Mechanisms in the regulation of aromatase in developing ovary and placenta

Abstract

During human gestation, the placental syncytiotrophoblast develops the capacity to synthesize large amounts of estrogen from C(19)-steroids secreted by the fetal adrenals. The conversion of C(19)-steroids to estrogens is catalyzed by aromatase P450 (P450arom), product of the CYP19 gene. The placenta-specific promoter of the hCYP19 gene lies approximately 100,000 bp upstream of the translation initiation site in exon II. In studies using transgenic mice and transfected human trophoblast cells we have defined a 246-bp region upstream of placenta-specific exon I.1 that mediates placental cell-specific expression. Using transgenic mice, we also observed that as little as 278 bp of DNA flanking the 5'-end of ovary-specific hCYP19 exon IIa was sufficient to target ovary-specific expression. This ovary-specific promoter contains response elements that bind cAMP-response element-binding protein (CREB) and the orphan nuclear receptors SF-1 and LRH-1, which are required for cAMP-mediated stimulation of CYP19 expression in granulosa and luteal cells during the estrous cycle and pregnancy. In this article, we review our studies to define genomic regions and response elements that mediate placenta-specific expression of the hCYP19 gene. The temporal and spatial expression of LRH-1 versus SF-1 in the developing gonad during mouse embryogenesis and in the postnatal ovary also will be considered.

Figure 1. Schematic representations of the hCYP19 (aromatase) gene and of hCYP19I.1:hGH/hGX fusion genes introduced into transgenic mice

Exons II-X (white boxes), which encode the aromatase protein, and their introns (black lines) comprise a region of ∼34 kb in size. The heme binding region (HBR) and two polyadenylation signals in the 3′-untranslated region (striped box) are encoded in exon X. Exons IIa, I.4 and I.1 (black boxes) encode the 5′-UTRs of the aromatase P450 mRNAs in the gonads, adipose tissue and placenta, respectively. The region containing these alternative first exons encompasses ∼100 kb. The hCYP19I.1:hGH/hGX fusion genes are comprised of hCYP19 DNA sequences encoding 501, 246, 201 and 125 bp of DNA flanking the 5′-end of exon I.1 (solid line) and the first 103 bp of exon I.1 (grey box) fused to either the wild-type (hGH) or mutated, biologically inactive form (hGX) of the human growth hormone structural gene, as reporter (white box). The arrow indicates the position of the transcription initiation site and direction of transcription for all fusion gene constructs. Endocrinology, 146, (2005) 2481-2488 [6], with permission. Copyright 2005, The Endocrine Society.

Figure 2. 246 bp of hCYP19 exon I.1 5′-flanking sequence is sufficient to mediate placenta-specific expression in transgenic mice

Aliquots of total RNA (30 μg) isolated from placentae of a E17.5 F1 transgenic mice or from various tissues of an adult F1 male or female mice carrying the hCYP19I.1_-501:hGH, hCYP19I.1_-246:hGH, hCYP19I.1_-201:hGH or hCYP19I.1_-125:hGH transgenes were analyzed by northern blotting using a ³²P-labeled hGH cDNA probe. Endocrinology, 146, (2005) 2481-2488 [6], with permission. Copyright 2005, The Endocrine Society.

Figure 3. hCYP19I.1_-246:hGX transgene is expressed as early as E8.5 in trophoblast giant cells while hCYP19I.1_-501:hGH transgene expression is evident only at E10.5 specifically in the labyrinthine trophoblast layer

Placental tissues obtained from E7.5, E8.5, E9.5 and E10.5 fetal mice carrying either the -501 bp- (left panel) or -246 bp-containing transgene (right panel) were processed for in situ hybridization using an ³⁵S-labeled antisense hGH cRNA probe and exposed to photographic emulsion for 1-2 weeks. Bright and dark field microscopy was then performed. Left panels: Dark field micrographs of placental tissue sections from E7.5, E8.5, E9.5 and E10.5 transgenic mice carrying the hCYP19I.1_-501:hGH fusion gene. Right panels: Dark field micrographs of placental tissue sections from E7.5, E8.5, E9.5 and E10.5 transgenic mice carrying the hCYP19I.1_-246:hGX fusion gene. Bright field micrograph of the haematoxylin-stained E10.5 placental tissue section from mice carrying -501 bp- or -246 bp-containing transgene are shown below their respective dark field micrographs. gc, trophoblast giant cell; sp, spongiotrophoblast; lab, labyrinthine trophoblast. Endocrinology, 146, (2005) 2481-2488 [6], with permission. Copyright 2005, The Endocrine Society.

Figure 4. Region between -300 and -246 bp upstream of hCYP19 exon I.1 binds transcriptional repressors

Freshly isolated human cytotrophoblasts and lung A549 adenocarcinoma cells in culture were infected with 1 × 10⁶ recombinant adenoviral particles containing hCYP19I.1_-501:hGH, hCYP19I.1_-400:hGH, hCYP19I.1_-350:hGH, hCYP19I.1_-300:hGH or hCYP19I.1_-246:hGH fusion genes. Culture media were harvested and replaced with fresh media every 24 h over a 4 day period. Shown here are the levels of hGH that accumulated in the culture medium between days 2 and 3 of culture. Values are the mean ± SEM (n = 3) of data from a representative of three independent experiments. Endocrinology 146, (2005) 2481-2488 [6], with permission. Copyright 2005, The Endocrine Society.

Figure 5. The pattern of expression of liver receptor homologue-1 (LRH-1) in the early postnatal and adult ovary correlates with the initiation of folliculogenesis

Contiguous sections of gonads collected from mice at various stages during postnatal development (P2-P24) were analyzed by in situ hybridization. Images are shown in darkfield after hybridization with probes specific for steroidogenic factor 1 (SF-1), LRH-1, P45017α, and P450arom. Labels are indicated in the images for granulosa cells (GC), theca interna (th), interstitium (int), and oviduct (ovi). Slides were hybridized for either 35 (LRH-1), 28 (SF-1), or 21 (P450arom/P45017α) days. Scale bars = 500 μm. Dev. Dyn. 234, (2005) 159-168 [32], with permission. Copyright 2005, John Wiley & Sons, Inc.