309 Identification of estradiol/ERa-regulated genes in the mouse pituitary Hyun Joon Kim1,2,*, Mary C Gieske1,3,*, Kourtney L Trudgen1,*, Susan Hudgins-Spivey1, Beob Gyun Kim4, Andree Krust5,6, Pierre Chambon5,6, Jae-Wook Jeong7, Eric Blalock8 and CheMyong Ko1,3 1Division of Reproductive Sciences, Department of Clinical Sciences, University of Kentucky, Lexington, Kentucky 40536, USA 2Department of Anatomy and Neurobiology, Institute of Health Sciences, Medical Research Center for Neural Dysfunction, School of Medicine, Gyeongsang National University, Jinju, Korea 3Department of Biology, University of Kentucky, Lexington, Kentucky 40536, USA 4Department of Animal Science and Environment, Konkuk University, Seoul 143-701, Korea 5Institut de Genetique et de Biologie Moleculaire et Cellulaire (CNRS, INSERM, ULP, College de France), 67404 Illkirch Cedex, Strasbourg, France 6Institut Clinique de la Souris, BP10142, 67404 Illkirch Cedex, Strasbourg, France 7Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University College of Human Medicine, Grand Rapids, Michigan 49503, USA 8Department of Molecular and Biomedical Pharmacology, University of Kentucky, Lexington, Kentucky 40536, USA (Correspondence should be addressed to C Ko who is now at Division of Clinical and Reproductive Sciences, College of Health Sciences, University of Kentucky, Lexington, Kentucky 40536, USA; Email: [email protected]) *(H J Kim, M C Gieske, and K L Trudgen contributed equally to this work) Abstract Estrogen acts to prime the pituitary prior to the GnRH- pathway in the pituitary. This approach substantiates ERa induced LH surge by undiscovered mechanisms. This study regulation of membrane potential regulators and intracellular aimed to identify the key components that mediate estrogen vesicle transporters, among others, but not the basic action in priming the pituitary. RNA extracted from the components of secretory machinery. Subsequent character- pituitaries of metestrous (low estrogen) and proestrus (high ization of six selected genes (Cacna1a, Cacna1g, Cited1, estrogen) stage mice, as well as from ovariectomized wild- Abep1, Opn3,andKcne2) confirmed not only ERa type and estrogen receptor a (ERa) knockout mice treated dependency for their pituitary expression but also the with 17b-estradiol (E2) or vehicle, was used for gene significance of their expression in regulating GnRH-induced expression microarray. Microarray data were then aggregated, LH secretion. In conclusion, findings from this study suggest built into a functional electronic database, and used for further that estrogen primes the pituitary via ERa by equipping characterization of E2/ERa-regulated genes. These data were pituitary cells with critical cellular components that potentiate used to compile a list of genes representing diverse biological LH release on subsequent GnRH stimulations. pathways that are regulated by E2 via an ERa-mediated Journal of Endocrinology (2011) 210, 309–321 Introduction (Smith et al. 1984). It has also been shown that estrogen downstream pathways include cytoskeleton rearrangement The ovarian steroid estradiol (E2) plays a critical physiological (Powers 1986, Sapino et al. 1986, DePasquale 1999), role in inducing the LH surge by acting on both the regulation of ion channels (Clarke 2002), and energy hypothalamus and the pituitary (Clarke 2002, Christian et al. metabolism (Simpson et al. 2005, Jones et al. 2006). The 2005). While much focus has been placed on the role of molecular mechanisms by which pituitary priming occurs estrogen in the hypothalamic GnRH surge, less work has remain largely unknown, but these functions may play a part. been done concerning the actions of estrogen on the pituitary. Both estrogen receptor (ER) subtypes ERa and ERb are Estrogen has been shown to be involved in priming or expressed throughout the pituitary (Mitchner et al. 1998). sensitizing the pituitary gonadotroph to GnRH stimulus However, diverse lines of evidence indicate that ERa is the (Clarke & Cummins 1984, Clarke 1995a) by increasing predominant mediator of estrogen action in the pituitary. expression of GnRH receptor (GnRH-R) in gonadotrophs Agonists for ERa, but not ERb, were capable of inducing (Liu & Yen 1983, Leung & Peng 1996, Strauss & Barbieri increased LH secretion in estrogen-primed GnRH- 2009), mobilizing secretory granules to the periphery of stimulated rat pituitaries in vitro (Sanchez-Criado et al. the cell (Thomas & Clarke 1997, Thomas et al. 1998), and 2004, 2005). ERa activation was shown to be primarily recruiting the number of gonadotrophs to the pool of responsible for the reorganization of the disrupted organelle those that are capable of responding to GnRH stimulation morphology seen in the gonadotroph after ovariectomy Journal of Endocrinology (2011) 210, 309–321 DOI: 10.1530/JOE-11-0098 0022-0795/11/0210–309 q 2011 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 10/03/2021 12:04:01PM via free access 310 HJKIM, M C GIESKE, K L TRUDGEN and others . ERa-induced genes in the female mouse pituitary (Sanchez-Criado et al. 2006). In addition to ERa knockout cardiac perfusion was performed on 10-week-old mice using (ERaKO) female mice exhibiting complete infertility and 4% neutralized buffered paraformaldehyde. After postfixation lack of ovulation (Dupont et al. 2000, Hewitt & Korach 2003), with the same fixative, tissues were stored in 20% sucrose and we recently reported that targeted deletion of ERa in the gona- then frozen in OCT compound (Tissue-Tek, Sakura Finetek, dotroph caused infertility in female mice (Gieske et al. 2008). Torrance, CA, USA). For primary pituitary culture, 10-week- In the gonadotroph, binding of GnRH to GnRH-R, old cycling WT female mice were used. a G-protein-coupled receptor, activates intracellular signaling pathways causing membrane depolarization and a rapid C Reagents change in intracellular Ca2 concentration, which sub- sequently elicits multiple intracellular events that facilitate LH Antibodies raised in rabbit for adipocyte enhancer binding secretion (Ghosh et al. 1996, Shacham et al. 2001). The well- protein 1 (AEBP1; ARP31592_P050, AVIVA Systems timed and rapid nature of LH secretion on GnRH stimulation Biology, San Diego, CA, USA), Cav2.1 (encoded by Cacna1a; at the time of surge suggests the need for synchronization of ACC-001, Alomone Labs, Jerusalem, Israel), Cav3.1 the cellular secretory machinery. Considering the evidence (encoded by Cacna1g; ACC-021, Alomone Labs), potassium that estrogen priming of the pituitary is required for the voltage-gated channel, Isk-related subfamily, gene 2 induction of the LH surge (Liu & Yen 1983, Strauss & (KCNE2; APC-054, Alomone Labs), Cbp/p300-interacting Barbieri 2009), we hypothesize that estrogen via ERa primes transactivator with Glu/Asp-rich carboxy-terminal domain 1 the pituitary by equipping gonadotrophs and other pituitary (Cited1; XAV-8490, ProSci, Inc., CA, USA), and encepha- cells with key regulators of the LH secretion machinery. lopsin, opsin 3 (Opn3; NLS2134, Novus Biologicals, Inc., This study aimed to identify components that play critical CO, USA) were purchased from indicated company. roles in estrogen priming of the pituitary. For this purpose, Polyclonal antiserum for mouse pituitary LH was purchased genes that are differentially regulated in the pituitary under from the National Hormone and Pituitary Program (Harbor– various estrogen and ERa backgrounds were identified, and UCLA Medical Center, Torrance, CA, USA). GnRH and E2 the expression patterns and functional roles of six selected were purchased from Sigma. Molecular reagents were genes were characterized. Future study on the functional roles purchased from Invitrogen. Cell culture reagents including of the identified genes will begin to reveal the molecular DMEM, gentamicin, BSA, HEPES, trypsin, trypsin inhibitor, mechanism of pituitary estrogen priming for the GnRH- and DNase I were purchased from Sigma. Other reagents induced LH surge. including ITS, fungizone, and fetal bovine serum were purchased from Gibco-BRL. u-Agatoxin TK (selective blocker of Cav2.1 channel), r-Kurtoxin (selective blocker of C Ca 3.1), and E-4031 (selective blocker of HERG K Materials and Methods v channel) were purchased from Alomone Labs. Animals and treatments Gene expression microarray Animal handling procedures were carried out in accordance with the University of Kentucky Animal Care and Use Gene expression microarray was performed with total RNA Committee. Mice were maintained with food and water made (5 mg/group) at the University of Kentucky DNA Microarray available ad libitum in a 14 h light:10 h darkness cycle at 24 8C. Core Facility using the Affymetrix Mouse 430 2.0 All mice used in this study were of C57BL/6 genetic oligonucleotide array set (Affymetrix, Santa Clara, CA, background. ERaKO mice were produced as described USA). Briefly, the total RNA was extracted from the previously (Gieske et al. 2008). For microarray analysis, wild- pituitaries of mice in six groups: naturally cycling WT mice type (WT) mice were divided into two subgroups at the age in either metestrus (group 1) or proestrus (group 2), OVX of 7 weeks after birth. The first group of WT mice was WT mice treated with vehicle (group 3) or E2 (group 4), and ovariectomized (OVX), kept for 3 weeks, and injected (s.c.) OVX ERaKO mice treated with vehicle (group 5) or E2 with 10 mgE2 per mouse or 100 ml sesame oil (vehicle (veh))