AMH Regulation by Steroids in the Mammalian Testis: Underlying Mechanisms and Clinical Implications

Abstract

Anti-Müllerian hormone (AMH) is a distinctive biomarker of the immature Sertoli cell. AMH expression, triggered by specific transcription factors upon fetal Sertoli cells differentiation independently of gonadotropins or sex steroids, drives Müllerian duct regression in the male, preventing the development of the uterus and Fallopian tubes. AMH continues to be highly expressed by Sertoli until the onset of puberty, when it is downregulated to low adult levels. FSH increases testicular AMH output by promoting immature Sertoli cell proliferation and individual cell expression. AMH secretion also showcases a differential regulation exerted by intratesticular levels of androgens and estrogens. In the fetus and the newborn, Sertoli cells do not express the androgen receptor, and the high androgen concentrations do not affect AMH expression. Conversely, estrogens can stimulate AMH production because estrogen receptors are present in Sertoli cells and aromatase is stimulated by FSH. During childhood, sex steroids levels are very low and do not play a physiological role on AMH production. However, hyperestrogenic states upregulate AMH expression. During puberty, testosterone inhibition of AMH expression overrides stimulation by estrogens and FSH. The direct effects of sex steroids on AMH transcription are mediated by androgen receptor and estrogen receptor α action on AMH promoter sequences. A modest estrogen action is also mediated by the membrane G-coupled estrogen receptor GPER. The understanding of these complex regulatory mechanisms helps in the interpretation of serum AMH levels found in physiological or pathological conditions, which underscores the importance of serum AMH as a biomarker of intratesticular steroid concentrations.

Keywords: androgen insensitivity; androgen response element; dihydrotestosterone; estradiol; estrogen response element; ketoconazole; testotoxicosis; triptorelin.

Figure 1

Onset and upregulation of AMH expression. Transcription factors SOX9, SF1, GATA4 and WT1 bind to specific response elements in the proximal AMH promoter and trigger AMH expression in early fetal life, independently of gonadotropin or steroid action, resulting in Müllerian duct regression. From the second trimester of gestation, FSH increases testicular AMH production by 2 different mechanisms: it promotes cell proliferation and upregulates AMH transcription via the transduction pathway involving the heterotrimeric (α/β/γ) G protein, adenylyl cyclase (AC), cyclic AMP and protein kinase A (PKA) resulting in increased expression and nuclear translocation of transcription factors SOX9, SF1, AP2 and NFκB. Modified with permission from Grinspon and Rey (14), ^© 2010 S. Karger AG.

Figure 2

Relationship between AMH production, androgen receptor (AR) expression and androgen levels in the human testis. During the postnatal period of activation of the hypothalamic-pituitary-testicular axis (0-6 months, first column, A–C), testosterone (T) levels are high (bottom graph, serum hormone levels); however, Sertoli cells do not show maturational changes because they do not express the AR (upper graph, second line, immunohistochemistry). Therefore, AMH expression is high (upper graph, third line, AMH immunohistochemistry) and germ cells do not enter meiosis (upper graph, first line, HE: hematoxylin-eosin staining). During the “quiescent” period of the gonadal axis (childhood, second column, D–F), androgen synthesis is negligible, which explains why Sertoli cells remain immature even though they have started to express the AR. In puberty and adulthood (third column, G–I), T increases, inducing Sertoli cell maturation, reflected in the decline of AMH expression and the onset of adult spermatogenesis. Bottom graph shows % AR+: percentage of Sertoli cells expressing the AR. AMH (pmol/l) and T (ng/dl): schematic representation of AMH and T serum levels from birth to adulthood in males.

Figure 3

Classical (genomic) and non-classical (non-genomic) androgen signaling in Sertoli cells. In cells not exposed to androgens, the androgen receptor (AR) is bound to heat-shock proteins (HSP) in the cytoplasm. Testosterone and other androgens are steroids that easily cross the cell membrane and bind to the AR, that is released from HSP. The ligand-bound AR can either translocate to the nucleus and interact with androgen response elements (ARE) or with other transcription factors (TF), thus activating the classical/genomic pathway, or migrate to the inner side of the cell membrane and interact with Src, thus activating the non-classical/non-genomic pathway. CREB, cAMP response element binding protein; EGFR, Epidermal growth factor receptor; ERK, Extracellular signal-regulated kinase; MEK, Mitogen-activated protein kinase; Src, Steroid receptor coactivator. Reproduced with permission from Edelsztein and Rey (18) ^© 2019 The Authors.

Figure 4

Molecular mechanisms involved in androgen-induced downregulation of AMH expression in Sertoli cells. A and B: luciferase assays in SMAT1 Sertoli cells transfected with a luciferase plasmid under control of the human AMH promoter (pGL2B-hAMH) of different lengths and co-transfected with an expression plasmid of the androgen receptor (pSG5-AR) or pSG5 devoid of the AR. Cells were exposed to testosterone or DHT 10^-7 M, or vehicle, and results were expressed as relative luciferase units (RLU) comparing cells exposed to androgens and those exposed to vehicle (* p<0.05, ** p<0.01, *** p<0.001). A 100% level indicates the basal AMH promoter activity. Testosterone and DHT induce an inhibition of AMH promoter activity when the AR is present but not in its absence, when 433 to 3076 bp of the promoter are present but not when the proximal 1916-bp sequences are lacking (A). The inhibition induced by androgen persists even if the binding sites for AP1 or GATA4 are mutated, but not when SF1 sites are mutated (B). (C, D) schematic of androgen regulation of AMH expression. In fetal and postnatal periods before the onset of puberty, the lack of androgen action allows the AMH promoter activity induced by SOX9, SF1 and GATA4, resulting in high AMH production. During puberty and adulthood, the steroid-bound AR represses AMH promoter activity by competition or by interaction with SF1. Modified with permission from Edelsztein et al. (80) ^© 2018 The Authors and Edelsztein and Rey (18) ^© 2019 The Authors.

Figure 5

Serum AMH as a biomarker of intratesticular androgen concentration in boys with central precocious puberty (A) or with gonadotropin-independent precocious puberty (B, C). Serum AMH is low at diagnosis, indicating the inhibitory effect of high androgen concentration reflected in high serum testosterone levels in all cases. When testosterone production is effectively curtailed by the adequate treatment, such as a GnRH analogue in the patient with central precocious puberty (A) or ketoconazole in the patient with gonadotropin-independent precocious puberty (B), Sertoli cells recover their immature status and increase AMH production until treatment is discontinued. Conversely, when adherence to treatment is erratic (C), intratesticular and serum testosterone concentration does not remain at prepubertal levels and Sertoli cells do not revert their pubertal status, which is reflected in low serum AMH. Modified with permission from Rey et al. (48) ^© 1993 The Endocrine Society.

Figure 6

Molecular mechanisms involved in estrogen-induced upregulation of AMH expression in Sertoli cells. (A–D) Luciferase assays in SMAT1 Sertoli cells transfected with a luciferase plasmid under control of the human AMH promoter (pGL2B-hAMH) of different lengths and co-transfected with an expression plasmid of the estrogen receptor alfa (ERα) or the G protein-coupled estrogen receptor (GPER) or the control plasmids devoid of the estrogen receptor sequences. Cells were exposed to estradiol (E2), the ERα/β antagonist ICI 182780, the ERα-specific agonist PPT or antagonist MPP, or the GPER-specific agonist G1 or antagonist G15. Results were expressed as relative luciferase units (RLU) comparing cells exposed to estrogens, agonists and/or antagonists (* p<0.05, *** p<0.001). A 100% level indicates the basal AMH promoter activity. E2 induce an upregulation of AMH promoter activity when the ERα or the GPER is present but not in their absence (A–C), and when the estrogen response element (ERE) at -1782 of the promoter is conserved (D). (E) Proposed model for E2 regulation of AMH expression in Sertoli cells. E2 upregulates AMH transcription via ERα binding to the ERE. GPER also upregulates AMH expression more modestly. The increased AMH expression results in a higher testicular AMH production. Another potential mechanism is the increase in Sertoli cell proliferation induced by membrane-bound ERα signaling through the PI3K/Akt pathway, and/or GPER via MAPK signaling. Reproduced with permission from Valeri et al. (19)^© 2020 The Authors.

Figure 7

Interaction between androgens, estrogens and FSH on the regulation of AMH expression in Sertoli cells. During childhood, the hypothalamic-pituitary-gonadal axis is quiescent, and the extremely low steroid levels do not exert any physiological regulation on AMH production that is mostly hormone-independent. At puberty, the reactivation of the GnRH neuron and the gonadotropes result in higher LH and FSH levels. LH induces testosterone secretion by testicular Leydig cells. FSH acts on its receptor in the Sertoli cell membrane, resulting in a direct upregulation of AMH expression, through the cyclic AMP-PKA pathway involving transcription factors SOX9, SF1, AP2 and NFκB, and in an indirect upregulation of AMH by inducing aromatase expression. Aromatase converts androgens into estrogens, which can upregulate AMH directly by binding to the nuclear ERα or indirectly acting through the GPER expressed in the membrane of the endoplasmic reticulum. Nonetheless, the inhibitory effect of androgens overrides the stimulatory effect of FSH and estrogens on AMH expression, resulting in a decreased AMH secretion. In hyperestrogenic states with abrogated androgen action, such as the androgen insensitivity syndrome, the inhibitory effect of androgens does not exist, FSH and LH increase resulting in high testosterone that is converted to estradiol. Consequently, AMH production is substantially boosted.