Family 19 of the P450 super family is responsible for the conversion of C19 androgenic steroids to the corresponding estrogens, a reaction known as aromatization because it involves conversion of the delta4-3-one A-ring of the androgens to the corresponding phenolic A-ring characteristic of estrogens. The gene encoding human aromatase has been cloned and characterized and shown to be unusual compared to genes encoding other P450 enzymes, because there are numerous untranslated first exons that occur in aromatase transcripts in a tissue-specific fashion due to differential splicing as a consequence of the use of tissue-specific promoters. Thus, expression in the ovary uses a proximal promoter that is regulated primarily by cAMP. On the other hand, expression in the placenta uses a distal promoter located at least 40 kb upstream of the start of transcription that is regulated by retinoids. Other promoters are used in brain and adipose tissue. In the latter case, class I cytokines such as IL-6 and IL-11, as well as TNF-alpha, are important regulatory factors. A common 3'-splice junction located upstream of the start of translation is used in all of the splicing events involved in the use of these various promoters. Thus, the coding region of the transcripts, and hence the protein, are identical regardless of the tissue site of expression; what differs in a tissue-specific fashion is the 5'-end of the transcripts. This pattern of expression has great significance both from a phylogenetic and ontogenetic standpoint, as well as for the physiology and pathophysiology of estrogen formation, as will be discussed in this review.

Ovarian granulosa cells are the primary site of estrogen and progesterone synthesis and play an essential role in the maturation of the developing ovum. Freshly isolated granulosa cells are often used to study the regulation of steroid and protein biosynthesis, but the small number of cells available for these cultures has proven inadequate for many detailed gene regulatory studies. The goal of this study was to develop human granulosa (HG) cell lines that maintain differentiated function. The E6 and E7 open reading frames of high risk strains of human papillomavirus have been used to produce immortalized cell lines. Primary cultures of human luteinized granulosa cells were infected with defective retroviruses containing the E6 and E7 regions of human papillomavirus 16 and with the neomycin phosphotransferase gene to confer G418 resistance. Three of eight clones that were isolated after selection in medium containing G418 were found to produce progesterone following treatment with forskolin or dibutyryl cAMP for 48 h. Forskolin caused these cells to retract in the characteristic rounding response, as described in primary HG cultures. One clone, HGL5, was used for a detailed characterization of differentiated function. HGL5 cells retained the ability to increase progesterone production and convert exogenously added androstenedione to estradiol in response to agonists of the protein kinase-A pathway (forskolin and dibutyryl cAMP), but were not responsive to FSH or LH treatment. A key enzyme in the production of estradiol, cytochrome P450 aromatase, has proven difficult to maintain in long term cultures of granulosa cells. For that reason, we examined the expression of aromatase in the transformed HGL5 clone by monitoring mRNA levels. Aromatase mRNA increased by 4- to 5-fold after forskolin treatment, as determined by Northern analysis. This human granulosa cell culture line maintains many of the functions of normal cells and should provide an important model to study the molecular events controlling granulosa cell differentiation and function.