Aromatase expression in the ovary: hormonal and molecular regulation

Abstract

Estrogens are synthesized by the aromatase enzyme encoded by the Cyp19a1 gene, which contains an unusually large regulatory region. In most mammals, aromatase expression is under the control of two distinct promoters a gonad- and a brain-specific promoter. In humans, this gene contains 10 tissue-specific promoters that are alternatively used in various cell types and tumors. Each promoter is regulated by a distinct set of regulatory sequences and transcription factors that bind to these specific sequences. The cAMP/PKA/CREB pathway is considered to be the primary signaling cascade through which the gonad Cyp19 promoter is regulated. Very interestingly, in rat luteal cells, the proximal promoter is not controlled in a cAMP dependent manner. Strikingly, these cells express aromatase at high levels similar to those found in preovulatory follicles, suggesting that alternative and powerful mechanisms control aromatase expression in luteal cells and that the rat corpus luteum represents an important paradigm for understanding alternative controls of the aromatase gene. Here, the molecular and cellular mechanisms controlling the expression of the aromatase gene in granulosa and luteal cells are discussed.

Fig. 1. Aromatase expression in the ovary of immature and adult rats

A and B, in situ hybridization analysis of ovaries at 15 days postnatal (dpn) and first proestrus: At 15 dpn, aromatase is expressed in primary, preantral, and antral growing follicles. At first proestrus, aromatase is observed only in preovulatory follicles (PoF). Immunohistochemistry analysis of the ovaries of adult animals (C and D) demonstrated that levels of expression of aromatase are not uniform throughout the antral follicles, being highest in the mural granulosa cells at the periphery of the follicle (arrows) and absent from the granulosa cells surrounding the antrum and within the cumulus (arrowheads). The intensity of immunostaining increases in the periovulatory follicle (D, proestrus) compared with less mature antral follicles (C, diestrus).O: oocyte. Panels A and B were adapted from Turner KJ, et al. (2002) Development and validation of a new monoclonal antibody to mammalian aromatase. Journal of Endocrinology;172:21−30. ^©Society for Endocrinology (2002). Panels C and D were adapted from Guigon CJ, et al. (2003) Unaltered development of the initial follicular waves and normal pubertal onset in female rats after neonatal deletion of the follicular reserve; Endocrinology;144:3651−62. Reproduced by permission.

Fig. 2. Hormone interactions in the regulation of aromatase expression in follicles

FSH activates the cAMP/PKA and PI3K/PKB signaling pathways, which are known to mediate its stimulatory effect on aromatase expression. Androgens, IGF-1, and estradiol potentiate this effect of FSH. Androgens have direct effects probably mediated by the activation of androgen receptors and indirect effects throughout its conversion to estradiol. IGF-1 stimulates the expression of the FSH receptor and probably synergizes with FSH in the activation of PKB. Estradiol also enhances the stimulatory effect of FSH. The positive feed-back of estradiol may be mediated by a cooperative effect on the activation of the PI3K/PKB and cAMP/PKA pathways. On the other hand, the oocyte (O) produces factors (GDF-9 and BMP-15) that block the stimulatory effect of FSH in cumulus cells and in antral granulosa cells in contact with the follicular fluid, limiting the expression of aromatase to mural granulosa cells. BM: basal membranes.

Fig. 3. Hormonal regulation of luteal aromatase during pregnancy

Bars represent aromatase mRNA levels in corpora lutea of rats on different days of pregnancy. Aromatase and L19 mRNA levels were determined by real-time PCR and the results expressed as aromatase mRNA molecules per μg of total RNA was determined by using a standard curve generated from known quantities of aromatase cDNA. Values represent average ± SEM. Luteal aromatase seems to be maintained by prolactin (PRL) at a low level during the first part of pregnancy, is modulated by LH at midgestation, and becomes highly expressed by the stimulatory action of placental lactogens (PL) and testosterone in coordination with the stimulatory effect of ovarian estradiol (E2). Prostaglandin F2α (PGF2α) is involved in the rapid downregulation of aromatase toward the end of pregnancy.

Fig. 4. Alignment of the nucleic acid sequences of the proximal promoter region of the human (h), rat (r), and mouse (m) aromatase genes

Bolded sequences indicate identical nucleotides among species. Solid lines indicate transcription factors' binding sequences. TATA binding sites are indicated by boxes.

Fig. 5. Dynamics of the activation of the aromatase promoter in ovarian cells

Aromatase mRNA levels and DNA protein binding in 26-day-old immature rats (d26), immature rats treated with PMSG (PMSG), and from corpora lutea of rats on days 4 (d4), 15 (d15), or 23 (d23) of pregnancy. Aromatase mRNA levels were determined as in figure 3 and expressed as the ratio between aromatase and ribosomal L19 mRNA. DNA protein binding was investigated using gel shift analyses with oligonucleotides spanning the cAMP response element-like sequence (CLS), the nuclear receptor elements a and b (NREa and NREb), the GATA binding sites, or the AP-3 binding site. Only shifted bands are shown.

Fig. 6. Scheme depicting hypothetical multiunit complexes on the aromatase promoter in granulosa and luteal cells

The participation and binding of CREB, GATA-4, β-catenin and AP-3 have been shown using in vitro approaches such as gel shift and gene reporter assays. The involvement of SF-1 has been inferred from knockout and over-expression experiments. Interactions between CREB, SF-1 and GATA-4 with CBP are deduced from experiments in cells other than granulosa or luteal cells. The spatial binding of transcription factors on the DNA does not necessarily represent an in vivo situation but highlights the complexity of this promoter and the differences between granulosa and luteal cells on the activation of the aromatase gene. GTM= general transcriptional machinery; TBP: TATA binding protein; RNApol: RNA polymerase. See the text for more details.