Factors regulating the bovine, caprine, rat and human ovarian aromatase promoters in a bovine granulosa cell model Fatiha Sahmi a , Edmir S. Nicola a , Gustavo O. Zamberlam a , Paulo D.B. Gonçalves a,1 , Jens Vanselow b , Christopher A. Price a,⇑ a Centre de recherche en reproduction animale, Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, QC, Canada b Reproductive Biology, Leibniz Institute for Farm Animal Biology, Dummerstorf, Germany article info Article history: Received 11 November 2013 Revised 21 January 2014 Accepted 10 February 2014 Available online 18 February 2014 Keywords: Aromatase Granulosa NR5A2 FOXL2 Ruminant abstract The ovarian promoter of the primate and rodent genes encoding cytochrome P450 aromatase (CYP19A1) are robustly responsive to forskolin in luteinized cell models, whereas the ruminant ovarian promoter is minimally active. We explored this discrepancy by investigating the activity of the bovine ovarian pro- moter in two bovine granulosa cell models, luteinizing and non-luteinizing cells in vitro. In non-lutein- izing cells, both FSH and IGF1 increased abundance of transcripts derived from the ovarian promoter. Comparison of the activity of promoters of several species in response to transcription factors (forskolin, NR5A2, FOXL2) in luteinizing cells demonstrated that a rat ovarian promoter-luciferase reporter was reg- ulated mainly by forskolin (18-fold increase over basal expression) and addition of NR5A2 or FOXL2 had no further effect. Activity of a human promoter was significantly increased by NR5A2 plus forskolin (153- fold) compared with forskolin alone (71-fold over basal); addition of FOXL2 did not significantly increase promoter activity. Forskolin alone provoked minor activation of caprine and bovine promoter reporters (3-fold over basal), and addition of NR5A2 increased activity (7- to 11-fold). When forskolin, NR5A2 and FOXL2 treatments were combined, the activity of the caprine and bovine promoters increased to 20- and 34-fold, respectively. These data suggest that a major reason why CYP19A1 is not expressed in luteinized cells (and the corpus luteum) of ruminants may be the stimulatory effect of FOXL2, which does not appear to be the case in the human and rat. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction Oestrogens are crucial for fertility in females, as they are neces- sary for granulosa cell proliferation, growth of the oocyte and acquisition of luteinizing hormone (LH) receptors (Knecht et al., 1985; Britt et al., 2004; Quirk et al., 2006). Loss of the enzyme responsible for the production of oestrogens, cytochrome P450 aromatase (CYP19A1), renders mice infertile (Fisher et al., 1998). Expression of ovarian CYP19A1 is stimulated by follicle-stimulating hormone (FSH) in human, rat and bovine granulosa cells (Steinkampf et al., 1987; Fitzpatrick and Richards, 1991; Silva and Price, 2000), and by insulin-like growth factor-1 (IGF1) in humans and cattle (Steinkampf et al., 1988; Spicer and Aad, 2007) although in rodents IGF1 may augment FSH-stimulated CYP19A1 expression rather than act alone (Zhou et al., 1997). FSH and IGF1 act through multiple pathways. FSH increases intracellular cAMP levels and activates protein kinase A (PKA), which in turn activates cAMP response element binding protein, a major transcription factor for FSH target genes (reviewed in Hunzicker-Dunn and Maizels, 2006). In rodent granulosa cells, PKA has also been shown to activate ERK1/2 and phosphoinositide 3-kinase (PI3K)/Akt pathways (Zeleznik et al., 2003; Hunzicker- Dunn et al., 2012), although these may not be as important in bo- vine granulosa cells (Silva et al., 2006). IGF1 acts through the PI3K/ Akt and ERK1/2 pathways independently of PKA in rodent and bo- vine granulosa cells (Gonzalez-Robayna et al., 2000; Mani et al., 2010). Transcription of CYP19A1 is driven by tissue-specific promoters, and there are numerous species differences in the number and structure of the CYP19A1 promoters. In rats, two promoters are de- scribed, in humans there are nine promoters, and in sheep and cat- tle there are five and seven promoters, respectively. In all species, the proximal promoter, also named PII in humans and P2 in http://dx.doi.org/10.1016/j.ygcen.2014.02.008 0016-6480/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Address: Faculté de médecine vétérinaire, C.P. 5000, St- Hyacinthe, QC J2S 7C6, Canada. E-mail address: christopher.price@umontreal.ca (C.A. Price). 1 Present address: Laboratório de Biotecnologia e Reproducão Animal, Universidade Federal de Santa Maria, Santa Maria, Brazil. General and Comparative Endocrinology 200 (2014) 10–17 Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen