucleotides generally ranging from zero to six (Fig. 2 and reviewed in Forman and Evans [14]).

Fig. 1.

Schematic representation of the modular structure of nuclear receptors. Top: The majority of nuclear receptors are composed of six domains: domain A/B contains an activation domain (AF-1; orange box) of variable length, domain C is the DBD (yellow box) that consists of two zinc fingers (represented by the circles), domain D is a hinge region (light green box), domain E (bright green box) contains the dimerization interface, LBD, and ligand-dependent activation domain (AF-2), and domain F is a variable region of unknown function (blue box). Bottom: In two atypical nuclear receptors belonging to the NR0B family, the A/B, C, and D domains are replaced by a 65- to 70-amino acid motif rich in alanine and glycine (blue arrow). This motif is repeated 3.5 times in NR0B1 and only once in NR0B2.

Fig. 1.

Schematic representation of the modular structure of nuclear receptors. Top: The majority of nuclear receptors are composed of six domains: domain A/B contains an activation domain (AF-1; orange box) of variable length, domain C is the DBD (yellow box) that consists of two zinc fingers (represented by the circles), domain D is a hinge region (light green box), domain E (bright green box) contains the dimerization interface, LBD, and ligand-dependent activation domain (AF-2), and domain F is a variable region of unknown function (blue box). Bottom: In two atypical nuclear receptors belonging to the NR0B family, the A/B, C, and D domains are replaced by a 65- to 70-amino acid motif rich in alanine and glycine (blue arrow). This motif is repeated 3.5 times in NR0B1 and only once in NR0B2.

Fig. 2.

DNA-binding properties of nuclear receptors. The HRE core sequence AGGTCA or AGAACA is represented by the black arrows. A) Steroid hormone receptors bind as homodimers to palindromic DNA sites (inverted repeats) separated by three nucleotides (IR3). B) Several nuclear receptors bind to DNA as heterodimers with NR2B members to direct repeats separated by zero to six nucleotides (DR1–6) or to inverted repeats spaced by 0 or 1 nucleotide (IR0–1). C) Some nuclear receptors (excluding steroid hormone receptors) bind as homodimers to DR0–6, or everted repeats separated by four to six (ER4–6) or 10 (ER10) nucleotides. D) A small group of nuclear receptors bind to DNA as monomers to HRE containing a three-nucleotide 5′ extension, represented by the black box left of the arrow. Recognition of these extra three nucleotides in the DNA-binding motif is mediated by amino acid residues located in the C-terminal extension (CTE) of the core DBD of the nuclear receptor.

Fig. 2.

DNA-binding properties of nuclear receptors. The HRE core sequence AGGTCA or AGAACA is represented by the black arrows. A) Steroid hormone receptors bind as homodimers to palindromic DNA sites (inverted repeats) separated by three nucleotides (IR3). B) Several nuclear receptors bind to DNA as heterodimers with NR2B members to direct repeats separated by zero to six nucleotides (DR1–6) or to inverted repeats spaced by 0 or 1 nucleotide (IR0–1). C) Some nuclear receptors (excluding steroid hormone receptors) bind as homodimers to DR0–6, or everted repeats separated by four to six (ER4–6) or 10 (ER10) nucleotides. D) A small group of nuclear receptors bind to DNA as monomers to HRE containing a three-nucleotide 5′ extension, represented by the black box left of the arrow. Recognition of these extra three nucleotides in the DNA-binding motif is mediated by amino acid residues located in the C-terminal extension (CTE) of the core DBD of the nuclear receptor.

Nuclear receptors can be separated into two families: the steroid receptor family (glucocorticoid, mineralocorticoid, androgen, estrogen, and progesterone receptors), and the nonsteroid receptor family (vitamin D, retinoic acid, thyroid hormone receptors, and several dozens more). All members of the steroid receptor family are activated upon ligand binding. The nonsteroid receptor family is further divided into two subfamilies: those with known ligands and those for which no ligand has been identified (orphan nuclear receptors).

Several nuclear receptors are present in Leydig cells. Although developmental and physiological roles have been determined for these nuclear receptors in Leydig cells, their target genes and molecular mechanisms of action have yet to be fully elucidated. This review presents an overview of nuclear receptors in Leydig cells (listed in Table 1) and their roles in the differentiation and function of these cells.

Table 1.

Nuclear receptors in Leydig cells.

Official nuclear receptor nomenclaturea  Official gene nameb  Common name  Common abbreviation 
NR1A1, NR1A2 Thra, Thrb Thyroid hormone receptor α, β TRα, TRβ 
NR1B1, NR1B2, NR1B3 Rara, Rarb, Rarg Retinoic acid receptor α, β, γ RARα, RARβ, RARγ 
NR1C1, NR1C2, NR1C3 Ppara, Ppard, Pparg Peroxisome proliferator activated receptor α, β/δ, γ PPARα, PPARβ/δ, PPARγ 
NR1H3 Nr1h3 Liver X receptor α LXRα 
NR1H4 Nr1h4 Farnesoid X receptor FXR 
NR2B1, NR2B2, NR2B3 Rxra, Rxrb, Rxrg Retinoid X receptor α, β, γ RXRα, RXRβ, RXRγ 
NR2C1 Nr2c1 Testicular orphan nuclear receptor 2 TR2 
NR2F2 Nr2f2 Chicken ovalbumin upstream promoter-transcription factor II COUP-TFII 
NR3A1, NR3A2 Esr1, Esr2 Estrogen receptor α, β ERα, ERβ 
NR3C1 Nr3c1 Glucocorticoid receptor GR 
NR3C2 Nr3c2 Mineralocorticoid receptor MR 
NR3C3 Pgr Progesterone receptor PR 
NR3C4 Ar Androgen receptor AR 
NR4A1 Nr4a1 Nerve growth factor induced-B NGFI-B, NUR77 
NR4A2 Nr4a2 Nur-related factor 1 NURR1, RNR1 
NR5A1 Nr5a1 Steroidogenic factor 1 SF1, Ad4BP 
NR5A2 Nr5a2 Liver receptor homolog 1 LRH1, FTF 
NR0B1 Nr0b1 Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 DAX1, AHCH 
NR0B2 Nr0b2 Small heterodimer partner SHP 
Official nuclear receptor nomenclaturea  Official gene nameb  Common name  Common abbreviation 
NR1A1, NR1A2 Thra, Thrb Thyroid hormone receptor α, β TRα, TRβ 
NR1B1, NR1B2, NR1B3 Rara, Rarb, Rarg Retinoic acid receptor α, β, γ RARα, RARβ, RARγ 
NR1C1, NR1C2, NR1C3 Ppara, Ppard, Pparg Peroxisome proliferator activated receptor α, β/δ, γ PPARα, PPARβ/δ, PPARγ 
NR1H3 Nr1h3 Liver X receptor α LXRα 
NR1H4 Nr1h4 Farnesoid X receptor FXR 
NR2B1, NR2B2, NR2B3 Rxra, Rxrb, Rxrg Retinoid X receptor α, β, γ RXRα, RXRβ, RXRγ 
NR2C1 Nr2c1 Testicular orphan nuclear receptor 2 TR2 
NR2F2 Nr2f2 Chicken ovalbumin upstream promoter-transcription factor II COUP-TFII 
NR3A1, NR3A2 Esr1, Esr2 Estrogen receptor α, β ERα, ERβ 
NR3C1 Nr3c1 Glucocorticoid receptor GR 
NR3C2 Nr3c2 Mineralocorticoid receptor MR 
NR3C3 Pgr Progesterone receptor PR 
NR3C4 Ar Androgen receptor AR 
NR4A1 Nr4a1 Nerve growth factor induced-B NGFI-B, NUR77 
NR4A2 Nr4a2 Nur-related factor 1 NURR1, RNR1 
NR5A1 Nr5a1 Steroidogenic factor 1 SF1, Ad4BP 
NR5A2 Nr5a2 Liver receptor homolog 1 LRH1, FTF 
NR0B1 Nr0b1 Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 DAX1, AHCH 
NR0B2 Nr0b2 Small heterodimer partner SHP 
a

 According to the Nuclear receptors nomenclature committee [166].

b

 According to the International Committee on Standardized Genetic Nomenclature for Mice and the Rat Genome and Nomenclature Committee, http://www.informatics.jax.org/mgihome/nomen/gene.shtml [167].

Thyroid Hormone Receptors (NR1A1 and NR1A2)

Thyroid hormone (T3) is known to influence male reproductive function (reviewed in Jannini et al. [15] and Wagner et al. [16]) and Leydig cell steroidogenesis [17]. More recently, dietary-induced prenatal hypothyroidism was found to delay differentiation of the ALC population without affecting the fetal population [18]. Thyroid hormone acts through two distinct thyroid hormone receptors: NR1A1 (TRα) and NR1A2 (TRβ). NR1A members can bind to DNA as monomers to an HRE with a three-nucleotide 5′ extension (Fig. 2D) and as homodimers (Fig. 2C) and heterodimers with NR2B members (Fig. 2B) to a variety of HREs (DR4, IR0, ER4–6; reviewed in Glass [13] and Desvergne [19]). Hardy et al. [20] reported that the mRNA for both thyroid hormone receptors is present in primary Leydig cells from rat, and Buzzard et al. [21] later confirmed that NR1A1 is the main thyroid receptor present in testis. Manna et al. [22] have also demonstrated the presence of thyroid hormone receptors in nuclei of the mLTC-1 mouse Leydig cells. This group also showed that T3 increases Star mRNA levels and steroid production in a dose-dependent manner [22]. Conversely, testosterone production was reduced in hypothyroid rat testis [23]. It remains to be determined whether the effects of T3 on Star transcription are the result of NR1A1 directly binding to the Star promoter.

Despite the fact that these data support a role for T3 in Leydig cell differentiation and function, an effect of T3 directly on Leydig cells is still a matter of debate. Indeed, although some groups have reported the presence of NR1A receptor in Leydig cells, others have concluded that Leydig cells do not express the thyroid hormone receptor and that the effects on these cells would be indirectly mediated through Sertoli cells (reviewed in Maran [17]).

The Retinoic Acid and Retinoid X Receptors (NR1B and NR2B Families)

Vitamin A (retinol) and its main biologically active metabolite, retinoic acid, have been shown to regulate testicular function, including steroidogenesis, in rodents [24]. Vitamin A deficiency, hypervitaminosis A, or treatment with 13-cis retinoic acid is known to affect testosterone production, interstitial volume, and seminal vesicle weight [24, 25]. During fetal life, retinoids were shown to negatively regulate steroidogenesis during FLC differentiation [24]. On the other hand, retinoic acid treatment of Leydig cell lines results in increased testosterone production and expression of Cyp17a1 and Star, which encode two enzymes involved in testosterone biosynthesis [26, 27].

Retinoic acid is known to mediate its effect through a family of well-conserved receptors. There are six receptors encoded by distinct genes: NR1Bs 1, 2, and 3 (retinoic acid receptors [RARs] α, β, and γ) and NR2Bs 1, 2, and 3 (retinoid X receptors [RXRs] α, β, and γ). Several RARs have been identified in the rodent testis (reviewed in Livera et al. [28]). Consistent with this, male mice in which the Nr1b1 (Raratm1Ipc/Raratm1Ipc and Raratm3.1Ipc/Raratm3.1Ipc), Nr1b3 (Rargtm1Blt/Rargtm1Blt, Rargtm1Ipc/Rargtm1Ipc, Rargtm3.1Ipc/Rargtm3.1Ipc), and Nr2b2 (Rxrbtm1Ipc/Rxrbtm1Ipc) genes have been inactivated are sterile (reviewed in Mark et al. [29]). Leydig cells were also found to express different RARs and RXRs, especially NR2B1 [30, 31]. A definitive assessment of the role of NR2B1 in male reproduction and in Leydig cells remains to be established, because Nr2b1-deficient mice (Rxratm1Rev/Rxratm1Rev, Rxratm2Ipc/Rxratm2Ipc) die early during fetal life due to cardiac defects [32]. NR2B1 regulates gene expression by binding to DNA as a homodimer to a direct repeat 1 (DR1) motif (Fig. 2C) and as heterodimers to DR1–5 motifs with other nuclear receptors (Fig. 2B), including some that have been shown to regulate gene transcription in steroidogenic cells (NR4A1, NR1H2, and NR1H3; reviewed in Germain et al. [33]). Despite their importance, no direct target genes for this family of nuclear receptors have been identified in Leydig cells.

The Peroxisome Proliferator-Activated Receptors (NR1C Family)

The peroxisome proliferator-activated receptors (PPARs) are transcription factors regulating the expression of genes implicated in lipid metabolism, cellular differentiation, and inflammation. The PPAR family is composed of three nuclear receptors encoded by distinct genes: NR1C1 (PPARα), NR1C2 (PPARβ/δ), and NR1C3 (PPARγ). Although all three NR1C receptors have been detected in MA-10 Leydig cells, NR1C1 appears to be the predominant form [34, 35]. Consistent with this, Nr1c1-deficient mice (Pparatm1Gonz/Pparatm1Gonz) have decreased testosterone levels [34]. In addition, NR1C members, and particularly NR1C1, have been reported to mediate part of the deleterious effects of the widely used endocrine disruptors phthalates in Leydig cells (reviewed in Corton and Lapinskas [36]).

Members of the NR1C family are known to regulate gene expression by binding as heterodimers with NR2B (RXR) members (Fig. 2B) to a peroxisome proliferator response element (PPRE), or DR1, AGGTCAnAGGTCA, where NR1C members occupy the 5′ half of the PPRE [37]. No direct target genes have been identified for NR1C family members in Leydig cells.

The Liver and Farnesoid X Receptors (NR1H Family)

The liver X receptor (LXR) family is composed of two members: NR1H2 (LXRβ) and NR1H3 (LXRα). Their natural ligands include a variety of cholesterol derivatives known as oxysterols. Once activated, they act as transcriptional regulators of lipid and carbohydrate metabolism. NR1H members regulate gene expression by binding as heterodimers with NR2B (RXR) members to a LXRE motif in the promoter of target genes [38]. LXRE consists of two nuclear receptor half-sites (AGGTCA) separated by four base pairs (DR4 element; Fig. 2B). The NR1H/NR2B complex may be independently activated by ligands for either partner [38].

Although they have mostly been studied in the liver (reviewed in Baranowski [39]), roles for NR1H members have recently emerged in the testis, including a role in Leydig cell steroidogenesis. Leydig cells were found to express NR1H3, whereas NR1H2 is found in Sertoli cells [40]. Mice deficient for either Nr1h2 (Nr1h2tm1Djm/Nr1h2tm1Djm) or Nr1h3 (Nr1h3tm1Djm/Nr1h3tm1Djm) are fertile, and only Nr1h2−/− mice show accumulation of lipid droplets in Sertoli cells [41]. Double-knockout mice (Nr1h2tm1Djm/Nr1h2tm1Djm-Nr1h3tm1Djm/Nr1h3tm1Djm), however, are infertile by 4 mo, and by 20 mo Leydig cells are conspicuously absent [41]. In these animals, serum testosterone has a strong tendency to decrease with age, whereas serum luteinizing hormone (LH) levels are elevated consistent with abnormal Leydig cell function [41]. Similar results were also reported by another group, with the exception that a decrease in testosterone levels in Nr1h3−/− mice was also observed [40]. This decrease, however, was found to be due to a central defect, because a significantly lower concentration of LH was found in Nr1h-deficient mice compared with controls [40]. Although no target genes have been identified for NR1H receptors in Leydig cells, treatment of mice with NR1H agonists was found to increase expression of several steroidogenic enzyme-encoding genes, including Star and Hsd3b1, leading to enhanced testosterone production [40]. In adrenal steroidogenic cells, NR1H factors were found to directly bind to and activate the Star promoter [42].

The farnesoid X receptor (FXR) NR1H4 is mainly expressed in the liver, intestine, kidney, and adrenal glands and is activated by bile acids (reviewed in Lefebvre et al. [43]). NR1H4 is known to bind to a DNA motif called FXRE as a monomer (Fig. 2D) or as heterodimers with NR2B (RXR; Fig. 2B and reviewed in Lefebvre et al. [43]). More recently, NR1H4 was identified in normal rodent testis and in Leydig cell lines, where it negatively regulates expression of the Cyp19a1 gene, which encodes the enzyme aromatase that converts androgens into estrogens. NR1H4 inhibits Cyp19a1 transcription through a mechanism that involves competition with the NR5A1 receptor for binding to common nuclear receptor response elements on the Cyp19a1 PII promoter [44]. However, no obvious reproductive abnormalities and no differences in expression levels of various steroidogenic genes (Star, Cyp11a1, Hsd3b1, Cyp17a1) were found in Nr1h4−/− mice (Nr1h4tm1Gonz/Nr1h4tm1Gonz) [45, 46]. Furthermore, Nr1h4−/− mice have normal plasma testosterone levels [46]. On the other hand, treatment of mice with an NR1H4 agonist led to a decrease in both plasma and testicular testosterone concentrations [46]. This was associated with a decrease in Star, Cyp11a1, and Hsd3b1 mRNA levels [46]. The repressive effects of NR1H4 on testicular steroidogenesis are mediated by the nuclear receptor NR0B2, whose expression is upregulated by NR1H4 in Leydig cells [46]. NR0B2 is a well-known transcriptional repressor (see the section on NR0B), and it mediates its repressive effects mainly by inhibiting the activity of the nuclear receptor NR5A2, an activator of steroidogenic gene expression in Leydig cells [46]. NR5A2 and NR0B2 are described in greater detail in the NR5A and NR0B sections below. Through a well-established cascade of nuclear receptor action, NR1H4 is unquestionably an important regulator of testosterone production in the adult testis.

Testicular Orphan Receptor (NR2C1)

The two NR2C family members, NR2C1 (TR2) and NR2C2 (TR4), can act as transcriptional activators or repressors by binding as homodimers to DR1, DR2, and DR4 elements (Fig. 2C and reviewed in Lee et al. [47]). Although they have been best studied in germ cell function [47], Leydig cells were reported to weakly express NR2C1 [48]. The exact role of NR2C1 in Leydig cells remains to be determined, although Nr2c1-deficient mice (Nr2c1tm1Chc/Nr2c1tm1Chc) display normal testis development and are fertile [49].

The Chicken Ovalbumin Upstream Promoter Transcription Factor (NR2F2)

The chicken ovalbumin upstream promoter-transcription factor (COUP-TF) family is composed of two members: NR2F1 (COUP-TFI) and NR2F2 (COUP-TFII). These orphan nuclear receptors are expressed in a wide variety of tissues, where they were shown to play important physiological roles (reviewed in Pereira et al. [50]). NR2F members can either activate or repress gene expression by binding as homodimers, preferably to an imperfect direct repeat separated by one nucleotide (GTGTCAaAGGTCA, or DR1). However, they can also bind to elements containing a variable number of nucleotides between the direct repeats (DR1 to DR6; Fig. 2C) [50]. Although NR2F factors preferentially form homodimers [51], they can also heterodimerize with NR2B (RXR) members (Fig. 2B) [52]. Of the two NR2F members, only NR2F2 is expressed in Leydig cells throughout development (Qin et al. [53] and Brousseau and Tremblay, unpublished data). Because Nr2f2−/− mice (Nr2f2tm1Tsa/Nr2f2tm1Tsa) die before Embryonic Day 10.5 due to angiogenesis and cardiovascular defects [54], an inducible inactivation strategy was used to investigate the function of this nuclear receptor in male reproductive function [53]. Prepubertal NR2F2 ablation causes male infertility, hypogonadism, and spermatogenic arrest due to defective testosterone biosynthesis [53]. NR2F2 was found to be essential for progenitor Leydig cell formation and the cells' further maturation into functional Leydig cells [53]. Interestingly, NR2F2 is not required for the maintenance of Leydig cell-differentiated function in adults because male reproduction and Leydig cell function were normal when NR2F2 was deleted in mature Leydig cells [53]. Therefore, NR2F2 is indispensable for the differentiation of both Leydig cell populations but not for the maintenance of their differentiated function. The morphogen Sonic hedgehog (SHH) was found to regulate Nr2f2 expression in neurons [55, 56]. Interestingly, another hedgehog family member, Desert hedgehog (DHH), is required for Leydig cell differentiation [57, 58]. Because hedgehog family members act through common receptors and signaling pathways [59], it is tempting to speculate that DHH might mediate its effects on Leydig cell differentiation by upregulating Nr2f2 expression. Despite its importance for Leydig cell differentiation and male reproductive function, no target genes have yet been identified for NR2F2 in Leydig cells.

Estrogen Receptor (NR3A1)

Despite species differences and some conflicting data, the consensus is that both estrogen receptor NR3A1 (ERα) and NR3A2 (ERβ) are present in Leydig cells [60, 61] (reviewed in O'Donnell et al. [62]). In Nr3a2−/− mice (βERKO, Esr2tm1Unc/Esr2tm1Unc), Leydig cell number per adult testis is increased, plasma and testicular testosterone levels are unchanged, and Leydig cell volume is decreased [63]. This suggests that testosterone production by each individual Leydig cell is decreased in Nr3a2−/− mice [63]. In Nr3a1−/− mice (αERKO, Esr1tm1Ksk/Esr1tm1Ksk), Leydig cell number is unchanged, plasma and testicular testosterone levels are increased, and Leydig cell volume is increased [63]. Consistent with this, Leydig cells from Nr3a1−/− (αERKO) mice have an increased steroidogenic capacity (increased testosterone production and steroidogenic gene expression, including Star, Cyp11a1, and Cyp17a1) compared with wild-type Leydig cells [6466]. Estrogen receptors are therefore important regulators of both Leydig cell steroidogenesis (NR3A1 and NR3A2) and proliferation (NR3A2).

NR3A members bind to DNA as homodimers or heterodimers to a classical estrogen response element (ERE) composed of two inverted repeats separated by three nucleotides (IR3) (Fig. 2A and reviewed in Gruber et al. [67]). No direct target genes have been identified for NR3A members in Leydig cells.

Leydig cells are particularly sensitive to estrogenic compounds (reviewed in Delbes et al. [68]). In utero exposure to estradiol (E2) and diethylstilbestrol (DES) reduces testosterone production and expression of several steroidogenic enzyme-encoding genes, including Star, Cyp11a1, and Cyp17a1. Expression of Insl3 is also dramatically affected (reviewed in Ivell and Hartung [69]). In agreement with a direct effect on Leydig cells, treatment of mouse MA-10 Leydig cells with E2 was found to repress insulin-like 3 (INSL3) transcription [70] and Star expression [71, 72]. Interestingly, all of these genes are regulated by the nuclear receptors NR5A1 and NR5A2 (see the section on NR5A). Expression of Nr5a2 is decreased after exposure to DES [72], whereas for Nr5a1, the general consensus is that its expression is not affected by exposure to estrogens [7275], although some have reported a decrease in Nr5a1 expression at higher doses [7678]. The deleterious effects of E2 and DES on FLC differentiation and function are mediated through the nuclear receptor NR3A1, because Nr3a1-deficient male mice are insensitive to these compounds [65, 74, 79]. None of the gene promoters affected by estrogens, however, contains an ERE, which indicates that the effect of estrogens on these genes is likely to be indirect through yet unknown mechanisms. Recent developments, however, indicate that the nuclear receptor NR0B2, a transcriptional repressor, is involved in estrogen-mediated male reproductive dysfunctions [72]. Nr0b2 expression is upregulated by estrogens in an NR3A1-dependent manner (see section on NR0B below) [80].

Glucocorticoid Receptor (NR3C1)

It is well recognized that elevated glucocorticoid levels resulting from diverse stressful conditions, whether physical or psychological, lead to suppression of serum testosterone levels [81]. Conversely, reduction of endogenous corticosterone levels (the main glucocorticoid in rodents) leads to increased testosterone production by Leydig cells, thus further supporting the repressive role of glucocorticoids on testosterone production [82]. Glucocorticoid levels in Leydig cells must therefore be tightly regulated. This is achieved by the metabolizing enzyme 11β hydroxysteroid dehydrogenase type 1 (HSD11B1), which has oxidase and reductase properties. In Leydig cells, HSD11B1 catalyses the oxidative inactivation of glucocorticoids (reviewed in Hu et al. [83]). When glucocorticoid accumulation overcomes degradation, testicular steroidogenesis is repressed. Glucocorticoids repress expression of several steroidogenic enzyme-encoding genes, including Star, Cyp11a1, Cyp17a1, Hsd3b1, and Hsd17b3 [8489]. In addition to repressing gene expression, glucocorticoids also induce Leydig cell apoptosis [90].

Glucocorticoids mediate their effects through the glucocorticoid receptor (GR, NR3C1), which is expressed in Leydig cells [84, 91, 92]. Two NR3C1 protein isoforms are produced in vivo by alternative translation initiation, with the second initiating at an ATG codon corresponding to methionine 27 [93]. Functional studies revealed that the shorter NR3C1 form is nearly twice as effective as the longer form in gene activation, but not in repression [93]. Once activated by glucocorticoids, NR3C1 binds as a homodimer to an IR3 DNA sequence AGAACAnnnTGTTCT (Fig. 2A) called the glucocorticoid response element (GRE) found in the promoter region of target genes, resulting in either transcriptional activation or repression (reviewed in Funder [94]). The promoters of steroidogenic genes repressed by glucocorticoids, however, do not contain GRE. Consistent with this, we recently reported that NR3C1-mediated repression of Star transcription involved an indirect mechanism. We found that glucocorticoids prevented the recruitment of the nuclear receptor NR4A1, an activator of Star transcription, to the Star promoter, leading to decreased transcription [85]. Nr3c1-deficient mice (Nr3c1tm1Gsc/Nr3c1tm1Gsc) die shortly after birth due to severe respiratory failure, thus precluding a direct assessment of the role of NR3C1 in Leydig steroidogenesis after exposure to stress [95].

Mineralocorticoid Receptor (NR3C2)

Aldosterone is known to increase testosterone production in rat primary Leydig cells [96]. This is prevented by the use of RU28318, a mineralocorticoid receptor (MR, NR3C2) antagonist, indicating that the effects of aldosterone are mediated by this receptor [96]. NR3C2 binds as a homodimer to the same DNA element as NR3C1; i.e., an IR3-type HRE (Fig. 2A and reviewed in Funder [94]). No direct target genes for NR3C2, however, have been identified in Leydig cells. The Nr3c2 gene was inactivated in mice by homologous recombination (Nr3c2tm1Gsc/Nr3c2tm1Gsc), and Nr3c2-deficient mice die around 10 days after birth due to massive renal sodium and water loss [95].

Nr3c2 expression in ALCs decreases after in utero treatment with diethylhexyl phthalate (DEHP) [97], an endocrine disruptor known to affect Leydig cells, leading to a decrease in testosterone production (reviewed in Foster [98]). Because mineralocorticoids enhance testosterone production through NR3C2, it is therefore possible that the inhibitory effect of DEHP on testosterone synthesis is caused in part by a decrease in NR3C2 levels.

Progesterone Receptor (NR3C3)

Progesterone stimulates Star transcription in MA-10 Leydig cells [84]. Progesterone acts through the progesterone receptor (NR3C3) that is present in Leydig cells [99]. NR3C3 binds to an IR3 HRE as a homodimer (Fig. 2A). There are two NR3C3 protein isoforms derived from a single gene by alternative translation initiation; the second ATG is located at residue 165 [100]. Disruption of the longer [101] (Pgrtm1Bwo/Pgrtm1Bwo) and shorter [102] (Pgrtm2Omc/Pgrtm2Omc) forms of NR3C3 in male mice, however, has no effect on fertility. This suggests that progesterone action in Leydig cells might involve a nonclassical pathway.

Androgen Receptor (NR3C4)

Testosterone is a well-established regulator of male sex differentiation and reproductive function, including spermatogenesis. The effects of testosterone are mediated by the androgen receptor (NR3C4). NR3C4 binds as a homodimer to specific DNA motifs called androgen response elements (AREs) located in the promoter of target genes. Two types of AREs have been described: classic AREs with the consensus sequence AGAACAnnnTGTTCTT (IR3; Fig. 2A) that are also recognized by the other steroid hormone receptors (NR3C1, NR3C2, and NR3C3), and selective AREs composed of a DR3 element that are specific for NR3C4 (reviewed in Verrijdt et al. [103]).

Within the testis, the NR3C4 receptor is expressed in various cell types, the best studied being Sertoli cells. NR3C4, however, is also present in peritubular myoid cells, perivascular smooth muscle cells, and Leydig cells (reviewed in Wang et al. [104]). Localization of NR3C4 in male germ cells, however, remains controversial, with several studies reporting the presence of NR3C4 in germ cells in different species and other groups reporting no NR3C4 in these cells (reviewed in Wang et al. [104]).

The first demonstration of a role for NR3C4 in Leydig cells came from natural mutation (testicular feminized mice, Tfm:ArTfm/Y) and gene targeting experiments in mice (Artm1.1Verh/Y- Tg(Pgk1-cre)1Lni). Both approaches revealed that although FLC development was normal, the ALC population failed to differentiate [105108]. Because this failure of ALC to differentiate could be caused by an indirect mechanism, a Leydig cell-specific Nr3c4 inactivation was generated using a Cre-Lox approach [109, 110]. At birth, these mice (Amhr2tm3(cre)Bhr/Amhr2+-Artm1Chc/Y) are phenotypically indistinguishable from wild-type male mice, indicating that fetal male sex differentiation occurs normally [109, 110]. As for global Nr3c4 knockout mice, Leydig cell-specific Nr3c4-deficient mice have smaller testes and epididymides, reduced circulating testosterone levels, and increased LH levels [109, 110]. These mice are also infertile, and spermatogenesis is arrested predominantly at the round spermatid stage [109, 110]. It is important to point out that the Amhr2 promoter used to drive Cre expression is also weakly active in Sertoli cells [111]. Therefore, some of the effects observed in the Amhr2tm3(cre)Bhr/Amhr2+-Artm1Chc/Y mice might be caused by decreased NR3C4 function in Sertoli cells. These results nonetheless suggest that the testosterone/NR3C4 pathway functions in an autocrine manner in Leydig cells to maintain steroidogenic function, and consequently spermatogenesis and male fertility.

Despite its importance in Leydig cells, no direct target genes have been identified for NR3C4 in Leydig cells. Androgens and NR3C4 were found to repress cAMP-induced transcription of Star and Cyp17a1 in Leydig cells [71, 112], whereas they were found to activate Insl3 transcription and promoter activity in Leydig cells [113]. In all of these cases, the mechanism is likely indirect, because the Star, Cyp17a1, and Insl3 promoters lack an ARE sequence.

Finally, the phthalic ester MEHP (the active metabolite of DEHP), a well-known endocrine disruptor with antiandrogenic properties, was found to partially antagonize the testosterone/NR3C4-activating effects on Insl3 transcription in Leydig cells [113].

The NR4A Family

NR4A1, also known as NUR77 or NGFI-B, is the prototypic member of the NR4A family of nuclear receptors, which also includes NR4A2 (NURR1) and NR4A3 (NOR1). The genes encoding these transcription factors are classified as immediate early-response genes because their expression is rapidly induced in several tissues by a variety of physiological stimuli, including fatty acids, prostaglandins, growth factors, calcium, cytokines, peptide hormones, phorbol esters, and neurotransmitters (reviewed in Eells et al. [114]). NR4A members are well-known transcriptional activators; they mediate their effects by binding to regulatory elements in promoter regions. NR4A family members were first reported to bind to DNA as monomers (Fig. 2D) to the sequence AAAAGGTCA, called the NGFI-B response element (NBRE), which is very similar to the NR5A1 element [115]. The three NR4A factors can also bind to DNA as homodimers or heterodimers to an imperfect ER10 element (Fig. 2C) called the NurRE [116, 117]. Finally, NR4A1 and NR4A2, but not NR4A3, can heterodimerize with the retinoid X receptor (NR2B), become responsive to retinoids, and regulate gene transcription by binding to a DR5 element (Fig. 2B) [118, 119]. The three different DNA-binding activities (monomer, homodimer, heterodimer) of NR4A family members allow for a unique versatility in mediating the transcriptional activation of different sets of genes in response to various stimuli.

In recent years, NR4A family members have received increased consideration as novel regulators of basal and hormone-induced gene transcription in Leydig cells. These cells express two NR4A family members, NR4A1 and NR4A2, although NR4A1 is considerably more abundant [120]. During mouse testis development, the increased LH secretion at the pubertal stage induces Nr4a1 expression in Leydig cells [120]. In adults, Nr4a1 expression is low in Leydig cells and becomes rapidly induced in response to LH/cAMP [120123]. Therefore, NR4A1 expression during Leydig cell development and in response to hormonal stimulation strikingly parallels the process of steroidogenesis, which requires increased expression of the various steroidogenic enzyme-encoding genes. Two distinct groups of transcription factors are involved in steroidogenic gene activation: those already present in the cell that are activated by posttranslational modifications, and those that are rapidly induced [124]. Several transcription factors belonging to the first group have been identified, whereas NR4A1 was the first member of the second group. In Leydig cells, NR4A1 activates numerous promoters, including HSD3B2 [121], Hsd3b1 [125], Cyp17a1 [125, 126], Star [124, 125, 127], INSL3 [128], and Giot1 [129]. GIOT1 is a corepressor that interacts with and represses Nr5a1-mediated activation of the Cyp17a1 promoter [129]. In addition, cAMP-induced NR4A1 expression precedes that of Star, further supporting a role for this nuclear receptor in hormone-induced steroidogenesis [124, 130].

NR4A1 also regulates steroidogenic gene expression by cooperating with other transcription factors. For instance, NR4A1 cooperates with the AP-1 factor cJUN to enhance Star transcription [130] and with the coactivator NCOA1 (SRC1) on the HSD3B2 and Cyp17a1 promoters [121, 125]. Other transcription factors are known to inhibit NR4A1 transactivation properties in Leydig cells, including the NR0B1 (DAX1) [131] and NR0B2 (SHP; Martin and Tremblay, unpublished data) nuclear receptors. NR0B1 represses NR4A1 transactivation by interacting with its AF-2 domain, thus competing for NCOA1 recruitment. Therefore, NR0B-dependent repression of steroidogenesis in Leydig cells involves, at least in part, inhibition of NR4A1 transactivation properties [131133]. Another repressor of NR4A1 activity is the NR3C1 receptor. As mentioned previously, stress-mediated repression of steroidogenesis involves the NR3C1 receptor, which is believed to interact with NR4A1 into a transcriptionally inactive complex [85]. This is supported by the fact that NR3C1 agonists significantly decrease NR4A1 association with the mouse Star promoter in a native chromatin environment in Leydig cells [85].

The lack of an overt reproductive phenotype in Nr4a1tm1Jmi/Nr4a1tm1Jmi mice, in which the Nr4a1 gene has been inactivated by homologous recombination [134, 135], would appear to argue against a role for NR4A1 in Leydig cells. However, no testicular histology was performed in Nr4a1-deficient mice. In fact, only the capacity to reproduce was analyzed in Nr4a1−/− mice as a measure of reproductive function [134]. The lack of gross phenotype is believed to be due to functional redundancy between NR4A family members [134, 136]. NR4A2, another member of the NR4A family, is also expressed in Leydig cells [120]. Moreover, its expression is increased significantly in adrenals of Nr4a1−/− mice [134]. No data are available regarding Nr4a2 levels in the testis of Nr4a1−/− mice. In agreement with a redundancy mechanism between NR4A1 and NR4A2, both factors were found to equally activate gene transcription in Leydig cells [121, 124, 128, 137]. The most recent piece of evidence demonstrating redundancy between NR4A family members is the first report of a double-null mouse for Nr4a1 and Nr4a3 [138]. These Nr4a1tm1Jmi/Nr4a1tm1Jmi-Nr4a3tm1Omc/Nr4a3tm1Omc (Nr4a1−/−/Nr4a3−/−) mice develop rapidly lethal acute myeloid leukemia (AML), which could not have been anticipated by the phenotype of the single-null mice. Furthermore, cells from 46 AML patients all showed downregulation of both Nr4a1 and Nr4a3, indicating that the redundancy mechanism between these two factors has been conserved throughout evolution [138]. Additional double-knockout (Nr4a1/Nr4a2 and Nr4a2/Nr4a3) and triple-knockout mice are clearly needed to fully decipher the roles of these nuclear receptors.

The NR5A Family

The NR5A family is composed of two members: steroidogenic factor 1 (NR5A1, SF1, Ad4BP) and liver receptor homolog 1 (NR5A2, LRH1). NR5A1 was originally identified as a tissue-specific transcriptional regulator of the cytochrome P450 steroid hydroxylases [139, 140]. In addition to the main steroidogenic cells (Leydig, theca, adrenal cortex), NR5A1 is also expressed in Sertoli, granulosa, and pituitary gonadotrope cells as well as in the hypothalamus (reviewed in Parker et al. [141]). The crucial role of NR5A1 in human reproductive function and steroidogenesis is underscored by mutations leading to disorders of sexual development with or without adrenal dysfunction (reviewed in Lin and Achermann [142]). The role of NR5A1 has also been studied using gene inactivation in the mouse. Three independent groups generated Nr5a1-deficient mice (Nr5a1tm1Enl/Nr5a1tm1Enl, Nr5a1tm1Jmi/Nr5a1tm1Jmi, Nr5a1tm1Klp/Nr5a1tm1Klp). In agreement with a role for NR5A1 in adrenal and testicular steroidogenesis, Nr5a1-deficient mice die shortly after birth from adrenocortical insufficiency, and males are pseudohermaphrodites (reviewed in Parker et al. [141]). In fact, in the absence of NR5A1, the adrenal glands and the gonads are missing. The onset of adrenal and gonadal development occurs normally in Nr5a1-deficient mice but fails to progress, causing their regression. Because gonadal regression occurs before male sex differentiation, the internal and external sex organs of Nr5a1-deficient mice are female (reviewed in Parker et al. [141]). Nr5a1-deficient mice also exhibit defects in gonadotrope cell gene expression and function, and the ventromedial hypothalamic nucleus is absent (reviewed in Parker et al. [141]). To better define the role of NR5A1 specifically in Leydig cells, a conditional inactivation approach in mice was used. These Leydig cell-specific, Nr5a1-deficient mice (Amhr2tm3(cre)Bhr/Amhr2+-Nr5a1tm1Klp/Nr5a1tm2Klp) have female external genitalia, are sterile, and exhibit no postnatal sexual maturation [143]. The testes are hypoplastic and remain at the level of the bladder within the abdominal cavity. In addition, the Wolffian duct derivatives are incompletely differentiated. Together, these data indicate that Leydig cell differentiation and/or function is impaired in the absence of NR5A1. Leydig cell precursors are, however, detected in the interstitial region of the testis of Leydig cell-specific, Nr5a1-deficient mice, indicating that Leydig cell specification occurs normally but fails to complete the differentiation process. Consequently, several steroidogenic enzyme-encoding genes, including Star and Cyp11a1, that are known to be regulated by NR5A1 are not expressed [143].

The NR5A1 nuclear receptor mediates its effects by binding to DNA as a monomer and recognizes variations of the DNA sequence (T/C)CAAGGTCA found in the promoter of target genes (Fig. 2D). NR5A1 regulates expression of numerous Leydig cell genes. These include Star, Cyp11a1, Cyp17a1, and Hsd3b1 (HSD3B2 in humans; reviewed in Parker et al. [141] and Val et al. [144]). The activity of NR5A1 is negatively regulated by the nuclear receptor NR0B1 (DAX1), which recruits the corepressor NCOR1 leading to decreased expression of NR5A1-regulated genes [132, 145]. NR5A1 is an essential regulator of Leydig cell gene expression and function, and additional information on this nuclear receptor can be found in excellent review articles [141, 142, 144, 146].

The second member of this family, NR5A2, was initially identified in the liver. It has since been identified in various tissues and cell types, including Leydig cells (reviewed in Fayard et al. [147]). NR5A2 is more abundant than NR5A1 in immature and mature rat Leydig cells [148, 149]. NR5A2 binds to the same DNA motif as NR5A1 and activates the promoters of several steroidogenic enzyme-encoding genes, including Cyp17a1 [149], Cyp11a1 [150], Star [151], HSD3B2 [152], and Cyp19a1 [148]. Despite this, the NR5A1 and NR5A2 nuclear receptors have nonredundant roles, because NR5A2 cannot rescue the phenotype of the Leydig cell-specific, Nr5a1-deficient mice. Nr5a2-deficient mice (Nr5a2tm1Auw/Nr5a2tm1Auw and Nr5a2tm1Bel/Nr5a2tm1Bel) were generated by two independent groups [153, 154]. These mice die early during embryonic development (Embryonic Days 6.5–7.5) because of visceral endoderm defects.

Interestingly, heterozygous Nr5a2+/− mice have lower plasma testosterone concentration and decreased testicular expression of several steroidogenic genes, including Star, Cyp11a1, and Hsd3b1 [46]. The weight of androgen-dependent organs (epidydimis and seminal vesicles) is lower in Nr5a2+/− [46]. Although these data firmly support a role for NR5A2 in Leydig cell steroidogenesis, a conditional inactivation approach in Leydig cells is warranted to better define the role of this nuclear receptor in these cells.

The Atypical Nuclear Receptors NR0B1 and NR0B2

The NR0B family is composed of two members: dosage-sensitive sex reversal, adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1 (DAX1, NR0B1); and small heterodimer partner (SHP, NR0B2). These two nuclear receptors have an atypical structure; they lack the canonical AF-1 activation domain, DBD, and hinge region (Fig. 1). In NR0B1, these domains are replaced by an N-terminal domain (NTD) consisting of 3.5 repeats of a 65- to 70-amino acid motif rich in alanine and glycine (Fig. 1). NR0B2 contains a short 70-residue NTD (Fig. 1) that is homologous to the NR0B1 NTD (reviewed in Iyer and McCabe [155] and Chanda et al. [156]). Both NR0B nuclear receptors are expressed in Leydig cells, albeit at different developmental stages. NR0B1 is mainly detected during fetal development, whereas NR0B2 is present in postnatal life [46, 157]. As mentioned previously, NR0B1 and NR0B2 are transcriptional repressors. Because they lack a classical DBD, they mediate their repressive effects by interacting with and inhibiting the activity of various nuclear receptors, including NR4A1, NR5A1, and NR5A2 (reviewed in Iyer and McCabe [155] and Bavner et al. [158]). In addition, NR0B1 represses Nr4a1 and Nr5a1 expression in Leydig cells [159]. Consequently, by repressing the activity and expression of these activators of steroidogenic gene expression in Leydig cells, NR0B1 and NR0B2 are active repressors of steroidogenesis. NR0B family members can also form homodimers (NR0B1/NR0B1 and NR0B2/NR0B2) and heterodimers (NR0B1/NR0B2) in mammalian cells [160], but the physiological significance of these dimers remains to be established. It nonetheless suggests novel function(s) for NR0B members that would be independent of other nuclear receptors.

In humans, inactivating mutations in NR0B1 cause AHC and hypogonadotropic hypogonadism, whereas duplication of the NR0B1 gene leads to male-to-female sex reversal in XY individuals [155]. In animal models, duplication of the Nr0b1 gene (Tg(Nr0b1)1812Rlb) causes male-to-female sex reversal on a specific mouse genetic background [161]. Nr0b1-deficient male mice (Nr0b1tm1.1Lja/Nr0b1tm1.1Lja) are infertile and have small testes due to a progressive degeneration of the testicular germinal epithelium [162]. These mice have normal levels of testosterone and gonadotropins and, from a histological perspective, appear to exhibit Leydig cell hyperplasia and hypertrophy [162]. Furthermore, expression of several steroidogenic enzymes is unaltered in Nr0b1−/− mice, including Star, Cyp11a1, Cyp17a1, Hsd3b1, and Hsd17b3. Only expression of Cyp19a1 is increased in Nr0b1-deficient Leydig cells [163]. Consistent with this, intratesticular estradiol levels are 40-fold greater in Nr0b1-deficient male mice compared with wild-type mice. Treatment of Nr0b1-deficient male mice with tamoxifen, an estrogen receptor antagonist, restores fertility and reduces the number of Leydig cells [163]. These studies indicate that in ALCs, the main role of NR0B1 is to ensure proper regulation of Cyp19a1 expression.

NR0B2 is mainly known for its role in the liver, where it is involved in the feedback inhibition of bile acid synthesis [158]. NR0B2 is also expressed in the testis, where it is first detected around Postnatal Day 10 in mice [46, 72]. This correlates with the process of Leydig cell maturation, thus supporting a role for this factor in Leydig cell function. This was indeed confirmed by gene inactivation experiments in the mouse. Three independent groups have generated Nr0b2-deficient mice (Nr0b2tm1.1Auw/Nr0b2tm1.1Auw, Nr0b2tm1Ddm/Nr0b2tm1Ddm, and Nr0b2tm1Rus/Nr0b2tm1Rus) [46, 164, 165]. Nr0b2−/− mice are viable and fertile. In fact, these mice have premature sexual maturation. In the absence of NR0B2, male mice can successfully impregnate females 8 days earlier than wild-type males [46]. This can be explained by the increased concentration of plasma and intratesticular testosterone in Nr0b2-deficient mice [46]. Expression of several steroidogenic enzyme-encoding genes, including Star, Cyp11a1, and Hsd3b1, is also increased in the absence of NR0B2 [46]. Consistent with this, the nuclear receptors NR5A1 and NR5A2, which are known activators of steroidogenic gene expression (see above), are expressed at higher levels in Nr0b2-deficient mice [46]. NR0B2 also represses expression of steroidogenic genes by inhibiting the transactivation properties of NR5A2 [46]. Together, these data establish a repressive role for NR0B2 in steroidogenic gene expression and androgen production in the postnatal testis, because Leydig cell differentiation and function as well as male sexual maturation are accelerated in Nr0b2-deficient mice.

As mentioned previously, Leydig cells are negatively affected by exposure to various estrogenic compounds, and this involves the estrogen receptor α NR3A1 [65, 74, 79]. Because all of the genes repressed by estrogen/NR3A1 in Leydig cells do not contain an ERE, estrogens are believed to act through an indirect mechanism that remains to be fully elucidated. The recent finding that NR0B2 plays a role in estrogen-mediated male reproductive dysfunctions constitutes an important contribution to our understanding of the molecular mechanisms of estrogen action in Leydig cells [72]. As for Nr3a1-deficient mice, Nr0b2−/− mice are resistant to the deleterious effects induced by various estrogens (pure estrogen agonist estradiol benzoate [EB] and DES) in Leydig cells. Indeed, in the absence of NR0B2, DES treatment did not decrease testosterone production, nor did it repress expression of Star, Cyp11a1, Cyp17a1, and Insl3 [72]. Interestingly, Nr0b2 expression is upregulated by estrogens through the NR3A1 estrogen receptor in the liver [80]. Similarly, Nr0b2 expression is upregulated by estrogens (EB and DES) in neonatal mouse testis and in MA-10 Leydig cells, which is followed by a decrease in steroidogenic gene expression [72]. It is therefore likely that estrogens mediate their effects in Leydig cells by upregulating expression of NR0B2, which in turn directly represses gene expression in these cells by inhibiting the activity of the nuclear receptor NR5A2 and the expression of Nr5a1 and Nr5a2, two important regulators of Leydig cell function.

Conclusions and Perspectives

As described in the present study, there has been tremendous progress in the recent years in the field of nuclear receptors in Leydig cells. However, the proposed physiological role of several nuclear receptors currently relies on promoter activation studies in cell culture and other in vitro approaches that will need to be validated using in vivo investigations. Nonetheless, these initial studies have demonstrated how important nuclear receptors are in Leydig cell development and function. In addition, experiments carried out in mammalian cell lines are essential for a fine dissection of the mechanisms of nuclear receptor action and of the gene regulatory sequences they target. We are just beginning to understand the intricate network of nuclear receptor action in Leydig cells, with some nuclear receptors regulating the expression and/or activity of others. A key challenge in deciphering nuclear receptor function in Leydig cells will be in defining the mechanisms by which physical and functional interactions between them either translate into the appropriate physiological response or hamper normal Leydig cell differentiation and function.

The genes encoding several nuclear receptors have been inactivated in mice, which has provided key information as to the role of certain nuclear receptors in Leydig cell development and/or function. For other nuclear receptors, however, global gene inactivation has failed to provide relevant physiological information due to embryonic lethality or redundancy by other family members. Alternative methodologies must therefore be used. These include gene inactivation of several family members, overexpression of dominant negative forms of nuclear receptors, and Leydig cell-specific gene inactivation using a conditional gene targeting approach. Gene inactivation at various developmental stages during fetal or adult Leydig cell differentiation and in adult animals is essential to completely understand the role of the various nuclear receptors. This requires the development of novel tools and mouse lines that express a Cre recombinase specifically in Leydig cells at the desired developmental stage. These temporally regulated knockouts will constitute unique models to examine the roles of nuclear receptors in Leydig differentiation and in Leydig cell-differentiated function. In addition, the use of nuclear receptor ligands, whether agonists or antagonists, might prove to be useful pharmacological treatments/therapies in improving male reproductive health or acting as new contraceptive methods.

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World Wide Web (URL: http://www.informatics.jax.org/mgihome/nomen/gene.shtml). (January 15, 2010)
.

Author notes

1Supported by Natural Sciences and Engineering Research Council of Canada grant 262224 and Canadian Institutes of Health Research grants MOP-81387 and MOP-93582 to J.J.T. J.J.T. is a holder of a Fonds de la Recherche en Santé du Québec Chercheur-boursier Scholarship.
3
Current address: Département de Biologie, Université de Moncton, Pavillon Rémi-Rossignol, Moncton, NB E1A 3E9, Canada. e-mail: Luc.Martin@umoncton.ca